The Interplay Between Metabolism, PPAR Signaling Pathway, and Cancer

PPAR Research

The Interplay between Metabolism, PPAR Signaling Pathway, and Cancer

Guest Editors: Daniele Fanale, Valeria Amodeo, and Stefano Caruso The Interplay between Metabolism, PPAR Signaling Pathway, and Cancer PPAR Research The Interplay between Metabolism, PPAR Signaling Pathway, and Cancer

This is a special issue published in “PPAR Research.”All articles are open access articles distributed under the Creative Commons Attribu- tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Khalid Al-Regaiey, USA Geoffrey Girnun, USA Richard P. Phipps, USA Rozalyn M. Anderson, USA Howard P. Glauert, USA Daniele Piomelli, USA P. R. Smith Baker, USA Youfei Guan, China Suofu Qin, USA Yaacov Barak, USA James P. Hardwick, USA Ruth Roberts, UK Marcin Baranowski, Poland Saswati Hazra, USA Stéphane Rocchi, France Josep Bassaganya-Riera, USA Weimin He, USA Enrique Saez, USA Abdulbari Bener, Turkey T. Hsun-Wei Huang, Australia Hervé Schohn, France Carlos Bocos, Spain Norio Ishida, Japan Henrike Sell, Germany Daniela Bonofiglio, Italy James Klaunig, USA Lawrence Serfaty, France Sandra Brunelleschi, Italy Joshua K. Ko, China Xu Shen, China Antonio Brunetti, Italy CarolynM.Komar,USA Xing-Ming Shi, USA Elke Burgermeister, Germany Bettina König, Germany Alexander Staruschenko, USA Norman Buroker, USA Markus P. Kummer, Germany Nguan Soon Tan, Singapore M. Paola Cerù, Italy Christopher Lau, USA Swasti Tiwari, India H. G. Cheon, Republic of Korea S. Mandard, France Vladimir T. Todorov, Germany Annamaria Cimini, Italy Harry Martin, New Zealand Antonella Trombetta, Italy Sharon Cresci, USA Andrew J. McAinch, Australia John P. Vanden Heuvel, USA Michael L. Cunningham, USA Jörg Mey, Germany Raghu Vemuganti, USA Paul D. Drew, USA Raghavendra G. Mirmira, USA Nanping Wang, China William Festuccia, Brazil Kiyoto Motojima, Japan Robert A. Winn, USA Brian N. Finck, USA Shaker Mousa, USA Wei Xu, USA Pascal Froment, France Elisabetta Mueller, USA Qinglin Yang, USA Yuchang Fu, USA Marcelo H. Napimoga, Brazil Tianxin Yang, USA Andrea Galli, Italy Dipak Panigrahy, USA Wei Zhao, USA C. Giaginis, Greece Hemang Parikh, USA Contents

The Interplay between Metabolism, PPAR Signaling Pathway, and Cancer Daniele Fanale, Valeria Amodeo, and Stefano Caruso Volume 2017, Article ID 1830626, 2 pages

Fatty Acids of CLA-Enriched Egg Yolks Can Induce Transcriptional Activation of Peroxisome Proliferator-Activated Receptors in MCF-7 Breast Cancer Cells Aneta A. Koronowicz, Paula Banks, Adam Master, Dominik Domagała, Ewelina Piasna-Słupecka, Mariola Drozdowska, Elżbieta Sikora, and Piotr Laidler Volume 2017, Article ID 2865283, 12 pages

Deciphering the Roles of Thiazolidinediones and PPAR𝛾 in Bladder Cancer Melody Chiu, Lucien McBeth, Puneet Sindhwani, and Terry D. Hinds Volume 2017, Article ID 4810672, 9 pages

PPAR Agonists for the Prevention and Treatment of Lung Cancer SowmyaP.Lakshmi,AravindT.Reddy,AsokaBanno,andRajuC.Reddy Volume2017,ArticleID8252796,8pages

Potential Role of ANGPTL4 in the Cross Talk between Metabolism and Cancer through PPAR Signaling Pathway Laura La Paglia, Angela Listì, Stefano Caruso, Valeria Amodeo, Francesco Passiglia, Viviana Bazan, and Daniele Fanale Volume 2017, Article ID 8187235, 15 pages

MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers Dorothea Portius, Cyril Sobolewski, and Michelangelo Foti Volume 2017, Article ID 7058424, 19 pages

Peroxisome Proliferator-Activated Receptor Modulation during Metabolic Diseases and Cancers: Master and Minions Salvatore Giovanni Vitale, Antonio Simone Laganà, Angela Nigro, Valentina Lucia La Rosa, Paola Rossetti, Agnese Maria Chiara Rapisarda, Sandro La Vignera, Rosita Angela Condorelli, Francesco Corrado, Massimo Buscema, and Rosario D’Anna Volume 2016, Article ID 6517313, 9 pages

PPARs and Mitochondrial Metabolism: From NAFLD to HCC Tommaso Mello, Maria Materozzi, and Andrea Galli Volume 2016, Article ID 7403230, 18 pages

PPAR𝛿 as a Metabolic Initiator of Mammary Neoplasia and Immune Tolerance Robert I. Glazer Volume 2016, Article ID 3082340, 7 pages

PPAR Gamma in Neuroblastoma: The Translational Perspectives of Hypoglycemic Drugs Serena Vella, Pier Giulio Conaldi, Tullio Florio, and Aldo Pagano Volume 2016, Article ID 3038164, 10 pages

Commonalities in the Association between PPARG and Vitamin D Related with Obesity and Carcinogenesis Borja Bandera Merchan, Francisco José Tinahones, and Manuel Macías-González Volume 2016, Article ID 2308249, 15 pages Hindawi PPAR Research Volume 2017, Article ID 1830626, 2 pages https://doi.org/10.1155/2017/1830626

Editorial The Interplay between Metabolism, PPAR Signaling Pathway, and Cancer

Daniele Fanale,1 Valeria Amodeo,2 and Stefano Caruso3

1 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, 90127 Palermo, Italy 2Samantha Dickson Brain Cancer Unit, UCL Cancer Institute, University College London, London WC1E 6DD, UK 3Genomique´ Fonctionnelle des Tumeurs Solides, INSERM, UMR 1162, 75010 Paris, France

Correspondence should be addressed to Daniele Fanale; [email protected]

Received 9 March 2017; Accepted 12 March 2017; Published 26 April 2017

Copyright © 2017 Daniele Fanale et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The peroxisome proliferator-activated receptors (PPARs) and other metabolic disorders, such as obesity, understanding belong to the ligand-inducible nuclear hormone receptor the potential molecular mechanisms underlying the interplay superfamily including three major members: PPAR𝛼 (also between metabolism, PPAR signaling, and cancer may rep- called NR1C1), PPAR𝛽/𝛿 (also called NR1C2), and PPAR𝛾 resent an interesting research field for the development of (also called NR1C3). Despite several similarities, each PPAR novel strategies useful for the prevention and treatment of isoform shows specific functions likely due to different cancer. Since there exists a strong correlation between obesity biochemical properties, variable tissue distribution, and dif- and diabetes and both have been also shown to increase ferential responses to different ligands. PPAR transcriptional cancer risk, the identification of the molecular mechanisms activity can be modulated through a nongenomic cross by which PPAR modulates these events may help us to better talk with phosphatases and kinases, including ERK1/2, p38- understand how the diet may affect the cancer susceptibility. MAPK, PKA, PKC, AMPK, and GSK3. PPARs can form In this special issue we have assembled different studies heterodimers with retinoid X receptor (RXR) modulat- describing possible intracellular pathways involved in these ing the expression of genes involved in lipid metabolism, processes, in order to increase the current knowledge about adipogenesis, maintenance of metabolic homeostasis, and the correlation between metabolic syndromes and cancer via inflammation and inducing also anticancer effects in a variety activation of different PPAR signaling pathways. In particular, of human tumors. Although all PPAR isoforms are impli- our endpoint was to provide an overview about the potential cated in several metabolic syndromes, PPAR𝛾 seems to be roles of PPARs in modulating the cancer risk induced by mostly involved in tumorigenesis regulation via activation metabolic disorders such as diabetes and obesity. Here, we of different pathways. Therefore, the modulation of PPAR gathered 1 original research paper and 9 review articles signaling pathways could be a potential novel strategy to that describe and discuss the various functions of PPARs in inhibit carcinogenesis and tumor progression. PPAR𝛾 can the context of several metabolic alterations associated with be activated by natural ligands, such as fatty acids and different types of cancer. their derivatives, as well as synthetic ligands, such as thia- In our review article titled “Potential Role of ANGPTL4 zolidinediones (TZDs), including ciglitazone, rosiglitazone, in the Cross Talk between Metabolism and Cancer through troglitazone, and pioglitazone. TZDs have been shown to be PPAR Signaling Pathway,” we speculated and discussed a class of drugs with potent insulin-sensitizing activity used the potential role of ANGPTL4 as key player in mediat- to improve lipid and glucose metabolism in obesity and type 2 ing the cross talk between metabolic syndromes, such as diabetes via PPAR. Since accumulating evidence highlighted diabetes and obesity, and cancer through regulation of its the role of PPAR signaling in carcinogenesis, type 2 diabetes, expression by PPARs. Indeed, since ANGPTL4 is involved 2 PPAR Research in several metabolic conditions, both physiological and PPAR agonists in lung cancer treatment. Finally, the authors pathological, including angiogenesis, glucose homoeostasis, discussed how PPAR ligands may be used in the context of lipid metabolism, and tumorigenesis, we hypothesized that novel therapeutic strategies against this disease. its transcriptional regulation by PPARs could represent a In “Deciphering the Roles of Thiazolidinediones and gateway between obesity, insulin sensitivity, and cancer. PPAR𝛾 in Bladder Cancer” M. Chiu and colleagues discussed ThereviewarticlebyS.G.Vitaleetal.“Peroxisome how some thiazolidinediones, synthetic ligands of PPAR𝛾 Proliferator-Activated Receptor Modulation during used in the treatment of diabetes mellitus type 2, could Metabolic Diseases and Cancers: Master and Minions” significantly increase the risk of developing bladder cancer. provided an overview on the different roles of PPARs Lastly, A. A. Koronowicz and collaborators in their in control of the expression of genes involved in energy research paper titled “Fatty Acids of CLA-Enriched Egg homeostasis, cell proliferation, and apoptosis, discussing the Yolks Can Induce Transcriptional Activation of Peroxisome involvement of these nuclear receptors in tumorigenesis and Proliferator-Activated Receptors in MCF-7 Breast Cancer development of metabolic diseases. Cells” investigated the effects in vitro of fatty acids from CLA- B. B. Merchan and collaborators in their review article enriched egg yolks (EFA-CLA) acting as potential ligands for titled “Commonalities in the Association between PPARG PPAR receptors in breast cancer cell line MCF-7. The authors and Vitamin D Related with Obesity and Carcinogenesis” showed that PPAR-responsive genes can be regulated by havefocusedontherecentadvancesthatledtohypothesizing EFA-CLA, leading to a reduction of tumor cell proliferation, a possible cross talk between PPARG, Vitamin D system, with a greater influence by EFA-CLA treatment compared to obesity, and cancer, suggesting a close cooperation between nonenriched FAs or single synthetic CLA isomers. the vitamin D/VDR system and PPARG signaling in order to In conclusion, understanding the different roles of PPARs maintain a correct metabolic homeostasis. Interestingly, the in the cross talk between metabolic syndromes, such as authors reported several studies describing how the shortage diabetes and obesity, and cancer could help us, in future, to ofVitaminDanddecreaseinPPARGlevelsmaybeinvolved identify novel potential therapeutic targets involved in the in obesity and cancer development. cancer development and different cancer-related metabolic In another manuscript titled “PPAR Gamma in Neu- syndromes. This special issue, thanks to the interesting roblastoma: The Translational Perspectives of Hypoglycemic contributions by various authors, attempts to provide a small Drugs” S. Vella et al., by reviewing literature data, discussed overview of the studies present in literature regarding the the potential beneficial effects of hypoglycemic drugs, such as involvement of PPAR Signaling Pathway in carcinogenesis thiazolidinediones and metformin, in treatment of neurob- and metabolic alterations, enriching the current knowledge lastoma patients. in the field and opening up new roads towards multidis- A noteworthy contribution to this special issue was the ciplinary approaches that promote the interaction between review article titled “PPAR𝛿 as a Metabolic Initiator of basic research and clinical research. Mammary Neoplasia and Immune Tolerance” by R. I. Glazer describing the involvement of PPAR𝛿 in the initiation and Daniele Fanale promotion of mammary tumorigenesis through its pivotal Valeria Amodeo role in regulating metabolism, inflammation, and immune Stefano Caruso tolerance. An interesting review article by T. Mello and colleagues titled “PPARs and Mitochondrial Metabolism: From NAFLD to HCC” described the functions of PPARs in the modulation of liver mitochondrial metabolism during the progression from nonalcoholic fatty liver disease to hepatocellular carcinoma, hypothesizing the possibility, in future, of using new therapeutic approaches able to selectively target the fuel requirements of HCC. In the review article titled “MicroRNAs-Dependent Reg- ulation of PPARs in Metabolic Diseases and Cancers,” D. Portius et al. stressed out the miRNA-mediated regulation of PPARs in the context of metabolic disorders, inflammation, and cancer. Moreover, the authors evaluated the reciprocal control of miRNA expression by PPARs, to finally speculate about the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNAs. Instead, S. P. Lakshmi et al. provided a review article titled “PPAR Agonists for the Prevention and Treatment of Lung Cancer,” where authors described the role of PPAR𝛼, PPAR𝛽/𝛿,andPPAR𝛾 in lung cancer pathogenesis and dissected the current literature on the multifaceted effects of Hindawi PPAR Research Volume 2017, Article ID 2865283, 12 pages https://doi.org/10.1155/2017/2865283

Research Article Fatty Acids of CLA-Enriched Egg Yolks Can Induce Transcriptional Activation of Peroxisome Proliferator-Activated Receptors in MCF-7 Breast Cancer Cells

Aneta A. Koronowicz,1 Paula Banks,1 Adam Master,2 Dominik DomagaBa,1 Ewelina Piasna-SBupecka,1 Mariola Drozdowska,1 Elhbieta Sikora,1 and Piotr Laidler3

1 Department of Human Nutrition, Faculty of Food Technology, University of Agriculture in Krakow, Balicka 122, 30-149 Krakow, Poland 2Department of Biochemistry and Molecular Biology, Medical Centre for Postgraduate Education, Marymoncka 99, 01-813 Warsaw, Poland 3Department of Medical Biochemistry, Jagiellonian University Medical College, Kopernika 7, 31-034 Krakow, Poland

Correspondence should be addressed to Aneta A. Koronowicz; [email protected]

Received 21 October 2016; Revised 2 February 2017; Accepted 12 February 2017; Published 26 March 2017

Academic Editor: Daniele Fanale

Copyright © 2017 Aneta A. Koronowicz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In our previous study, we showed that fatty acids from CLA-enriched egg yolks (EFA-CLA) reduced the proliferation of breast cancer cells; however, the molecular mechanisms of their action remain unknown. In the current study, we used MCF-7 breast cancer cell line to determine the effect of EFA-CLA, as potential ligands for peroxisome proliferator-activated receptors (PPARs), on identified in silico PPAR-responsive genes: BCAR3, TCF20, WT1, ZNF621,andTHRB (transcript TR𝛽2). Our results showed that EFA-CLA act as PPAR ligands with agonistic activity for all PPAR isoforms, with the highest specificity towards PPAR𝛾.In conclusion, we propose that EFA-CLA-mediated regulation of PPAR-responsive genes is most likely facilitated by cis9,trans11CLA isomer incorporated in egg yolk. Notably, EFA-CLA activated PPAR more efficiently than nonenriched FA as well as synthetic CLA isomers. We also propose that this regulation, at least in part, can be responsible for the observed reduction in the proliferation of MCF-7 cells treated with EFA-CLA.

1. Introduction cancer cell lines [5–7]. In particular, PPAR𝛾 isoform was shown to reduce cancer cell proliferation as well as regulate Peroxisome proliferator-activated receptors (PPARs) are celldifferentiation,activateapoptosis,andinhibitangiogen- ligand-activated transcription factors. Various fatty acids esis [8–10]. Specifically, the administration of specific PPAR𝛾 and their metabolic derivatives act as natural ligands for agonist resulted in cells arrest in G1 phase and inhibited PPARs [1]. Some, including linoleic, linolenic, and arachi- proliferation [5, 11]. However, available literature presents donic acid, were found to activate PPARs even at micro- also contradicting results. In some studies, PPAR𝛾 specific molar, physiologically relevant concentrations [2]. Hydrox- antagonist, T0070907, significantly reduced proliferation and yoctadecadienoic acids (HODEs), products of linoleic acid migration of breast cancer cells [12, 13]. oxidation as well as arachidonic acid metabolite 15d-PGJ2 Conjugated linoleic acid (CLA) term includes several (15-deoxyprostaglandin J2), were also associated with PPAR isomers of linoleic acid, with two main isomers: cis9,trans11 activation [3, 4]. (80–90% of total CLA) and trans10,cis12. Available literature It has been suggested that ligand-dependent activation of shows that CLA acts as a potent PPARs ligand and is involved PPARs results in the inhibition of proliferation in some model in modulating lipid metabolism through PPAR-mediated 2 PPAR Research pathways [14]. However, data showed isomer-specific activity and PPAR𝛾 (pioglitazone (PIO), troglitazone, and T0070907) of CLA; specifically, cis9,trans11 was characterized as PPAR were prepared as per appropriate protocols of the manufac- agonist [15, 16] while trans10,cis12 was shown to inhibit the turer. Respective concentrations were selected based on their activity of synthetic PPAR agonists [15]. In addition, studies EC/IC50 characteristics and confirmed for MCF-7 cell line showed potential antitumor properties of cis9,trans11 [17–20] using Cytotoxicity LDH Test (Roche, Poland). while the opposite effect was observed for trans10,cis12 isomer [18]. 2.4. Cell Cultures. The human breast adenocarcinoma cell PPARs act as transcription factors and regulate the line MCF-7 (ATCC5 HTB22TM) was purchased from the expression of dependent genes by binding to their PPREs. A American Type Culture Collections. Cells were cultured in significant number of genes regulated by PPARs have been appropriate medium (Sigma-Aldrich, MO, USA) as per the described; however, the list is not exhaustive and is constantly ATCC protocol with the addition of 10% FBS (Sigma-Aldrich, being updated as new results are being published from both MO, USA). experimental data and bioinformatics analyses of promoter Cell viability was determined by Crystal Violet Assay regions and PPRE consensus sequences. In the current study, (Sigma-Aldrich, MO, USA). we applied those tools to identify in silico PPRE selected genes involved in cell cycle progression and proliferation. 2.5. Fatty Acid Treatment. The experimental medium con- Next, we analyzed the effect of synthetic cis9,trans11CLA and tained MEM supplemented with 10% FBS and appropri- trans10,cis12CLA isomersaswellasamixtureoffattyacids ate treatment: (a) fatty acids extract at 0.5 mg/mL from extracted from CLA-enriched and nonenriched egg yolk on CLA-enriched egg yolks (EFA-CLA), (b) fatty acids extract the expression of those genes. To the best of our knowledge, at 0.5 mg/mL from nonenriched egg yolks (EFA), (c) our study is the first to address the effect of CLA incorporated cis9,trans11 synthetic isomer (final concentration at 35 𝜇M), in fatty acids profile of the egg yolk; we expect that activity of (d) trans10,cis12 synthetic isomer (final concentration at CLA in such a “bioorganic” form may deviate from that of 13 𝜇M), (e) untreated cell control (empty control, EC), and a synthetic form. The presence of other fatty acids in an egg (f) negative control (NC; ethanol at final concentration yolk, which themselves can act as potential ligands for PPARs, 0.1%). Synthetic PPARs agonists and antagonist were used may modulate the action of CLA; therefore, our data may as positive controls for PPAR𝛼 (10 𝜇MWY14643and10𝜇M be particularly important for the evaluation of CLA-enriched GW-6471), PPAR𝛿 (2 𝜇MGW-0742and1𝜇MGSK0660), food products. and PPAR𝛾 (40 𝜇MPIO,10𝜇M troglitazone, and 10 𝜇M T0070907). Each treatment included 3 biological and 3 2. Materials and Methods technical replicates.

2.1. Production of CLA-Enriched Egg Yolks. Production of CLA- 2.6. Plasmids. PPAR expression vectors were prepared using enriched egg yolks was performed in the National Research Gateway5 Cloning System (Thermo Fisher, USA). Briefly, Institute of Animal Production in Krakow (Poland), as per PPARA (CR456547_1), PPARD (NM_006238.4), and PPARG the recommendations of the Local Animal Ethics Committee (NM_015869.4) ORF sequences were synthesized, opti- (approval number: 851/2011) as described previously [21]. mized for the expression in human cells, and cloned into ∘ Eggs were collected and stored at 4 C, and yolks were the pDONR221 Entry Vectors (GeneArt, Thermo Fisher, ∘ separated from albumen, homogenized, and frozen at −20 C. USA). Subsequently, the ORF inserts were transferred into ∘ Samples were then lyophilized and again stored at −20 Cuntil pcDNA6.2/N-EmGFP-DEST Destination Vectors (Thermo further analyses. Fisher, USA) under the CMV promoter control via Clonase II Recombination Reaction. 2.2. Extraction and Analysis of Fatty Acids Composition. Lipids from control and CLA-enriched yolks were extracted 2.7. Cell Transfection with PPAR Encoding Plasmids. Cell by using modified Folch method [22] as described previously lines with PPARA, PPARD, and PPARG overexpression [23]. 10 mg of each lipid extract was subjected to saponifi- were obtained via transient transfections with pcDNA6.2/N- cation with 0.5 M KOH/methanol followed by methylation EmGFP-DEST vectors containing respective human PPAR with 14% (v/v) BF3/methanol and extraction with hexane. ORF. MCF-7 cells were seeded on 12-well plates, at 1× 5 Fatty acid methyl esters (FAME) were analyzed by GC/MS as 10 cells per well. 24 h after seeding, cells were transiently described previously [23]. transfected with 1.5 𝜇g of PPAR encoding plasmids using Lipofectamine (Thermo Fisher Scientific, MA, USA) in 2.3. CLA Isomers and Agonists/Antagonists of PPAR. OPTI-MEM medium (Thermo Fisher Scientific, MA, USA). cis9,trans11CLA and trans10,cis12CLA isomers (Nu-Chek 24 h after transfection, the growth medium was replaced with Prep, USA) were dissolved in ethanol and stored under selective MEM medium with 10% FBS and 5.0 𝜇g/mL blas- ∘ nitrogen in −20 C and were introduced to cell cultures at ticidin (BioShop, Canada). Transfected cells were cultured final concentrations corresponding to their concentration until confluency. in CLA-enriched egg yolk: cis9,trans11 at 30 𝜇Mand Real-time PCR and western blot method were performed trans10,cis12 at 12 𝜇M. to confirm the presence of PPAR plasmids after transfection The synthetic agonists and antagonists for PPAR𝛼 (Figure S1 and Table S2, Supplementary Material available (WY14643 and GW-6471), PPAR𝛿 (GW-0742 and GSK0660), online at https://doi.org/10.1155/2017/2865283). PPAR Research 3

2.8. Transfection with PPRE Plasmid. Cell lines overexpress- 120 ing, respectively, PPARA, PPARD, and PPARG were seeded 100 ∗ 1×105 ∗ ∗ on the 12-well plates, at cells per well. After 24 ∗ ∗ hours, cells were transfected with 0.7 𝜇g X3 PPRE-TK- 80 ∗ ∗ 𝜇 ∗ ∗ luc plasmid (Cat. # 1015, Addgene, USA) and 0.7 gpRL 60 ∗ control (Cat. # E2261, Promega, WI, USA) using Lipofec- tamine (Thermo Fisher Scientific, MA, USA) in OPTI-MEM 40 medium (Thermo Fisher Scientific, MA, USA). 20 Cell Viability (% of Control) (% of Cell Viability 2.9. Dual-Luciferase Assay. 24 hours after transfection with 0 PPRE plasmid, the medium was again replaced with MEM EFA medium containing 10% FBS and appropriate experimental treatment as described above. 24 hours after treatment, cells EFA-CLA were harvested for isolation of protein luciferase. cis9,trans11CLA

The luciferase protein (Photinus pyralis and Renilla reni- trans10,cis12CLA formis) detection was performed using Dual-Luciferase5 Reporter Assay System (Promega, WI, USA) in GloMax5 24 h 20/20 Single Tube Luminometer (Promega, WI, USA), 48 h according to the manufacturer’s instructions. 72 h Figure 1: Effect of fatty acids on MCF-7 cells viability. Values are 2.10. In Silico Selection and Experimental Confirmation of expressed as means ± SD for 𝑁≥9, standardized to control (NC) as ∗ PPAR-Dependent Genes (PPAR-Responsive mRNAs). PPAR- 100%. Statistical significance was based on 𝑡-test; 𝑝 < 0.05 versus responsivegeneswereselectedinsilicobysearching control. for peroxisome proliferator hormone response elements 󸀠 (PPREs, AGGTCANAGGTCA) within promoters and/or 5 - cis-regulatory regions of the promoters of genes involved in cell cycle progression and proliferation. This search was transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by performed with NCBI Gene and Blast tools. wet electroblotting. Subsequently, the immobilized proteins Experimentally, 24 hours after transfection with respec- were incubated with appropriate primary antibody, specific tive PPAR plasmids, the medium was replaced with MEM for PPAR𝛼 (SAB2101852), PPAR𝛾 (SAB2101853), and PPAR𝛿 medium containing 10% FBS and appropriate experimental (AV32880) as well as for selected in silico WT1 (SAB2102716), treatment as described above. 48 hours after treatment, cells THRB (AV36994), and TCF20 (SAB2106444) from Sigma- were harvested for mRNA isolation and RT-qPCR. Aldrich, MO, USA, or 𝛽-actin (#8457) or 𝛽-tubulin (#2128) from Cell Signaling Technology, MA, USA. Finally, appro- 2.11. RNA Isolation, cDNA Synthesis, and RT-qPCR Analysis. priate secondary antibody conjugated with horseradish per- Total RNA was isolated from the cells using RNA isolation oxidase (#7074, Cell Signaling Technology, MA, USA) was kit for cell cultures (A&A Biotechnology, Poland). Reverse applied. Detection was executed by chemiluminescence, 𝜇 transcription was performed on 1 g of total RNA using Max- using Clarity6 Western ECL Substrate (Bio-Rad, CA, USA). ima First-Strand cDNA Synthesis kit for RT-qPCR (Thermo To remove the antibodies from the membrane, we used Scientific, MA, USA). Quantitative verification of genes was western blot stripping buffer (Thermo Scientific, MA, USA). performed using CFX96 Touch6 Real-Time PCR Detection System instrument (Bio-Rad, CA, USA) and SYBR Green 2.13. Statistical Analysis. All experiments were performed Precision Melt Supermix kit (Bio-Rad, CA, USA). Conditions at least three independent times and measured in tripli- of individual PCR reactions were optimized for given pair of cate. Shapiro-Wilk’s test was applied to assess normality of oligonucleotide primers (Table S1, Supplementary Material). 𝑡 ∘ distribution. An independent samples -test was applied to Basic conditions were as follows: 95 Cfor10min,45PCR 𝑝 < 0.05 ∘ ∘ ∘ compare unpaired means between two groups. cycles at 95 C, 15 s; 59 C, 15 s; 72 C, 15 s, followed by melting ∘ ∘ was considered statistically significant. All analyses were curve analysis (65–97 Cwith0.11Cramprateand5acqui- ∘ performed using Statistica ver.12 (StatSoft, Tulsa, OK, USA). sitions per 1 C). Results were normalized using at least two reference genes (GAPDH, HPRT1, ACTB,orHSP90AB1)and −ΔΔC 3. Results were calculated using the 2 Tmethod[24]. 3.1. Cell Viability. Treatment with both extracts, EFA and 2.12. Protein Isolation and Western Blot Analysis. Cell lysis EFA-CLA, decreased viability of MCF-7 breast cancer cell was carried out using Cell Lysis Buffer (Cell Signaling Tech- line compared to the control; however, the effect of EFA-CLA nology, MA, USA) as per the manufacturer’s protocol. Total was more evident compared to EFA. 72 h after treatment, protein quantification was performed using Pierce6 BCA cell viability in EFA-CLA-treated group decreased by 50% Protein Assay Kit (Thermo Fisher Scientific, MA, USA). while for EFA the decrease in viability reached 32% (Figure 1). Each western blot followed a similar procedure. Pro- Treatment with synthetic trans10,cis12CLA reduced cell via- tein extract was separated on a polyacrylamide gel and bility in a linear manner with incubation time, reaching 43% 4 PPAR Research

PP oter ARA (1) First transfection om or Transactivation r P P with a P P d PA A efficiency i R Overexpression of m R s g D a e PPAR PPAR PPAR l or or n o P e r receptor proteins

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N of MCF-7 cells A

Figure 2: Experimental mechanism for studying the activity of EFA-CLA as a ligand for PPAR. DBD: DNA-binding domain specific for PPRE sequence in promoter regions of genes regulated by PPAR; LBD: ligand-binding domain (e.g., EFA-CLA).

at 72 h. The reductive effect of cis9,trans11CLA isomer was 3.3. Selective Effect of FA on Transcriptional Activity of less evident and statistically significant only after 72 h (overall PPARs. The selective effects of the studied FA as potential reduction in viability by 15%). PPAR ligands are shown in Figures 4(a)–4(d). EFA-CLA was determined to be the most specific for PPAR𝛾 (3.5-fold 3.2. Effects of EFA-CLA on Transcriptional Activity of PPARs. increase in activity, 𝑝 < 0.001; Figure 4(a)). EFA extract acted To analyze the activity and specificity of various CLAs as as an antagonist towards both PPAR𝛼 and PPAR𝛿, while it potential PPAR ligands, we applied the PPAR-dependent exhibited only negligible agonist activity on PPAR𝛿 (1.44- luciferase expression model (Figure 2). We used specific fold increase in activity, 𝑝 > 0.05, Figure 4(b)). cis9,trans11 agonists and antagonists for each isoform of PPARs as isomer showed agonist properties towards all PPAR isoforms, positive controls. Our results confirmed the expected effects with the strongest effect on PPAR𝛾 (2.37-fold increase in of selected agonists and antagonists (Figures 3(a)–3(c)). The activity, 𝑝 < 0.005; Figure 4(c)). trans10,cis12 isomer showed effect of experimental FA extracts varied. Compared to the no significant effect on transactivation of both 𝛼PPAR and negative control, EFA-CLA significantly increased the activity PPAR𝛿 (𝑝 > 0.05, Figure 4(d)), while it showed an antagonist of PPAR𝛼 (202% of NC; 𝑝 < 0.05; Figure 3(a)), PPAR𝛿 activity towards PPAR𝛾 (𝑝 < 0.01, Figure 4(d)). (187.10% of NC; 𝑝 < 0.01;Figure3(b)),andPPAR𝛾 (353% of NC; 𝑝 < 0.001; Figure 3(c)). Compared to EFA extract, 3.4. Prediction of Potential PPRE-Dependent Genes In Silico. EFA-CLA also showed statistically significant activation of The prediction of potential PPRE-responsive genes was per- all PPAR isoforms (Figures 3(a)–3(c)). Synthetic cis9,trans11 formed in silico. NCBI database was searched for the presence isomer also activated significantly all PPARs, PPAR𝛼 (211% of specific PPRE (peroxisome proliferator response element) of NC; 𝑝 < 0.05; Figure 3(a)), PPAR𝛿 (221.88% of NC; consensus sequences (AGGTCAAAGGTCA, AGGTCA- 𝑝 < 0.01;Figure3(b)),andPPAR𝛾 (237% of NC; 𝑝 < 0.01; GAGGTCA, AGGTCACAGGTCA, or AGGTCATAGGTC- 󸀠 Figure 3(c)). trans10,cis12CLA isomer had little or no effect A) in the 5 region of genes linked to oncogenesis and on the activation of PPAR𝛼 and PPAR𝛿 (Figures 3(a) and cell cycle (Figure 5). Seven genes were identified: BCAR3, 3(b)); however, it reduced the activity of PPAR𝛾 (85% of NC; LZTS, SLC5A1, TCF20, WT1, ZNF621,andTHRB (transcript 𝑝 < 0.05; Figure 3(c)). TR𝛽2), potentially regulated by PPARs (Table 1). THRB PPAR Research 5

600% 300% ∗ ∗ 500% 250% ∧∗ ∗ 400% 200%

300% ∗ 150% ∧∗ ∗ 200% 100% ∗ 100% ∗ 50% 0%

0% control) (% of activity Luciferase Luciferase activity (% of control) (% of activity Luciferase NC NC EFA EFA agonist agonist EFA-CLA EFA-CLA antagonist cis9,trans11CLA cis9,trans11CLA trans10,cis12CLA 0742 GW- trans10,cis12CLA WY 14643 GSK0660 antagonist GSK0660 6471 GW-

PPAR PPAR (a) (b) 800% ∗ 700% 600% 500% ∧∗ 400% ∗ 300% ∗ 200% ∗ ∗ 100% ∗ 0% Luciferase activity (% of control) (% of activity Luciferase NC EFA EFA-CLA antagonist PIO agonist PIO cis9,trans11CLA trans10,cis12CLA Troglitazone agonist Troglitazone T 0070907

PPAR (c)

Figure 3: Effect of EFA-CLA on the activity of (a) PPAR𝛼,(b)PPAR𝛿,and(c)PPAR𝛾 basedonmeasuredluciferaseactivityindual-luciferase assay. Values are expressed as means ± SEM for 𝑁≥12, standardized to control (NC) as 100%. Statistical significance was based on 𝑡-test; ∗ ∧ 𝑝 < 0.05 versus NC or 𝑝 < 0.05 versus EFA.

Table 1: Identification of in silico putative PPAR-responsive genes.

Gene symbol Transcript Position NCBI reference sequence BCAR3 AGGTCAGAGGTCA 93663502–93663514 NC_000001.11 LZTS1 AGGTCAAAGGTCA 20248971–20248983 NC_000008.11 SLC5A1 AGGTCACAGGTCA 32033858–32033870 NC_000022.11 TCF20 AGGTCATAGGTCA 42271609–42271621 NC_000022.11 WT1 AGGTCAGAGGTCA 32470961–32470973 NC_000011.10 32470822–32470834 ZNF621 AGGTCAGAGGTCA 41052623–41052635 NC_000003.12 THRB (TR𝛽2) AGGTCACAGGTCA 24169753–24169765 NC_000003.12 BCAR3: breast cancer antiestrogen resistance 3; LZTS1: leucine zipper putative tumor suppressor 1; SLC5A: solute carrier family 5 member 1; TCF20: transcription factor 20; WT1:Wilmstumor1;ZNF621: zinc finger protein 621; THRB: thyroid hormone receptor beta. 6 PPAR Research

EFA-CLA 4.5 2.5 EFA 4.0 3.5 2.0 3.0 1.5 2.5 2.0 1.0 1.5 Luciferase activity Luciferase Luciferase activity Luciferase 1.0 0.5 PPAR PPAR PPAR 0.5 2.02 1.87 3.53 PPAR PPAR PPAR 0.87 0.78 1.44 0.0 0.0

EFA-CLA EFA (a) (b)

c9,t11CLA 3.0 t10,c12 1.8 CLA 2.8 1.6 2.6

2.4 1.4

2.2 1.2

2.0 1.0

1.8 0.8 Luciferase activity Luciferase 1.6 PPAR PPAR PPAR Luciferase activity Luciferase 0.6 1.23 1.27 0.85 1.4 0.4 1.2 0.2 1.0    PPAR PPAR PPAR 0.0 2.11 2.22 2.37

cis9,trans11CLA trans10,cis12CLA

Figure 4: Selective effect of FA on PPARs expressed as fold difference versus control (100%), based on data from Figure 3. Values are expressed as means ± SEM for the 𝑁≥12.

gene was identified by the presence of the PPRE consensus EFA-CLA added to the PPAR𝛾-overexpressing cells ele- sequence in a region of the alternative promoter for TR𝛽2 vated the expression of TCF-20 over 3.2-fold and ZNF621 over isoform (intron between the 4th and 5th exon). Among 3.1-fold, while decreasing the expression of WT1 gene 1.2- identified potential PPRE-dependent genes, few were selected fold. However, the latest may be explained, at least in part, for further experimental analyses, including TCF20, WT1 from the fact that WT1 gene is cotranscribed with interfering ZNF621,andTHRB. long, noncoding antisense RNA (WT1-AS) from the same bidirectional promoter. For cells overexpressing PPAR𝛿,EFA- 3.5. Effects of EFA-CLA on the Expression of PPAR-Regulated CLA treatment resulted in the elevated expression of TCF-20 Genes. Expression of selected PPAR-responsive genes (con- over 3-fold, while for the PPAR𝛼-overexpressing cells ZNF621 taining PPRE) has been tested in response to various exper- gene was upregulated 1.8-fold. imental fatty acids as potential ligands for PPARA, PPARD, The strongest enhancement of TCF-20 expression (over or PPARG. Our results showed both agonist and antagonist 13-fold) was observed in PPAR𝛾-andPPAR𝛿-overexpressing effects of studied experimental FA. cells after treatment with trans10,cis12CLA. Interestingly, the PPAR Research 7

Extraction of CLA-enriched CLA fatty acids

Translation CDS

PPAR PPAR r.p. Splicing P P pre-mRNA A CLA PPAR CDS R -

RXR R

PPAR PPAR N A

n o ti ip Cancer cell r sc n ra DNA T R A PPAR as transcription factors P Recruitment and activation RNA pol II P y es −)? b n ( d e RXR te

g CLA +)? a ( ul

R eg

A r PPAR r.p.

P CoA s P CoR e PPAR en G PPAR r.p. CDS E Promoter PPR mRNA

Figure 5: Molecular aspects of CLA-induced accumulation of PPAR-responsive transcripts. PPAR-r. mRNA: PPAR-regulated mRNAs; PPAR- r.p.: PPAR-regulated proteins; PPRE: peroxisome proliferator hormone response element (AGGTCANAGGTCA); RXR: retinoid X receptor; ORF: open reading frame (coding sequence).

expression of THRB (TR𝛽2variant)genewasalsostrongly studies showed additional beneficial properties of CLA- increased by the treatment with trans10,cis12CLA over enriched eggs in reducing proliferation of breast cancer and 1 8 . 1 5 - , 1 7. 2 - , a n d 7. 9 - f o l d i n P PA R 𝛿-, PPAR𝛾-, and PPAR𝛼- melanoma cells [23, 27]. The current manuscript supports overexpressing cells, respectively, but not observed for EFA- those findings as our new results showed that fatty acids CLA-treatedcells.Thoseresultsshowthatthepresenceof extract from CLA-enriched egg yolks (EFA-CLA) reduced other fatty acids in EFA-CLA mixture contributes to the theviabilityofMCF-7breastcancercellline(Figure1). overall effect of FA treatment. However, the molecular mechanism is not fully understood. It is clear that the expression of the selected genes (TCF- Comparison of the effect on cancer cell proliferation between 20, WT1, ZNF621,andTHRB), which were identified for the extracts from CLA-enriched and nonenriched egg yolks first time in this work as putative PPAR-responsive genes, was could lead to the conclusion that it is simply the result of altered in the presence of the used agents (Table 2) and that the presence of CLA isomers incorporated in the egg yolk among them TCF-20 was affected the most by EFA-CLA. lipids. Available literature would support such a hypothesis asnumerousstudiesshowedaninhibitoryeffect,especially 4. Discussion for cis9,trans11CLA isomer, on tumor cells [28–32]. Indeed, ouranalysisofFAprofileofCLA-enrichedeggyolkshowed Chicken egg enriched with conjugated linoleic acid (CLA) that cis9,trans11CLA was incorporated more efficiently (3 :1 via feed modification meets the criteria of the functional ratio) than trans10,cis12 isomer [21] and therefore could food product. Based on Roberfroid’s [25] classification, CLA- predominate in EFA-CLA. Interestingly, comparison of the enriched egg can be considered as a conventional food effect of synthetic CLA isomers with CLA-EFA from egg product that is intended to be consumed as a part of a normal yolk showed the advantage of the latter in reducing cancer diet but is modified to contain biologically active substances, cell viability (Figure 1). The analysis of fatty acids profiles that is, CLA isomers. It has been shown to have a beneficial between enriched and nonenriched egg yolks revealed not effectonphysiologicalfunctionsofthehumanbody,inaway only CLA incorporation but also unexpected, significant that goes beyond its nutritional value, specifically by lowering change in SFA/MUFA ratio, specifically an increase in total theriskofdevelopingatherosclerosis[26].Ourprevious SFA concentration at the expense of MUFA. Thus, a question 8 PPAR Research ± 0.23 ± 0.00 ± 0.03 ± 0.03 ± 0.00 ± 0.01 ± 0.01 ± 0.01 ± 0.10 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 2.36 ± 0.08 1.81 1.81 2.00 3.76 −2.11 ± 0.18 −1.20 ± 0.24 −1.47 −1.58 −3.09 −1.77 −1.23 l FA or specific agonist/antagonist . ± 0.01 ± 0.01 ± 0.04 ± 0.04 ± 0.03 ± 0.20 ∗ ∗ ∗ ∗ ∗ ∗ <0.05 𝑝 1.48 ± 0.07 1.34 ± 0.26 1.24 ± 0.14 2.54 ± 0.22 ∗ 1.90 1.90 1.92 5.97 −1.06 ± 0.18 −1.76 ± 0.11 −1.43 −1.91 Agonist versus NC Antagonist versus NC NC versus 𝛾 , troglitazone/T0070907. ± 0.08 ± 0.11 ± 0.09 ± 0.13 ± 0.14 ± 0.34 ∗ ∗ ∗ ∗ ∗ ∗ 1.09 ± 0.19 1.71 ± 0.29 1.46 ± 0.07 1.70 7.98 −1.26 ± 0.19 −1.02 ± 0.08 −1.31 ± 0.23 13.02 18.15 13.48 17.22 SD ± trans10,cis12CLA 𝛾 -dependent genes 𝛼 -dependent genes FC values 𝛿 ,GW-0742/GSK0660;forPPAR versus NC ± 0.04 ± 0.05 ± 0.03 ± 0.07 ± 0.09 ± 0.11 ± 0.09 ± 0.16 ± 0.10 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1.09 ± 0.16 1.10 ± 0.24 7.05 6.67 6.66 1.74 9.96 −1.13 ± 0.05 − 1,86 −1.52 −2.51 −1.32 cis9,trans11CLA (B) mRNA expression of𝛿 -dependent PPAR genes (C) mRNA expression of PPAR (A) mRNA expression of PPAR ,WY14643/GW-6471;forPPAR 𝛼 ± 0.11 ± 0.03 ± 0.02 ± 0.20 ± 0.03 ± 0.01 ± 0.04 ± 0.17 ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ 1.02 ± 0.03 1.15 ± 0.12 1.14 ± 0.01 1.80 3.08 1.33 3.21 3.12 −1.24 ± 0.06 −1.38 ± 0.02 ± 0.04 ± 0.03± 0.02 ± 0.03 −1.29 ± 0.01 ∗ ∗ ∗ ∗ ∗ ∗ 1.36 ± 0.16 2.49 ± 0.08 1.18 ± 0.26 1.09 ± 0.01 2.03 1.61 2.99 1.37 2.09 −1.32 ± 0.11 −1.49 −1.02 ± 0.04 −1.00 EFA versus NC EFA-CLA versus NC (TR 𝛽 2) (TR 𝛽 2) (TR 𝛽 2) ZNF621 TCF-20 THRB TCF-20 Gene symbol WT1 WT1 THRB ZNF621 ZNF621 WT1 THRB TCF-20 of PPAR for 48 h. Table 2: mRNA expression of PPARs-responsive genes in PPAR-transfected MCF-7 cells (with overexpression of PPARs) after treatmentwith experimenta FC: fold change; NC: negative control. Agonist/antagonist: for PPAR PPAR Research 9 arisesofwhetheritisanindividualorcombinedeffectof isconstantlyupdatedasnewresultsarebeingpublished CLA and modified SFA/MUFA ratio in enriched egg yolks on from both experimental data and bioinformatics analyses MCF-7cellline[23].WeobservedthatresultsofCLA-EFAare of promoter regions containing PPRE consensus sequences most likely achieved by the effect of both: incorporated CLA (AGGTCANAGGTCA) (Figure 5). isomers and other fatty acids in eggs modified organically In the current study, we applied bioinformatic tools through hens’ diet [23]; however, this issue requires further to find genes with PPRE and analyze the effect of CLA research. ontheexpressionofthesegenes.Toourknowledge,we It has been shown that PPAR agonists have different prop- proposed several new genes that could be potentially PPAR- erties for individual PPAR isoforms, with different absorption regulated: BCAR3, LZTS, SLC5A, TCF20, WT1, ZNF621,and and distinctive gene expression profiles. To our knowledge, THRB (transcript TR𝛽2) (Table 2). Since preliminary data this is the first study focused on the effect of FA from CLA- showed that some of them were strongly regulated by PPARs, enriched egg yolks on transcriptional activation of PPARs we studied the expression of TCF201, WT , THRB (TR𝛽2), (PPAR𝛼,PPAR𝛾,andPPAR𝛿). All experiments included as and ZNF621 genes in the context of various PPAR ligands, controls synthetic CLA isomers as well as standard agonists including EFA-CLA. and antagonists of different PPARs. Our results showed that First one TCF20 can act as a phosphoserine-specific EFA-CLA extract exhibits the properties of agonists for all repressor of estrogen receptors (ER) in estrogen-dependent PPAR isoforms (Figures 3(a)–3(c)); however, those properties tumors [47]. MCF-7 human breast carcinoma cell line is seem to be most selective towards PPAR𝛾 (Figure 4). Interest- estrogen receptor (ER) positive; thus, the expression of ingly, PPAR𝛾 has been associated with the greatest impact on TCF20 should inhibit ER and consequently impair the viabil- cancer cell proliferation, survival, and differentiation, and its ity of the tumor cells. Our results confirm these assumptions, ligands are associated with anticancer properties [33, 34]. In showing elevated TCF20 mRNA level in cells treated with addition, as observed for EFA-CLA, transactivation of PPAR EFA-CLA. This effect was much stronger than for EFA receptors is more effective compared to fatty acids extracted (Table 2). Interestingly, the most pronounced effect was found from a nonenriched egg yolk (EFA) (Figures 3(a)–3(c)). for trans10,cis12CLA isomer (Table 2), which may explain its Since cis9,trans11CLA isomer showed PPAR agonist activity advantages over the cis9,trans11CLA in reducing the viability (Figures 3(a)–3(c)) and since this isomer was 3-fold more of MCF-7 (positively correlates with its effect on the reduction efficiently incorporated into egg yolks than trans10,cis12CLA in cell viability) (Figure 1). In contrast to Pariza et al. [18], [23], itcouldbehypothesizedthatcis9,trans11CLA plays a this result also suggests that trans10,cis12CLA isomer could significant role in EFA-CLA-mediated activation of PPARs. support antiproliferative action of cis9,trans11CLA in EFA- TheeffectofsyntheticCLAisomersprovidedus CLA via transcription-enhancing effects on TCF20. with important information about their specificity. While Available literature addresses the relationship between cis9,trans11 isomer acted as a PPAR agonist (Figures receptors encoded by PPAR and THRB genes [48–50]. THRB 3(a)–3(c)), the antagonist effect was observed for trans10,cis12 encodes three isoforms of human thyroid hormone receptor: isomer, specifically on PPAR𝛾 (Figure 3(c)). Available TR𝛽1 and tissue-specific TR𝛽2andTR𝛽4, which are thought literature is consistent with our results. cis9,trans11 isomer to be engaged in cell cycle control and metabolism [51]. hasbeenreportedtoinhibitcellgrowth[15,16]showing Recently, THRB has been studied as a tumor suppressor [52]. antitumor properties [17–20]. It has been found as well Although TR𝛽1isoformhasbeenfoundtoplayaroleinthe that the presence of trans10,cis12 isomer may abrogate competitive inhibition of the PPAR transactivation [53], there the antiproliferative activity of cis9,trans11 [18] and even is limited information on the relationships between TR𝛽2 inhibit the activity of synthetic PPAR agonists [15]. Thus, and PPAR receptors. TR𝛽 and PPAR receptors are linked by it is even more interesting that our results showed more the same obligatory coreceptor, retinoid X receptor (RXR), efficient reduction in cancer cells proliferation for EFA-CLA that binds to their heterodimeric partners before binding to treatment than using a pure synthetic cis9,trans11CLA DNA. Although RXR plays a central role in regulating the isomer that may suggest other factors including modified activity of a number of nuclear hormone receptors including SFA/MUFA ratio in enriched egg yolks [23], supporting TR𝛽 and PPARs by acting as a heterodimeric partner, this antiproliferative action of cis9,trans11CLA isomer. receptorisknowntobeconstitutivelyexpressedincells[53]; PPARs act as transcription factors and regulate the therefore, focusing on PPARs, we do not show the expression expression of dependent genes by binding to their PPREs. of RXR in this paper. Nevertheless, it has been reported Available literature gives a number of genes regulated by that TR𝛽 and PPAR receptors can compete for binding PPARs; the ligand-dependent transcription factors [35] and to RXRs in the nucleus [54]. Since we have found PPRE the expression of those genes can be both inhibited or within the sequence of TR𝛽2-specific promoter, located in activated depending on the ligand, suggesting selectivity [36]. intron IV of THRB gene, the bidirectional regulation of CLA isomers have been found to act as PPAR ligands and TR𝛽2 and PPARs is thought to be more complex. Results showntobeinvolvedintheinhibitionoftranscriptionof presented in the current manuscript indicated enhanced genes including TNF [37], NFKB1 [38], and NR1I3 [39] as transactivation of TR𝛽2byallPPARsisoformsinresponse well as transactivation: TGFB1 [40], BRCA1 [41], PTEN [42], to the treatment with experimental FA (Table 2) that may be p21/WAF1/CDKN1A [43], CEBPA [44], ABCB4 [45], and AOX evidence of the functional activity of the TR𝛽2-specific PPRE; [46]. Although a significant number of genes regulated by however, this needs further studies. The most significant PPARs have been described, the list is not exhaustive and effect was measured for the synthetic CLA isomers, especially 10 PPAR Research trans10,cis12 (Table 2). Taken together, our findings showed agonist activity, specifically towards the PPAR𝛾 isoform. that transcription levels of TR𝛽2areelevatedbyPPARsand Control, synthetic cis9,trans11 isomer of CLA exerted an their agonists. Simultaneously, TR𝛽1isoformhasbeenshown agonist effect on all PPAR receptors, while trans10,cis12 to compete with PPAR for access to the RXR coreceptor showed no effects or even acted as an antagonist of PPAR𝛾. or for PPRE binding sites in promoter regions of regulated However, this isomer was able to regulate some specific genes [50] that could suggest TR𝛽1-mediated inhibitory role genes containing PPREs such as TCF20 involved in cell cycle in expression of TR𝛽2isoformandpossiblyotherPPAR- arrest. Simultaneously, cis9,trans11 isomer upregulated THRB responsive genes. suppressor and downregulated WT1 oncogene showing a WT1 gene, as a transcription factor, directly or indirectly small part of a PPAR action that in case of EFA-CLA leads to interacts with a number of genes involved in cell cycle the observed reduction in proliferation of the breast cancer and neoplasia, including HIF1A, AREG, SRY, NROB1, SOX9, cells. It seems therefore that CLA-enriched eggs could be IGF2, MDM4, BRCA1, TP53,andSP1 (NCBI Gene). Available considered as food products with anticancer potential. literature suggests an oncogenic nature of WT1 and has shown itsoverexpressioninvarioustumorsandtumorcelllines, Conflicts of Interest especially in breast cancer cells and melanoma [55, 56]. In addition, decreased levels of WT1 gene expression correlated The authors report no financial or other conflicts of interest with reduced cell proliferation in both melanoma and breast relevant to the subject of this article. cancer cells [57, 58]. WT1 has also been linked with malignant transformation in breast cancer, and its overexpression asso- Acknowledgments ciated with reduced susceptibility to drug treatment. Indeed, it has been shown for estrogen-dependent lines that WT1 This work was supported by the Polish National Science Cen- positively regulates the expression of EGFR and HER2 [55], ter (Grant no. 2011/03/B/NZ9/01423) “Conjugated Linoleic contributing to the resistance to hormone therapy [59, 60]. In Acid (CLA) Induced Transcriptional Activation of PPAR: melanoma, in vitro WT1 silencing resulted in decreased cell An Investigation of Molecular Mechanisms of Putative Anti- proliferation, followed by apoptosis induction with caspase- cancer Action of Fatty Acids of CLA-Enriched Egg Yolks” and 3 activation [61], while in vivo it reduced the melanoma by the Ministry of Science and Education (Grant no. N N312 metastatic to lungs [56]. On the other hand, some studies 236038) “The Influence of CLA-Enriched Hen’s Egg Yolk indicate that pharmacologic activation of PPAR𝛿 by its Lipids on the Proliferation of Selected Tumor Cell Lines.” agonists (GW0742 and GW501516) inhibited proliferation of the murine melanoma cells, accompanied by downregulation References of WT1 [62]. It was suggested that PPAR𝛿 can act via the PPRE in the WT1 promoter and directly suppress its activity; [1] B. 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Review Article Deciphering the Roles of Thiazolidinediones and PPAR𝛾 in Bladder Cancer

Melody Chiu,1 Lucien McBeth,1 Puneet Sindhwani,2 and Terry D. Hinds1,2

1 Center for Hypertension and Personalized Medicine, Department of Physiology & Pharmacology, University of Toledo College of Medicine, Toledo, OH 43614, USA 2Department of Urology, University of Toledo College of Medicine, Toledo, OH 43614, USA

Correspondence should be addressed to Terry D. Hinds; [email protected]

Received 13 October 2016; Accepted 12 February 2017; Published 28 February 2017

Academic Editor: Stefano Caruso

Copyright © 2017 Melody Chiu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The use of thiazolidinedione (TZD) therapy in type II diabetic patients has proven useful in the lowering of blood glucose levels. However, recent investigations have shown that there may be potential health concerns associated, including the risk of developing bladder cancer as well as complications in the cardiovasculature. TZDs are ligands for the nuclear receptor PPAR𝛾,andactivation causes lipid uptake and insulin sensitization, both of which are critical processes for diabetic patients whose bodies are unable to utilize insulin effectively. Several studies have shown that PPAR𝛾/TZDs decrease IGF-1 levels and, thus, reduce cancer growth in carcinomas such as the pancreas, colon, liver, and prostate. However, other studies have shed light on the potential of the receptor as a biomarker for uroepithelial carcinomas, particularly due to its stimulatory effect on migration of bladder cancer cells. Furthermore, PPAR𝛾 may provide the tumor-promoting microenvironment by de novo synthesis of nutrients that are needed for bladder cancer development. In this review, we closely examine the TZD class of drugs and their effects on PPAR𝛾 in patient studies along with additional molecular factors that are positive modulators, such as protein phosphatase 5 (PP5), which may have considerable implications for bladder cancer therapy.

1. Introduction adults and children have been continuously rising. In the 2014 National Diabetes Statistics Report, the Centers for The predominant type of bladder cancer diagnosed among Disease Control and Prevention estimated that 29.1 million individuals in the United States is urothelial (transitional cell) people (9.3% of the population) are diagnosed with diabetes carcinoma [1]. Bladder cancer is the fourth most common in the United States [9]. Worldwide, an estimated 382 million type of cancer found among men in the United States and an adults were diagnosed with diabetes in 2013 [10], with type important cause of death worldwide [2, 3]. In 2015 alone, the II diabetes accounting for nearly 90–95% of these diabetic American Cancer Society predicted a total of 74,000 newly individuals [11]. diagnosed cases and 16,000 deaths from bladder cancer in There has been increasing evidence showing that antidi- the United States [4]. The cause of bladder cancer appears abetic TZDs are linked to the risk of bladder cancer as to be multifactorial; both exogenous environmental and well as other complications such as cardiovasculature (CVD) endogenous molecular factors may potentially play a role events. TZDs, such as pioglitazone and rosiglitazone, are syn- in cancer development [5, 6]. Environmental factors such thetic ligands of peroxisome proliferator-activated receptors ascigarettesmokingandoccupationalexposuretochemical gamma (PPAR𝛾) used in therapeutic treatments for patients carcinogens are among the top risk factors; however, family diagnosedwithtypeIIdiabetesmellitus[12,13].Theseligands history and genetics also increase the susceptibility to bladder bind to PPAR𝛾 and play a role in metabolism through carcinogenesis [7]. Moreover, evidence has suggested an induction of genes that control glucose and lipid uptake association between diabetes mellitus and the increased risk [13]. Through a series of metabolic pathways, PPAR𝛾 also of bladder cancer [8]. Rates of type II diabetes mellitus among activates adipogenesis, which is the process of transforming 2 PPAR Research a preadipocyte stem cell into fully mature adipocyte [14]. adiponectin and leptin. These adipokines regulate insulin Eventually, this process reduces insulin resistance by assist- activity in peripheral tissues to maintain glucose sensitivity ing in glucose uptake [15]. Potentially, PPAR𝛾 signaling in in the body. In addition, PPAR𝛾 regulates genes involved bladder cancer cells may provide a tumor microenvironment in fatty acid transport, release, and storage by increasing thatallowsfordenovolipogenesisfortheuseofincreasing expression of genes involved in fatty acid import such as tumor mass and energy usage. However, the role of PPAR𝛾 in cluster of differentiation 36 (CD36) and adipocyte protein 2 bladder cells is unknown. (aP2) [21, 26]; therefore, PPAR𝛾 has a major role in lipid and PPAR𝛾 is expressed in white and brown adipose tissues carbohydrate metabolism. as well as in the urinary bladder [16, 17]. More notably, high TZDs have long been a common therapeutic method to levels of PPAR𝛾 are selectively expressed in the transitional treat patients with type II diabetes mellitus. TZDs are used to epithelium of the ureter and urinary bladder, the area where treat hyperglycemia and insulin resistance, lowering fasting bladder cancer typically arises. PPAR𝛼 is another member of blood glucose and insulin, as well as HbA1C levels [27]. the PPAR family that is expressed in the ureter and bladder Previously, up to 20% of antidiabetic medications prescribed epithelium, but at a significantly lower level compared to in the USA were TZDs [28]. In the past, it has been shown that PPAR𝛾 [17]. Despite the prominent differences between the TZDs are effective in therapy as a second-line treatment after two receptors, there has also been evidence depicting a metformin, the current first-line agent in type II diabetes [27, degree of crosstalk between the receptors in urinary bladder 29]. They are high-affinity synthetic agonists of PPAR𝛾 [12], epithelium. A combination of synthetic ligands, known as and PPAR𝛾 activation affects lipid metabolism and ultimately “dual-acting agonists,” includes PPAR𝛼 and PPAR𝛾 agonists enhances lipid storage and promotes insulin sensitivity in adi- and has been shown to have a carcinogenic impact in rodents, posetissue,liver,andmuscle[16,23].Despitemanybenefits, primarily affecting the bladder epithelium [18]. In this review, TZDs have also been shown to induce weight gain among we discuss the functions of PPAR𝛾 and the effects of TZD diabetic patients on long-term therapy [30], which occurs therapy in the urinary bladder and to a lesser extent the role from activation of adipogenesis and the expansion of fat of PPAR𝛼. cells. Of the TZD class, rosiglitazone and pioglitazone are the most prevalently used drugs in clinical settings [31]. Studies 2. PPAR𝛾 Function have reported the adverse health effects of these medications, including the possible risk of developing bladder cancer The PPAR𝛾 gene is located on chromosome 3 in humans and or cardiovascular events [12, 32, 33]. However, there is a is alternatively spliced to produce two major proteins; how- conundrum for the effects of PPAR𝛾 and its ligands in cancer. ever, alternative usage of the promoter provides four different Several cancers have shown reduced growth with PPAR𝛾 transcripts [19, 20]. The mRNAs of transcripts PPAR𝛾1, activation with the TZD troglitazone such as in carcinomas PPAR𝛾3, and PPAR𝛾4 result in identical protein products that ofthebreast,kidney,liver,colon,pancreas,andprostate[34– we refer to as PPAR𝛾1.TheproteinproductfromthemRNA 39] as well as in non-small-cell lung cancer [40] and ACTH- of PPAR𝛾2iscomparabletothatofPPAR𝛾1; however, the secreting pituitary adenomas [41]. However, most of the product contains 30 additional amino acids located at the antigrowth properties of TZDs have been with troglitazone NH2-terminal region (reviewed in [20]) [21]. Not surpris- and not pioglitazone or rosiglitazone. Rosiglitazone may be ingly, the isoforms have varying expression levels in cells; associated with a lower risk of breast cancer [42], thyroid PPAR𝛾1 is expressed in nearly all cells, whereas PPAR𝛾2 cancer [43], and nonmelanoma skin cancer [44]. On the other is principally expressed in adipocytes [22]. However, it is hand, pioglitazone seems to be neutral or slightly (possibly unknown whether there is a difference in PPAR𝛾1and not significant) associated with various cancers including PPAR𝛾2 expression levels in bladder cancer cells. PPAR𝛾 bladder cancer [45], ovarian cancer [46], oral cancer [47], is also involved in regulating inflammatory processes [23]. kidney cancer [48], and thyroid cancer [49]. Analysis of There is evidence that shows PPAR𝛾 activation in endothelial specific TZDs and their actions on growth and migration are cells reduces systemic inflammation [24]. While the role in important for understanding the impact they may have in a adipocytes and insulin sensitivity is well understood, the specific cancer. effects of PPAR𝛾 activation in many other cell types remain SomeTZDshavebeenshowntoreducelevelsofthe unclear including bladder cancer. insulin-like growth factor-1 (IGF-1) in the blood, which is a PPARs are ligand-activated transcription factors that known growth factor that may induce cancer [50]. Plasma belong to the nuclear receptor superfamily [22]. When a levels of IGF-1 and IGF binding protein-3 (IGFBP-3) have ligand binds to an isoform of the PPAR family, the receptor been shown to be an association with bladder cancer risk [51]. is activated, translocates to bind regulatory regions on DNA, It is not known how PPAR𝛾 affects the expression of IGF-1, and then combines with retinoid X receptors (RXRs) to form IGFBP-3, or the IGF receptor (IGFR) in the bladder or differ- heterodimers (Figure 1). Consequently, these heterodimers ences among the TZD drug class. The use of pioglitazone, and serve as transcriptional activators for various genes by bind- not rosiglitazone, has been associated with an increased risk ing to specific PPAR response elements (PPREs) [13]. Of of bladder cancer in a population-based cohort study, sug- the PPARs, PPAR𝛾 is found to have the highest expression gesting the risk is TZD specific and not a particular class [52]. levels in adipose tissue. Once activated in adipocytes by Investigations on the consequences of troglitazone, rosiglita- TZDs or natural ligands, such as essential fatty acids and zone, and pioglitazone on the IGF system in uroepithelial eicosanoids [25], PPAR𝛾 is involved in the secretion of carcinomas may reveal differences between the drugs. PPAR Research 3

TZDs

EETs Fatty acids

PPAR𝛾 z z

RXR PPAR𝛾 aP2↑ CD36 ↑ z z z z

Figure 1: PPAR𝛾 heterodimerizes with RXR for transcriptional regulation. PPAR𝛾 ligands such as eicosanoids (EETs), fatty acids, or thiazolidinediones (TZDs) bind to PPAR𝛾 to cause transactivation resulting in the binding to regulatory regions on DNA. PPAR𝛾 combines with retinoid X receptors (RXRs) to form heterodimers, which together serve as transcriptional activators for various genes by binding to specific PPAR response elements (PPREs) in their promoters.

3. TZDs and Bladder Cancer 2.93 (2 or more years) [27]. Similarly, odds ratios for patients on rosiglitazone therapy were 0.98 (<1year),1.78(between An interim longitudinal cohort study using the Kaiser Perma- 1-2 years), and 2.00 (2 or more years) [31]. The increased nente Northern California Registry analyzed a sample size of duration of pioglitazone or rosiglitazone therapy is associated 193,099 diabetic patients and observed a correlation between with increased risk of bladder cancer, with the highest risk pioglitazone therapy and bladder cancer [12]. The increased among diabetic patients on therapy for 2 or more years [32]. dosage and duration of pioglitazone treatment show rises in However, this observation may only apply to specific TZDs bladder cancer incidence rates, with a 30% risk of developing and not all of them [29], as there appears to be a weaker bladder cancer among patients on pioglitazone therapy after association between bladder cancer and rosiglitazone. 12–24 months. Furthermore, the risk increases to 50% for There is some debate as to the association of TZDs with patients on pioglitazone therapy for 2 or more years [12]. In bladder cancer. Two meta-analyses show only moderate to the 10-year follow-up, however, the statistical significance was no risk of developing bladder cancer. Monami et al. found not found while there was a numerical increased adjusted that the overall risk of malignancies (regardless of location) risk of 78% (0.93–3.4, 95% CI) for patients on pioglitazone was decreased by TZD treatments [56]. However, there treatment for 1.5–4 years [53]. Additionally, Hsiao et al. was a numerical, but not statistically significant, increase in showed current users of both pioglitazone and rosiglitazone the risk of bladder cancer development from pioglitazone had increased risks of developing bladder cancer [32]. The treatment (2.05 Mantel-Haenszel odds ratio, 𝑝 = 0.12) correlation between pioglitazone and bladder cancer is con- but no association with rosiglitazone treatment (0.91, 𝑝= sistent with the previous Kaiser cohort study. However, the 0.62). Interestingly, the odds ratio was associated with a large use of rosiglitazone was not associated with an increased risk confidence interval, 0.84–5.02, which the authors attributed of bladder cancer in any analysis [52], but it has been linked to a small sample size, three studies, due to potential bias to increased risk of cardiovascular events [54]. However, from incomplete disclosure of negative results. In addition, rosiglitazone was not increased in bladder cancer risk [55]. the second meta-analysis conducted by Bosetti et al. showed Pioglitazone may be the only TZD to enhance cases of only a modest increased risk of developing bladder cancer bladder cancer, as results from the National Health Insur- when treated with TZDs for less than two years (relative risk ance Research Database (NHIRD) group also presented an 1.20, CI 1.07–1.34) [57]. There was a moderate increased risk association with uroepithelial carcinomas [32]. Through the for treatment longer than two years (relative risk 1.42, CI NHIRD study, it was shown that increased exposure period to 1.17–1.72), which the authors led to claim that the short-term both pioglitazone and rosiglitazone is related to an increased (less than two years) treatment with TZDs in type II diabetes risk of bladder cancer. Regardless of whether patients have mellitus might be worth the modest risk of developing been on pioglitazone or rosiglitazone treatment, the highest bladder cancer. risk of bladder cancer is among diabetic patients with the longest exposure to either treatment. The NHIRD cohort 4. PPAR𝛾 and Bladder Cancer showed the odds ratios for the risk of bladder cancer among diabetic patients on pioglitazone therapy in the exposure To provide a closer look at the impact of PPAR𝛾 on bladder groups were 1.45 (<1year),1.74(between1and2years),and cell progression, Yang et al. analyzed samples of both benign 4 PPAR Research bladder and bladder cancer mucosal samples by fluorescence 5. An Independent Microenvironment in situ hybridization (FISH) assay for expression of PPAR𝛾, through PPAR𝛾 and the authors found 31% (8/21 samples) of the bladder cancer mucosal samples and 4.3% (1/23 samples) of benign In general, tumor development in the urinary bladder is bladder samples showed amplification [58]. In addition, dependent upon complex interactions with host molecular lower levels of PPAR𝛾 amplification were detected in non- factors that are part of its surrounding microenvironment muscle-invasive bladder cancer samples compared to muscle- [60, 61]. Furthermore, there are signaling interactions of a invasive samples (16.7% versus 46.7%, resp.) [58]. Yang et al. certain level in the microenvironment that are capable of also observed different rates of cell migration and invasion in inducing malignant transformation of cells, such as factors that promote angiogenesis, abnormal development, and pro- various bladder cancer cell lines that have PPAR𝛾 expression. liferation. Neoangiogenesis, or the formation of new blood The 5637 bladder cell line had a considerably higher mRNA vessels from preexisting vessels, is required for tumor growth, and protein expression of PPAR𝛾 compared to other bladder and vascular endothelial growth factor (VEGF) has been cancer cell lines such as UMUC-3. Moreover, the 5637 cancer shown to play a critical role as a proangiogenic factor in cell line displayed higher cell migration and invasion than bladder cancer progression [62]. VEGF-A is the primary the UMUC-3 cell line [58]. Another study showed that the proangiogenicfactorthatservestomaintainadequatelevels 𝛾 T24 bladder cancer cell line expresses PPAR and high levels of oxygen and nutrient supply in growing adipose tissue and 𝛽 𝛽 of the nuclear receptor glucocorticoid receptor (GR ), is positively regulated by PPAR𝛾 [63]. The levels of VEGF which also showed higher migration rates than the UMUC- found in the urine and bladder tissue are significantly ele- 3 cells that have low PPAR𝛾 and GR𝛽 expression [59]. These vated in patients diagnosed with urinary bladder carcinoma results suggest that PPAR𝛾 may be a potential biomarker of compared to cancer-free patients [64]. Additionally, it has bladder cancer aggressiveness, where high levels of receptor been shown that VEGF-A is found in bladder tumors and expression correlate with higher rates of cancer cell migration is upregulated in patients with invasive bladder cancer [65]. and invasion. Potentially, VEGF-A may also be enhanced by PPAR𝛾 in Rosiglitazone treatments have been shown to have vary- bladder tumor tissue consequently enhancing tumor growth ing effects on 5637 and UMUC-3 cancer cells [58]. The and migration through angiogenesis. However, the specific 𝛾 5637 bladder cancer cells display significantly enhanced cell TZDs that may enhance VEGF-A or if PPAR induces VEGF- migration and invasion with rosiglitazone treatment. On A in bladder are yet to be determined. the other hand, there are minimal rates of cell migration In order to continue to proliferate indefinitely, cancer and invasion in UMUC-3 cells, and rosiglitazone has less of cells require molecular factors that increase both glucose an effect. The difference in the levels ofPPAR𝛾 expression uptake and rates of glycolysis for energy. Elevated rates of glycolysis produce higher amounts of lactic acid, and this between the two cancer cell lines may account for this pathway enhances lipogenesis through fatty acid synthase observation, as the 5637 cell line has a considerably higher (FAS). FAS is the key enzyme involved in de novo synthesis PPAR𝛾 expression than UMUC-3 cell line [58]. Lubet et of fatty acids for lipid storage, and high expression levels are al. performed a series of experiments using rosiglitazone frequently limited in tissues with lipogenic activity, such as and hydroxybutyl(butyl)nitrosamine (OH-BBN), which is a adiposetissueandliver[66].However,ithasbeenshownthat carcinogen that is known to induce urinary bladder cancer FAS is overexpressed in numerous human cancers, including in rats [13]. Interestingly, rats treated with rosiglitazone had bladder cancer, and its expression level is positively correlated 100% incidence of bladder cancer, while the untreated control with tumor progression [67]. Similar to FAS, fatty acid group had a 57% incidence of bladder cancer. There were binding proteins (FABPs) are involved in lipid metabolism also increased levels of PPAR𝛾 expression in the presence and facilitate the transfer of lipids, including lipid droplets of rosiglitazone treatment compared to those that were not for storage, across various cellular membranes and compart- treated. Furthermore, rats that were exposed to OH-BBN ments [68–70]. Adipocyte-type FABP (A-FABP), also known and treated with the highest dosage of rosiglitazone have the as adipocyte protein 2 (aP2) and fatty acid binding protein 4 highest incidence of bladder cancer. Rats on rosiglitazone (FABP4), binds to long chain fatty acids and PPAR𝛾 agonists therapy had earlier cancer onsets and larger tumor sizes in [69].TheseligandsbindandactivateA-FABPsinthecytosol, the bladders, and a dose-dependent response existed between and A-FABPs then transfer the ligands to PPAR𝛾 upon rosiglitazone and bladder cancer incidence. TZDs may not entering the nucleus to drive adipogenic activities [71]. Unlike have an effect in the earlier stages but may promote cancer FAS, low expression levels of A-FABP are correlated with the progression at the later stages of bladder cancer [13]. However, progression of human bladder transitional cell carcinoma. itisimportanttonotethatinhumansrosiglitazonehasnot When comparing specific types of bladder tumor tissue, A- been associated with higher risk, but this has been observed FABP was mainly detected in cells that were papillary in with pioglitazone. Regardless, decreasing PPAR𝛾 expression origin and not invasive urothelial carcinoma [72]. Evidence may potentially alter bladder cancer migration and invasive suggests low-grade bladder tumors have higher levels of A- abilities. Therefore, regulating levels of PPAR𝛾 expression FABP compared to high-grade bladder tumors [73]. On the in the urinary bladder may have implications for targeting other hand, high expression of A-FABP has been observed bladder cancer, particularly regarding metastasis and cancer in tongue squamous cell carcinoma [70]. The differences in cell progression. tissue types, such as bladder and tongue, may partially PPAR Research 5 account for the discrepancy in the effects of A-FABP expres- both PPAR𝛼 and PPAR𝛾, thereby combating diabetes melli- sion. tus and the metabolic syndrome among patients diagnosed Metabolic changes may occur in nonadipose tissues when with both conditions [81]. Examples of such dual agonists they receive fatty acids released by hypertrophic dysfunc- include ragaglitazar and muraglitazar, which would be of tional adipose tissue, commonly seen among obese and type interests for the treatment of obesity and diabetes. However, II diabetic patients [74]. Nonadipose tissues are not equipped muraglitazar has been shown to induce gallbladder adenomas with adequate cellular machinery for excessive amounts of in male mice, and ragaglitazar has been demonstrated to lipid deposits. Therefore, an overload of lipids in these tissues induce urinary bladder and renal pelvis tumors in both male causes a series of organ-specific toxic reactions and results in andfemalerats[82]. lipotoxicity, which is lipid-induced metabolic tissue damage It is worth noting that certain combinations of PPAR𝛼 and death [75]. Glucuronidation is important for detoxifying and PPAR𝛾 synthetic dual-acting agonists may have a car- the bladder from toxins [76] and may be regulated differ- cinogenic impact on rodents, especially targeting urinary entially by fatty acid accumulation. While tissues, such as bladder epithelium. In a recent study, Egerod et al. found skeletalmuscleandliver,areknowntobehighlysusceptible that rat bladder epithelium expresses both PPAR𝛾 and PPAR𝛼 to lipotoxicity [77], little is known regarding the effects of throughacrosstalklinkthatinvolvestheearlygrowth lipid accumulation in the bladder. Presumably, the functional response-1 (Egr-1) factor [18]. Egr-1 is a transcription factor impairment will occur in most healthy nonadipose tissues; and has been previously shown to play a role in bladder cancer however, this observation may not entirely apply to bladder among different species, including humans [83]. When either tissue. PPAR agonist is used alone, there is only slightly increased It may be possible that, in bladder tissue, lipid accumu- Egr-1 expression in the rat bladder epithelium [18]. High Egr- lation modifies metabolic functions in a way that strongly 1 induction is dependent on the coactivation of PPAR𝛼 and upregulates PPAR𝛾 and enhances lipid uptake, similar to PPAR𝛾 by their respective synthetic ligands fenofibrate and adipose tissue. Eventually, sufficient amounts of free fatty rosiglitazone. Together, fenofibrate and rosiglitazone appear acids (FFAs) will be present in the bladder due to ectopic to exert a positive interaction in the bladder epithelium, fat accumulation, and the bladder may no longer require upregulating high Egr-1 expression. However, this positive A-FABP to import additional extracellular FFAs but will interaction is not observed in other tissues, such as the heavily utilize FAS for lipid production. FFAs bind PPAR𝛾 liver, where there are high expression levels of Egr-1 and the andotherPPARisoformsandactivatetranscriptionalactivity. absence of carcinogenic effects of dual-acting agonists on rats Other dysregulated metabolic pathways, including those that [18]. involve glycolysis [78], can cause a metabolic switch regulated It has also been demonstrated that ragaglitazar treatment by oncogenes and tumor suppressor genes to favor tumor has a carcinogenic impact on rat bladder epithelium and growth and play a role in bladder carcinogenesis. Together, involves the induction of Egr-1 [82, 84]. Importantly, the these observations are consistent with evidence showing fenofibrates that are PPAR𝛼 agonists have not been shown to lower expression levels of A-FABP and higher expression lev- induce bladder cancer. PPAR𝛼 agonists with a different struc- els of FAS in more invasive forms of bladder cancer. Increased ture,theclofibrates[85],havebeenshowntoweaklyenhance levels of PPAR𝛾 activity may alter the microenvironment in BBN-induced bladder carcinogenesis [86]. However, a sec- a way that allows for the cells to autonomously synthesize ond report indicated that clofibrates are not carcinogenic nutrients within the bladder through lipid accumulation and [87]. The differences in these studies may be from clofibrate angiogenesis. However, more studies need to be performed to potentially having off-target effects or through possible weak understand the role of PPAR𝛾 in bladder cancer. interactions with PPAR𝛾. Furthermore, it is rather a unique characteristic of bladder epithelium to express high levels 6. The Impact of Dual-Acting PPAR Agonists of both PPAR𝛼 and PPAR𝛾. While the exact mechanism behind the interactions of PPAR agonists and bladder cancer Despite evidence showing PPAR𝛾 as the predominant PPAR remains unknown, these studies provide further insight into in urinary bladder epithelium, PPAR𝛼 has also been found to the relevance of PPAR activation, particularly in bladder be expressed in both rabbit and human bladder epithelium. cancer development. PPAR𝛼 is activated by a class of synthetic ligands known as fibrates (i.e., fenofibrate) and is predominantly expressed 7. PP5, a Positive Modulator of PPAR𝛾 in the liver, heart, brain, skeletal muscle, and kidney. Also, endogenous ligands such as fatty acids can bind PPAR𝛼 PPAR𝛾 activity is inhibited by the phosphorylation of serine to increase transcriptional activity. Recently, bilirubin was 112, and, currently, only one phosphatase, protein phos- also shown to function as an endogenous PPAR𝛼 agonist phatase 5 (PP5), has been shown to bind directly to the by direct binding [25] and was shown to decrease mRNA receptor [26]. PP5 belongs to the PPP-family consisting of expression of PPAR𝛾.Onceactivated,PPAR𝛼 regulates genes serine/threonine protein phosphatases [88, 89]. Evidence has that encode for mitochondrial and peroxisomal 𝛽-oxidation, indicated that PP5 activation requires the binding of its which reduces dyslipidemia. In addition, activated PPAR𝛼 tetratricopeptide repeat (TPR) domain to the heat shock functions to hinder hepatic fatty acid synthesis through protein 90 (Hsp90) chaperone complex [26, 89] (Figure 2). inhibition of FAS and SREBP1 and therefore lower lipid levels PP5 is a positive modulator of PPAR𝛾 inthepresenceof [21, 79, 80]. Dual agonists are a class of drugs that activate proadipogenic activity, with PP5 described as a “prolipogenic 6 PPAR Research

TZDs

Insulin sensitization S112 S112 S112 Glucose/lipid uptake P PPAR𝛾 PPAR𝛾 PPAR𝛾 P HSP90 TPR HSP90 HSP90 HSP90

Bladder cancer PP5 TPR P TPR HSP90 HSP90

Figure 2: Theoretical model of PPAR𝛾 and PP5 in bladder cancer. Activation of PPAR𝛾 byTZDsrecruitsPP5topositivelymodulate and dephosphorylate Ser-112 (S112). PPAR𝛾 is activated once the phosphate group is removed, and a series of PPAR𝛾-mediated activities commence shortly thereafter, including insulin sensitization. PP5 has been shown to mediate𝛾 PPAR activity by controlling phosphorylation of S112 in an adipogenic model, and targeting PP5 in the bladder epithelium may potentially affect PPAR𝛾 and its carcinogenic effects on the bladder.

phosphatase” [26]. Upon activation by the adipogenic stimu- bladder epithelium and cancer development. It will be of lus rosiglitazone, PP5 is recruited to positively modulate the therapeutic importance to determine if the same relationship activity of PPAR𝛾 by dephosphorylating PPAR𝛾 at serine-112 exists between PP5 and PPAR𝛾 in the bladder epithelium as residue[26,90].Oncedephosphorylated,PPAR𝛾 becomes for adipose tissue in the presence of TZD therapy. Bilirubin active and regulates genes in metabolic processes, such as adi- may offer a therapeutic potential because it activates PPAR𝛼 pogenesis. Not only is PP5 a potential target in the treatment and suppresses PPAR𝛾, and fenofibrate has not been associ- of obesity [26], but it may also provide an effective therapeutic ated with bladder cancer. In the future, therapies that target intervention for bladder cancer. Other studies have suggested PPAR𝛾, or possibly PP5, may prove to be useful in bladder that PP5 plays a role in tumorigenesis. PP5 mRNA levels cancer treatment, particularly among diabetic patients that are remarkably elevated in malignant ascites hepatomas in require long-term health management. rats [91]. Also, increased levels of PP5 protein have been observed in human tumor breast tissue and have been linked Disclosure to the promotion of breast cancer development [92]. It is unknown whether a similar association exists between PP5 The content is solely the responsibility of the authors and does and human bladder cancer. The mechanism of PP5 expression not necessarily represent the official views of the National and tumorigenesis has yet to be determined, but it may Institutes of Health. potentially regulate PPAR𝛾 in the bladder epithelium similar to adipose, as high levels of PPAR𝛾 are also associated with Competing Interests bladder cancer. If PP5 is a positive modulator of PPAR𝛾 in the bladder epithelium, then reducing PP5 expression may serve The authors declare that they have no competing interests. as an alternative therapeutic target to hinder bladder cancer progression. However, these studies are yet to be conducted. Acknowledgments This work was supported by the University of Toledo deArce- 8. Conclusion Memorial Endowment Fund (Terry D. Hinds Jr.). Research reported in this publication was also supported by the Long-term TZD therapy may increase the risk of developing National Heart, Lung, and Blood Institute of the National bladder cancer, especially pioglitazone. Rosiglitazone does Institutes of Health under Award nos. K01HL125445 (Terry not appear to have the long-term effects on the bladder. D. Hinds Jr.) and L32MD009154 (Terry D. Hinds Jr.). Prolonged and higher PPAR𝛾 activity levels are associated with higher incidences of bladder cancer, potentially due to the downstream effects of PPAR𝛾-mediated metabolism. References 𝛾 In addition to incidence rates, PPAR activity is associated [1]J.C.Park,D.E.Citrin,P.K.Agarwal,andA.B.Apolo, with increased bladder cancer cell migration and invasion. “Multimodal management of muscle-invasive bladder cancer,” Further understanding of the roles of the PPARs and their Current Problems in Cancer,vol.38,no.3,pp.80–108,2014. agonists in the bladder may uncover additional strategies [2] G. M. Dancik, “An online tool for evaluating diagnostic and in bladder cancer therapy. Previously, there have not been prognostic gene expression biomarkers in bladder cancer,” BMC studies examining the interaction of PP5 with PPAR𝛾 in Urology,vol.15,no.1,article59,2015. PPAR Research 7

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Review Article PPAR Agonists for the Prevention and Treatment of Lung Cancer

Sowmya P. Lakshmi,1,2 Aravind T. Reddy,1,2 Asoka Banno,1 and Raju C. Reddy1,2

1 Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA 2Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, PA 15240, USA

Correspondence should be addressed to Raju C. Reddy; [email protected]

Received 15 August 2016; Accepted 8 December 2016; Published 20 February 2017

Academic Editor: Valeria Amodeo

Copyright © 2017 Sowmya P. Lakshmi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Lung cancer is the most common and most fatal of all malignancies worldwide. Furthermore, with more than half of all lung cancer patients presenting with distant metastases at the time of initial diagnosis, the overall prognosis for the disease is poor. There is thus a desperate need for new prevention and treatment strategies. Recently, a family of nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs), has attracted significant attention for its role in various malignancies including lung cancer. Three PPARs, PPAR𝛼,PPAR𝛽/𝛿,andPPAR𝛾, display distinct biological activities and varied influences on lung cancer biology. PPAR𝛼 activation generally inhibits tumorigenesis through its antiangiogenic and anti-inflammatory effects. Activated PPAR𝛾 is also antitumorigenic and antimetastatic, regulating several functions of cancer cells and controlling the tumor microenvironment. Unlike PPAR𝛼 and PPAR𝛾, whether PPAR𝛽/𝛿 activation is anti- or protumorigenic or even inconsequential currently remains an open question that requires additional investigation. This review of current literature emphasizes the multifaceted effects of PPAR agonists in lung cancer and discusses how they may be applied as novel therapeutic strategies for the disease.

1. Introduction squamous cell carcinoma, and large cell carcinoma, while the more aggressive, neuroendocrine tumor SCLC represents Approximately 1.8 million people were newly diagnosed with most of the rest [5, 6]. Because more than half of lung cancer lung cancer and approximately 1.6 million died from it in patients are diagnosed in an advanced stage, with distant 2012, making lung cancer the most common and most fatal metastases [2] and a 5-year survival rate of approximately malignancy in the world [1]. In the USA alone, 224,390 2% [7], the overall 5-year relative survival rate for all lung new cases and 158,080 deaths are estimated for 2016 [2]. A cancer patients combined falls below 18% [2, 7]. This dire state number of risk factors such as hereditary genetic mutations, stresses the need for novel approaches in prevention, early occupational exposure to lung carcinogens, poor diet, and air detection, and therapy for the disease. pollutionhavebeenassociatedwithlungcancer[3].Chronic A family of nuclear hormone receptors, the peroxi- lung inflammation and certain pulmonary infections have some proliferator-activated receptors (PPARs), has recently also shown a positive association [3, 4]. Nevertheless, tobacco attracted interest as potential therapeutic targets for a variety smoke is the single, major contributor to the pathogenesis of of malignancies, including lung cancer [8]. Besides being lung cancer, increasing the lifetime risk even in those who key regulators of lipid and glucose metabolism [9, 10], quit smoking. Lung cancer is categorized into two major PPARs, as ligand-activated transcription factors, are also histological subtypes, small cell lung cancer (SCLC) and non- involved in cellular processes including cell differentiation, small cell lung cancer (NSCLC). NSCLC accounts for as much proliferation, survival, apoptosis, and motility [11–13]. Since as 85% of all lung cancers and includes adenocarcinoma, many tumors result from dysregulation of these cellular 2 PPAR Research processes and metabolic disorders have been associated with that mice lacking PPAR𝛼 are resistant to the agonist WY- increased cancer risk [14], the role of PPARs in cancer biology 14,643-induced increase in DNA synthesis and formation is not surprising. PPARs have indeed been implicated in of hepatic neoplasia [28]. Interestingly, epidemiological data the regulation of various solid cancers as well as leukemias suggest this tumorigenic effect of PPAR𝛼 activation is absent [8, 12]. in humans [11, 16], perhaps due to significantly lower expres- The PPAR family comprises three members, PPAR𝛼, sion of PPAR𝛼 in human hepatocytes and/or inefficient PPAR𝛽/𝛿,andPPAR𝛾 [15]. Each PPAR subtype is unique ligand activation of human PPAR𝛼 [11]. Another plausible in its structure and function [10]. All three PPAR receptors explanation suggests that human PPAR𝛼 does not exert are found in many cells and tissues throughout the body carcinogenic effects, as activation of a humanized PPAR𝛼 [16–18]. While sharing some common ligands, PPAR family in transgenic mice does not induce hepatic tumors [14]. In members also respond to distinct repertoires of natural and sum, although the between-species variation in effects of synthetic ligands, as might be expected from their specific PPAR𝛼 activation on liver carcinogenesis requires further biological activities [13]. PPAR𝛼 can be activated by fatty acids elucidation, humans appear to be protected from the harmful and eicosanoids (e.g., 8(𝑆)-hydroxyeicosatetraenoic acid and outcomes of PPAR𝛼 agonists [11]. leukotriene B4) as well as synthetic fibric acid derivatives The involvement of PPAR𝛼 in lung cancer biology has (e.g., clofibrate and fenofibrate) and pirinixic acid (WY- been extensively investigated within the past decade. A study 14,643) [13, 19]. Saturated and unsaturated fatty acids and using a mouse xenograft model showed that absence of eicosanoids such as prostacyclin can activate PPAR𝛽/𝛿 [13, PPAR𝛼 expression in the host animals suppresses tumor 20]. In addition, synthetic compounds with higher affini- growth of Lewis lung carcinoma (LLC) cells and lung and ties for the receptor have been developed [8, 13]. Natural liver metastasis of B16 melanoma cells [29]. This suppres- PPAR𝛾 ligands include saturated and unsaturated fatty acids, sion of tumorigenesis and metastasis reflects an increase in 12,14 eicosanoid derivatives such as 15-deoxy-Δ -prostaglandin leukocyte infiltration of the tumor that is associated with J2 (15d-PGJ2), and nitrated fatty acids such as nitrated linoleic host tissues’ antitumor inflammatory responses as well as acid and nitrated oleic acid [21–23]. Synthetic molecules, a reduction in tumor angiogenesis. Intriguingly, the same most notably thiazolidinediones (TZDs) such as pioglitazone, research group found that PPAR𝛼 agonists such as fenofi- rosiglitazone, troglitazone, and ciglitazone, are potent PPAR𝛾 brate and WY-14,643 have the same antitumorigenic and agonists [23]. Upon binding to their respective receptors, antiangiogenic effects via host PPAR𝛼 [30]. Together, these these agonists induce dissociation of corepressors that other- two seemingly contradictory observations imply that the wise maintain PPARs in their inactive state [10]. Corepressor antitumor effect of PPAR𝛼 may be two-pronged; complete dissociation allows the receptors to heterodimerize with absence of PPAR𝛼 expression allows tumor clearance by the retinoid X receptors and initiate transcription by binding to host’s immune system while agonist-induced stimulation of specificPPARresponseelementsinthepromoterregions PPAR𝛼 prohibits the exaggerated inflammatory responses of of their target genes [10]. Emerging evidence suggests that the host that can aggravate tumor development [29, 30]. each PPAR regulates tumorigenesis of different cancer types. WY-14,643 has also demonstrated a similar antiangio- Moreover, it has been reported that expression of all three genic effect, consequently inhibiting tumor formation in a isotypes is altered during lung carcinogenesis [24–26]. Thus, mouse xenograft model established with A549 NSCLC cells PPAR agonists hold potential as novel chemopreventive and as well as in a mouse model of spontaneous NSCLC [31, therapeutic agents for lung cancer, warranting a review of 32]. In addition to suppressing primary tumor development, 𝛼 current literature and further investigation. PPAR activation by WY-14,643 inhibits metastasis to the contralateral lung and to the liver in an orthotopic NSCLC model [32]. This negative effect of PPAR𝛼 stimulation during 2. PPARs in Lung Cancer carcinogenesis is directed toward proliferation of endothelial 2.1. PPAR𝛼. PPAR𝛼 was the first PPAR subtype to be iden- cells, rather than tumor cells, via suppression of epoxye- tified [27]. Its primary function is to regulate energy home- icosatrienoic acid biosynthesis [31, 32]. Epoxyeicosatrienoic acids have been shown by both in vitro and in vivo studies ostasis, controlling fatty acid catabolism and lipoprotein to be proangiogenic [32]. Lastly, fenofibrate treatment was metabolism, especially in the liver, as well as metabolism of found in mice to significantly abrogate neoplasia formation glucose and amino acids [10, 11, 14]. In vitro and in vivo studies induced by the potent carcinogen 4-nitroquinoline 1-oxide have shown that PPAR𝛼 agonists also play a regulatory role in 𝛼 [33]. These studies supporting the antitumorigenic effect of inflammatory responses [10]. The function of PPAR during PPAR𝛼 agonists, combined with clinical efficacy and safety of carcinogenesis has not been extensively defined, with most these molecules in treating hyperlipidemia, certainly warrant available studies focusing on its role in hepatocarcinogenesis closer investigation of PPAR𝛼 as a therapeutic target in lung 𝛼 in rodents. In this context, long-term PPAR activation cancer. leads to the development of tumors via induction of DNA replication and cell proliferation and suppression of apoptosis 2.2. PPAR𝛽/𝛿. PPAR𝛽/𝛿 is involved in a variety of physi- [11, 16]. Reactive oxygen species that are byproducts of fatty ological processes including embryonic development, lipid acid metabolism mediated by PPAR𝛼 are also thought to metabolism, wound healing, and inflammation [11, 14, 16, contribute to tumorigenesis [11, 16]. Further supporting the 34]. Its critical role in regulation of cellular functions such involvement of PPAR𝛼 in hepatocarcinogenesis is the finding as adhesion, proliferation, differentiation, and survival has PPAR Research 3

also been well characterized, especially in keratinocytes [11, induction of G1 cell cycle arrest as a result of reduced 16, 34], strongly suggesting its involvement in carcinogen- cyclin D expression, rather than induction of apoptosis, as esis. The biological function of PPAR𝛽/𝛿 in cancer has the underlying mechanism. This antiproliferative effect of perhaps been most studied in colon cancer. However, its PPAR𝛽/𝛿 activation parallels several in vivo observations. effect during carcinogenesis remains highly controversial Although involvement of PPAR𝛽/𝛿 was not directly assessed due to lack of consensus in clinical and experimental data. and a PPAR𝛽/𝛿-independent mechanism remains a viable One controversy revolves around expression of PPAR𝛽/𝛿, possibility, one research group used multiple lung cancer with some studies reporting enhanced expression in colon models to demonstrate that lung tumorigenesis is suppressed tumors compared to nontransformed colonic epithelium in by increased synthesis of the PPAR𝛽/𝛿 agonist prostacyclin which PPAR𝛽/𝛿 expression is normally high. However, most [38, 39]. Mice lacking PPAR𝛽/𝛿 expression also display of these studies are associated with significant limitations increased tumor incidence in a RAF-induced lung cancer such as small sample size, lack of appropriate controls, or model [40]. These in vitro and in vivo data suggest a protective inadequate experimental methods and thus need to be inter- role of PPAR𝛽/𝛿 against lung cancer. preted with some caution [14]. The most robust findings to It has also been postulated that PPAR𝛽/𝛿 may prevent date are provided by the recent retrospective clinical analy- lung cancer via its anti-inflammatory function, as it does sis of 141 subjects, showing that higher PPAR𝛽/𝛿 expres- with colorectal cancer. In two independent studies, one sion in primary colorectal tumors is associated with lower using a lipopolysaccharide-induced pulmonary inflamma- expression of a marker related to cell proliferation rate, more tion model [41] and the other using a carrageenan-induced differentiated cells, reduced rate of lymph node metastasis, pleurisy model [42], the potent PPAR𝛽/𝛿 agonist GW0742 and better patient survival following radiation treatment was shown to reduce neutrophil infiltration into the lungs [35]. This report supports the protective role of PPAR𝛽/𝛿 and suppress expression of proinflammatory cytokines such in human colorectal cancer. In contrast, an in vivo study as interleukin-6 (IL-6), IL-1𝛽, and tumor necrosis factor-𝛼 using a colorectal cancer cell xenograft model found that (TNF-𝛼) [41, 42]. Although these studies did not assess the PPAR𝛽/𝛿 deficiency in the grafted tumor cells suppresses effect of PPAR𝛽/𝛿’s anti-inflammatory activity on lung car- tumor growth, suggesting a protumorigenic role [36]. How- cinogenesis, pulmonary inflammation has been implicated ever, when interpreting these expression data, it is important as a contributing factor [4, 12, 43]. The anti-inflammatory to note that PPAR𝛽/𝛿 expression does not indicate the function of PPAR𝛽/𝛿 agonists in the context of lung cancer receptor is functionally active; the receptor’s activity can be biology is therefore worthy of further investigation. modulated by a variety of factors such as ligand availability In contrast to these studies, however, others have pro- and the presence or absence of other proteins [14] as well as by vided evidence that PPAR𝛽/𝛿 activation promotes lung can- posttranslational modifications. Thus, future studies should cer; the agonist GW501516 stimulates proliferation, inhibits examine and compare activity state in addition to expression apoptosis, and supports anchorage-independent growth of of PPAR𝛽/𝛿 intumorsandnormaltissuecounterparts. A549, H157, and H23 NSCLC cells [25]. The proliferative Evidence regarding functional outcomes of PPAR𝛽/𝛿 effect is mediated through PDPK1 overexpression, increased activation is similarly contradictory; some studies show AKT phosphorylation, and PTEN suppression, while resis- activated PPAR𝛽/𝛿 promotes tumor development by stim- tance to apoptosis results from enhanced expression of B-cell ulating cell proliferation and preventing apoptosis while lymphoma-extra large (Bcl-xL) and cyclooxygenase-2 (COX- others propose receptor activation attenuates tumorigene- 2). PPAR𝛽/𝛿 can potentiate tumor formation by modulation sis by inducing differentiation and suppressing exaggerated notonlyofcancercellsbutalsoofnontransformedcellsin inflammatory responses [14, 16]. Several molecular mecha- the tumor microenvironment. In a mouse xenograft model nismshavebeenproposedtounderliePPAR𝛽/𝛿’s effect on with LLC cells, absence of PPAR𝛽/𝛿 in the host animals tumorigenesis. Two pathways implicated in its protumori- significantly reduced tumor volume and improved survival genic effect are increased expression of vascular endothelial of the animals [44]. This suppression of LLC cell tumor growth factor (VEGF) and enhanced prosurvival signaling growth in the PPAR𝛽/𝛿-deficient mice is a consequence of involving integrin linked kinase (ILK), 3-phosphoinositide- dysregulated angiogenesis and reduced blood flow. dependent-protein kinase 1 (PDPK1), phosphatase and tensin Other studies suggest that PPAR𝛽/𝛿 may not influence homolog deleted on chromosome 10 (PTEN), and AKT [14]. tumorigenesis at all. One such study observed that GW501516 It has been shown that the antitumorigenic effect is mediated or GW0742 had no effect on expression of PTEN or PDPK1, through enhanced activity of prodifferentiation genes and/or or on AKT phosphorylation, in A549 or H1838 NSCLC cells, suppression of proinflammatory signals mediated primarily implying that PPAR𝛽/𝛿 activation does not influence these by the NF-𝜅B pathway [14]. cells’ proliferation [45]. No change in the percentage of cells Similar discrepancies are present in cancers of other in each phase of the cell cycle was observed either. Likewise, tissues, with the exception of skin cancer where there seems to the PPAR𝛽/𝛿 antagonist GSK3787 did not affect proliferation be general agreement on the protective role of PPAR𝛽/𝛿 [14]. of A549 or H1838 cells [46]. The involvement of PPAR𝛽/𝛿 in lung cancer was first reported Thus, based on our current knowledge, it is difficult to in an in vitro study showing that the agonist L-165041 draw a definite conclusion regarding the biological effect induces growth inhibition of A549 cells, as evidenced by of PPAR𝛽/𝛿 activation in lung cancer. There are several decreased expression of the proliferation marker proliferating possible explanations for these discrepancies, however. First, cell nuclear antigen (PCNA) [37]. The authors identified the contradictory results may be related to PPAR𝛽/𝛿’s ability 4 PPAR Research to repress as well as induce target gene expression; it has nitric oxide synthase, MMP-9, scavenger receptor A, TNF- been observed that PPAR𝛽/𝛿 can repress the transcription 𝛼,IL-1𝛽, and IL-6 [53–55]. Importantly, these inflammatory of its target genes when not bound by its ligands, whereas molecules have been shown to promote tumorigenesis in ligand-bound PPAR𝛽/𝛿 induces expression [16]. Secondly, severalcancers[56].Thenegativeregulationofinflammatory PPAR𝛽/𝛿 activity may be affected by the presence or absence responses is mediated by the inhibition of transcription of cofactors and repressors [16]. Therefore, it is conceivable factors, for example, NF-𝜅B, activator protein-1 (AP-1), mem- that the between-study variability in cell culture conditions bers of the signal transducer and activator of transcription and genetic background of model animals creates differential (STAT) protein family, and nuclear factor of activated T cellular environments and thereby leads to the contradictory cells (NFAT), often via a mechanism termed transrepression observations [16]. Finally, many PPAR ligands demonstrate [17, 57]. During transrepression, PPAR𝛾 interacts with tran- PPAR-dependent and -independent activities, which makes scription factors and sequesters them from their response data interpretation more challenging [16]. In summary, fur- elements, preventing inflammatory responses [57]. PPAR𝛾 ther careful analyses are required to delineate the complexi- also regulates pathways essential to expression and activity of ties of PPAR𝛽/𝛿 expression and activation in lung cancer. these transcription factors. 15d-PGJ2, troglitazone, ciglitazone, and rosiglitazone [58, 2.3. PPAR𝛾. PPAR𝛾 is an established regulator of adipocyte 59], as well as constitutively active PPAR𝛾 [59], also suppress differentiation, glucose metabolism, and lipid homeostasis differentiation of human lung fibroblasts into myofibroblasts [10, 23]. Its involvement in inflammation has also been [58, 59]. Myofibroblasts within the tumor microenvironment recognized [10]. More recently, PPAR𝛾’s role in cancer has are the predominant source of tumor-supporting extracellu- become apparent; PPAR𝛾 hinders tumor development and lar matrix and also produce molecules that facilitate tumor progression, in most cases by modulating differentiation, growth and progression [17, 60] and are considered more proliferation, apoptosis, and motility of cancer cells through carcinogenic than normal fibroblasts [60]. PPAR𝛾 agonists a variety of molecular pathways [8, 17, 47, 48]. In addition to also prevent the myofibroblast-associated increase in collagen regulating the oncogenic activities of cancer cells, PPAR𝛾 can secretion [58, 59] that can result in remodeling of the control the tumor microenvironment; the receptor creates tumor microenvironment and facilitate cancer pathogenesis a hostile environment for tumor growth and metastasis via [61]. Furthermore, PPAR𝛾 agonists demonstrate suppressive multiple mechanisms [8, 17]. In the context of lung cancer, effects on neutrophils’ chemotactic response and neutrophil with general agreement on its role as a tumor suppressor, cytokine production [62]. As predicted by this concept, in the biological effects of activated PPAR𝛾 are perhaps better a mouse model of pulmonary inflammation, endothelial defined than those of PPAR𝛼 or PPAR𝛽/𝛿 [8]. As its influence cell PPAR𝛾 deficiency enhanced neutrophil infiltration into on cancer cells has been extensively reviewed elsewhere [8, 17, the lungs and exacerbated tissue injury [63]. These studies, 18], this review will focus on how the microenvironment may providing a link between PPAR𝛾, inflammation, and cancer, be affected by PPAR𝛾’s anti-inflammatory function. highlight the significance of inflammation-associated cells as Lung carcinogens such as tobacco smoke and inhaled a trigger of tumorigenesis as well as of PPAR𝛾 as a tumor asbestos are known to cause chronic pulmonary inflamma- suppressor acting via multiple mechanisms. tion that is associated with lung carcinogenesis [4, 43]. Key It may prove beneficial to pursue PPAR𝛾 activation as players in the cancer-associated inflammatory responses are a novel chemopreventive strategy [64]. The chemopreven- cellular constituents of the tumor microenvironment such as tive effects of PPAR𝛾 agonists in lung cancer have been tumor-associated macrophages, neutrophils, and fibroblasts reported by several studies. Troglitazone and pioglitazone thatsecretegrowthfactors,cytokines,chemokines,reactive as well as sulindac sulfide, a nonsteroidal anti-inflammatory oxygen species, and matrix metalloproteinases (MMPs) [4]. drug known to activate PPAR𝛾,significantlyreduceprimary The influence of the resulting inflammatory microenvi- tumor formation by A549 cells in a xenograft mouse model ronment on tumor formation and metastasis is multifold [65, 66]. Pioglitazone also decreases tumor volume and and often involves the NF-𝜅B signaling pathway [4]. For significantly deters disease progression in mouse models instance, inflammation has been shown to increase the of spontaneous lung adenocarcinoma and squamous cell rate of genetic mutation within the adjacent epithelial cells carcinoma induced by vinyl carbamate and N-nitroso-tris- and the proliferation of those mutated cells [4]. Tumor- chloroethylurea, respectively [67]. These findings suggest that associated macrophages can facilitate tumor angiogenesis, PPAR𝛾 agonists can inhibit epithelial cell transformation a process that supplies microscopic tumors with nutrients in the early stages of tumorigenesis. Most significantly, one and provides cancer cells with ready access to the circulation epidemiologic analysis of diabetic patients from 10 Veterans required for metastasis [4, 49–51]. The cells mediating cancer- Affairs medical centers, comparing 11,289 TZD users with associated inflammatory responses also promote metastasis 76,389 nonusers, observed a 33% reduction in subsequent by contributing MMPs, the key regulator of extracellular lung cancer diagnosis in the former group [68], thus under- matrix remodeling and disruption [4, 52]. scoring the chemopreventive potential of PPAR𝛾 agonists. A PPAR𝛾 has been shown to affect multiple aspects of these clinical trial (NCT00780234) designed to assess the ability cancer-associated inflammatory responses. Agonist activa- of pioglitazone to prevent lung cancer in a more general, tion of PPAR𝛾, whose expression increases upon macrophage nondiabetic population has been initiated, and its results may and monocyte activation [10], suppresses these leukocytes’ provide additional justification for the application of PPAR𝛾 production of inflammatory mediators such as inducible agonists as a chemopreventive strategy against lung cancer. PPAR Research 5

3. Therapeutic Application of PPAR Ligands modulators may become a valuable tool in the prevention and for Lung Cancer treatment of lung cancer. All three members of the PPAR family demonstrate involvement in carcinogenesis, although their mode of action differs. Abbreviations 12,14 These receptors are therefore attractive targets for lung cancer 15d-PGJ2:15-Deoxy-Δ -prostaglandin J2 prevention and treatment. Indeed, the therapeutic appli- AP-1: Activator protein-1 𝛾 cability of PPAR agonists is evident in several studies. Bcl-xL: B-cell lymphoma-extra large 𝛾 Besides experimental data supporting the use of PPAR ago- COX-2: Cyclooxygenase-2 nists as a monotherapy for lung cancer, as discussed above, HMG-CoA reductase: 3-Hydroxy-3-methyl-glutaryl- 𝛾 multiple PPAR agonists demonstrate synergy with com- coenzyme A reductase monly used traditional chemotherapeutic drugs such as cis- IL: Interleukin platin, carboplatin, and paclitaxel, inhibiting proliferation of ILK: Integrin linked kinase multiple NSCLC cell lines and suppressing tumor growth in LLC: Lewis lung carcinoma a xenograft lung cancer model [69, 70]. This synergistic effect MMP: Matrix metalloproteinase 𝛾 has also been observed between PPAR agonists and targeted NFAT: Nuclear factor of activated T cells therapies such as gefitinib, an epidermal growth factor recep- NSCLC: Non-small cell lung cancer tor inhibitor, and lovastatin, an inhibitor of 3-hydroxy-3- PCNA: Proliferating cell nuclear antigen methyl-glutaryl-coenzyme A reductase (HMG-CoA reduc- PDPK1: 3-Phosphoinositide-dependent- tase) [71, 72]. These data substantiate the chemotherapeutic proteinkinase1 potential of PPAR𝛾 agonists. Unlike PPAR𝛾 agonists, how- PPAR: Peroxisome proliferator-activated ever, the clinical applicability of PPAR𝛼 and PPAR𝛽/𝛿 ligands receptor in lung cancer has not been assessed. Nevertheless, the PTEN: Phosphatase and tensin homolog PPAR𝛼 fibrate agonists have proven relatively safe and effec- deleted on chromosome 10 tive for treatment of dyslipidemia and cardiovascular disease SCLC: Small cell lung cancer [11] and clinical assessment of PPAR𝛽/𝛿 ligands should be STAT: Signal transducer and activator of starting soon. In this context, reported studies showing their transcription physiological effects in lung cancer make a strong argu- TNF-𝛼: Tumor necrosis factor-𝛼 ment for further investigation of their chemopreventive and TZD: Thiazolidinedione chemotherapeutic potentials. VEGF: Vascular endothelial growth factor. SomePPARagonistsactivatetwoorallthreePPARrecep- tors [14, 73]. Recently, an intriguing concept has emerged Disclosure suggesting that use of these dual- or pan-PPAR agonists may be more beneficial than using agents targeting a single The contents in this article do not represent the views of PPAR subtype. For instance, one recent clinical trial observed the US Department of Veterans Affairs or the United States bezafibrate, a pan-PPAR agonist, reduced development of Government. new colon cancer by 53%, although there was no comparison to more selective PPAR agonists [74]. This finding can be Competing Interests interpreted as showing that lower-affinity pan-PPAR agonists maybeusefulasanovelchemopreventivestrategy[14]. The authors declare that they have no conflict of interests. Simultaneous activation of PPAR𝛼 and/or PPAR𝛽/𝛿 may also alleviatetheknownsideeffectsofPPAR𝛾 agonists (weight Acknowledgments gainandbonefractures)bystimulatinglipidmetabolismand boneformation[14,73].Thus,activationofmorethanone This work was supported by a Merit Review Award from the PPAR receptor should also be pursued as a new therapeutic US Department of Veterans Affairs and National Institutes of approach in lung cancer. Health Grants HL093196 and AI125338 (RCR).

4. Conclusions References

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Review Article Potential Role of ANGPTL4 in the Cross Talk between Metabolism and Cancer through PPAR Signaling Pathway

Laura La Paglia,1 Angela Listì,2 Stefano Caruso,3 Valeria Amodeo,4 Francesco Passiglia,2 Viviana Bazan,2 and Daniele Fanale2

1 ICAR-CNR, National Research Council of Italy, 90146 Palermo, Italy 2Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, 90127 Palermo, Italy 3Genomique´ Fonctionnelle des Tumeurs Solides, INSERM, UMR 1162, 75010 Paris, France 4Samantha Dickson Brain Cancer Unit, UCL Cancer Institute, University College London, London WC1E 6DD, UK

Correspondence should be addressed to Daniele Fanale; [email protected]

Received 20 October 2016; Accepted 19 December 2016; Published 15 January 2017

Academic Editor: Stephane´ Mandard

Copyright © 2017 Laura La Paglia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The angiopoietin-like 4 (ANGPTL4) protein belongs to a superfamily of secreted proteins structurally related to factors modulating angiogenesis known as angiopoietins. At first, ANGPTL4 has been identified as an adipokine exclusively involved in lipid metabolism, because of its prevalent expression in liver and adipose tissue. This protein regulates lipid metabolism by inhibiting lipoprotein lipase (LPL) activity and stimulating lipolysis of white adipose tissue (WAT), resulting in increased levels of plasma triglycerides (TG) and fatty acids. Subsequently, ANGPTL4 has been shown to be involved in several nonmetabolic and metabolic conditions, both physiological and pathological, including angiogenesis and vascular permeability, cell differentiation, tumorigenesis, glucose homoeostasis, lipid metabolism, energy homeostasis, wound healing, inflammation, and redox regulation. The transcriptional regulation of ANGPTL4 can be modulated by several transcription factors, including PPAR𝛼,PPAR𝛽/𝛿,PPAR𝛾, and HIF-1𝛼, and nutritional and hormonal conditions. Several studies showed that high levels of ANGPTL4 are associated with poor prognosis in patients with various solid tumors, suggesting an important role in cancer onset and progression, metastasis, and anoikis resistance. Here, we have discussed the potential role of ANGPTL4 in mediating the cross talk between metabolic syndromes, such as diabetes and obesity, and cancer through regulation of its expression by PPARs.

1. Peroxisome Proliferator-Activated actions are limited to specific tissue types [3, 4]. PPAR𝛼, Receptors (PPARs): Structure and Functions the first PPAR to be cloned, is highly expressed in tissues characterized by elevated fatty acid oxidation such as liver, Peroxisome proliferator-activated receptors (PPARs) are heart, skeletal muscle, brown adipose tissue, kidney, adrenal ligand-activated transcription factors belonging to the steroid gland, and intestinal mucosa, where it plays a key role in the hormone receptor superfamily, identified for the first time fatty acid catabolism [5, 6]. PPAR𝛽/𝛿 is expressed in most in 1990 by Issemann and Green [1]. There are three distinct of human tissues, mainly in the liver, adipose tissue, skeletal PPAR subtypes, PPAR𝛼,PPAR𝛽/𝛿 (also known as PPAR𝛽 or muscle, heart, brain, kidney, skin, and intestine, characterized PPAR𝛿), and PPAR𝛾, encoded by specific genes located on by an increased lipid metabolism. However, the function of different chromosomes. Although these three members show this isoform remains to be elucidated [7–10]. PPAR𝛾 is highly a significant homology, they differ from each other for tissue expressedinwhiteandbrownadiposetissue(WATandBAT) distribution, affinity for ligands, and biological functions and plays a pivotal role in the regulation of adipogenesis, fat [2]. All subtypes are activated by endogenous ligands and storage, and glucose metabolism [11–15]. In addition, PPAR𝛾 participate in the regulation of several genes involved in also regulates the expression of proinflammatory cytokines, glucose and lipid metabolism. However, other specific PPAR such as tumor necrosis factor-𝛼 (TNF-𝛼), as well as genes 2 PPAR Research involved in insulin sensitivity. For this reason, PPAR𝛾 is the fibrinogen-like globular domain and an N-terminal coiled- main target of thiazolidinediones (TZDs), a class of drugs coil domain [35] except for ANGPTL8. Indeed, this last used to improve lipid and glucose metabolism in type 2 ANGPTL family member is considered an atypical member, diabetes [16, 17]. since it lacks the main structural features present in all other PPARs show a DNA binding domain (DBD) in the N- proteins of the group, such as the fibrinogen-like domain, terminal and a ligand binding domain (LBD) in the C-ter- glycosylation sites, and amino acids requested for formation minalseparatedbyahingeregionactingasadockingsite of disulfide bonds [32] (Figure 2). Unlike the angiopoietins, for cofactors [18]. Three PPAR isoforms exhibit an 80% ANGPTLs are considered orphan ligands, as they do not bind homology and are more divergent in the LBD, confirming to either the angiopoietin receptor tyrosine kinase Tie2 or the their different response to various ligands. After activation by related protein Tie1 [36–38]. endogenous or synthetic ligands, PPARs undergo a confor- The first four family members (ANGPTL1-4) and mational change that causes the translocation to the nucleus ANGPTL6/angiopoietin-related growth factor (AGF) have and the heterodimerization with another nuclear receptor, beenshowntomodulateangiogenesis.ANGPTLs3,4,5,and the retinoid X receptor (RXR) [19]. The PPAR-RXR het- 8 and ANGPTL6/AGF seem to be involved also in regulation erodimer then binds a DNA portion in the promoter region of of other processes such as lipid metabolism and glucose and target genes, called peroxisome proliferator response element energy homeostasis [39–46]. Another study showed that (PPRE), modulating the expression of several genes involved ANGPTLs 3 and 4 control lipid metabolism by inhibiting in different physiological or pathological processes [20]. the activity of lipoprotein lipase (LPL) [47], an enzyme Interestingly, the PPAR functions also depend on the binding responsible for hydrolysis of triglycerides (TG) contained with different coactivator and corepressor proteins [21]. in lipoproteins, such as chylomicrons and very low-density Indeed, after interaction with agonists, the conformational lipoproteins (VLDL), fatty acids, and cholesterol, whereas change of the PPAR structure causes also the attachment of ANGPTL6/AGF antagonizes obesity and related metabolic coactivators and detachment of corepressors. Usually, PPAR- diseases, including insulin resistance, by enhancing systemic RXR heterodimers are packed with a corepressor molecule energy expenditure [45]. in PPRE and the binding with ligands causes an exchange ANGPTLs show different tissue expression patterns. of corepressors for coactivators. One of the more studied ANGPTL1 is mostly detected in liver, heart, skeletal muscle, PPAR corepressors is histone deacetylase (HDAC). Among kidney, and vessel-rich endocrine organs (adrenal glands, the different coactivators, there are PGC-1, p300, and CREB thyroid, and pituitary gland) but also to a lesser extent in that are involved in regulation of metabolism as well as in uterus and gastrointestinal tract [48]. ANGPTL2 shows high cancer development [22, 23] (Figure 1). expression levels in heart, stomach, adipose tissue, skeletal muscle, and uterus [49], whereas ANGPTL3 is predomi- nantlyexpressedinliver[50,51].ANGPTL4shows∼30% of 2. Angiopoietin-Like 4 (ANGPTL4): Structure sequence homology with ANGPTL3. It is abundantly present and Expression Patterns in the liver, adipose tissue, and skeletal muscle and, to a lesser extent, in placenta, small intestine, heart, and pituitary gland The angiopoietin-like 4 (ANGPTL4) protein was discovered [52–56]. ANGPTL5 is mainly expressed in adult human heart for the first time in 2000 by three independent research [57], whereas ANGPTL6/AGF expression is restricted to liver groups. They simultaneously identified this molecule asa and plasma [58]. Lastly, ANGPTL7 exhibits high expression fasting-induced adipose factor (Fiaf) in different tissues. levels in the cornea, neural tissues, and trabecular meshwork ANGPTL4 is mainly expressed in liver and adipose tissue, as as well as uterine endometrial cancer and melanoma [59]. shown by Kersten et al. [24] that highlighted its upregulation The human gene encoding ANGPTL4 is evolutionarily in these tissues during fasting and a PPAR-dependent mRNA 𝛼 𝛾 conserved among species and shares a sequence homology of regulation, using PPAR / wild-type and mutant mice. Also, ∼77% with mouse. It is located on chromosome 19p13.3 and Kim et al. [25] identified a novel angiopoietin-like protein consists of seven exons encoding a 406-amino acid glycopro- mainly expressed in hepatocytes. Finally, Cliff Yoon et al. tein. Like other proteins of the ANGPTL family, ANGPTL4 [26] proved the regulative relation between PPAR proteins contains a C-terminal fibrinogen-like domain (cANGPTL4) and ANGPTL4, demonstrating that ANGPTL4 is a target of and an N-terminal coiled-coil folding domain (nANGPTL4), PPAR𝛾 in adipose tissue. ANGPTL4 belongs to a superfamily of secreted proin which a highly hydrophobic region that acts as a signal teins structurally related to factors modulating angiogen- peptide for protein secretion is present. In addition, it exhibits esis known as angiopoietins (ANG). This protein family several potential N- and O-glycosylation sites and was found includes eight members encoded by eight genes (ANGPTL1- to be N-glycosylated at amino acid position 177 [37, 60]. 8) identified in humans and mice, except ANGPTL5, that is a The same domains of full-length ANGPTL4 (flANGPTL4) human orthologue [25, 27–29]. Only recently, in 2012, a new protein structure were found in plasma [61, 62]. Higher-order feeding-induced hepatokine was identified and called RIFL/ oligomeric structures can be formed by native flANGPTL4 lipasin/ANGPTL8 (also known as betatrophin) [30–33]. Just through the formation of intermolecular disulfide bonds. in 2015, the HUGO gene nomenclature defined the official ANGPTL4 contains several conserved cysteine residues that name of the protein as ANGPTL8 [34]. Like angiopoietins, all contribute to the formation of variable-sized multimeric angiopoietin-like proteins (ANGPTLs) exhibit a C-terminal structures. The N-terminal oligomerization of ANGPTL4 PPAR Research 3

Coactivators: Corepressors: PGC-1 mRPD3 P/CIP NCoR CBP/p300 Ligands mSin3 P/CAF

PPAR PPAR RXR target genes

AGGTCA N (N) AGGTCA PPRE

Figure 1: Interaction between PPARs and PPRE. The figure shows PPAR structure and related coactivator and corepressor molecules involved in activation and repression mechanisms. The activation signaling of PPAR-RXR heterodimer and PPRE allows the expression modulation of target molecules such as ANGPTL4.

requires the presence of two cysteine residues (Cys-76 and The transcriptional regulation of ANGPTL4 and its Cys-80) in the N-terminal portion [63]. resulting expression can be determined by several transcrip- Different studies showed that nANGPTL4 domain is used tion factors, including PPAR𝛼,PPAR𝛾,andHIF-1𝛼. A deeper to modulate lipid metabolism, whereas cANGPTL4 domain investigation about transcription activation of ANGPTL4 may be a modulator of tumorigenesis process [54]. Indeed, and all these TFs was done by Inoue et al. [71]. Starting from ANGPTL4 can exert its function of LPL activity inhibitor the evidence that all these factors are important angiogenic thanks to an oligomerization process mediated by the N- molecules, the authors assessed whether there could be a terminal region responsible for its assembly into tetrameric synergic action mechanism of these two different signals in or dimeric structures [64–66]. It was hypothesized that LPL stimulating ANGPTL4 as angiogenesis-related target gene. blockage is due to 12 highly conserved amino acids that are Indeed, microarray and ChiP-seq analyses showed a cross- near the N-terminus of the protein. Indeed, mutations in enhancement of ANGPTL4 expression dependent on the three polar amino acid residues within this region abolished conformational proximity of two response elements [71]. theabilityofANGPTL4toinhibitLPL[66].Experimental Different evidences showed that upregulation of evidence showed that several proprotein convertases, includ- ANGPTL4 expression is strongly linked to fasting in a ing furin, PC5/6 (proprotein convertase 5/6), and PCSK3 variety of tissues [72, 73]. Different actors likely mediate the (proprotein convertase subtilisin/kexin type 3), catalyze the “fasting effect” [74]. Several findings showed that PPARs proteolytic processing of the human flANGPTL4 protein via nuclear receptors induce an increase in ANGPTL4 expres- recognition of a specific amino acid sequence, causing the sion. Glucocorticoids, whose circulating levels are high dur- release of the N-terminal region and monomeric C-terminal ingfasting,alsoseemtomediatethisevent[75–78],and, portion [66, 67]. ANGPTL4 is cleaved in a tissue-dependent in addition to fasting, chronic caloric restriction or free fatty manner and can be secreted into the bloodstream from acids (also called NEFA) have been shown to increase plasma adipose tissue and liver in nativeandcleaved,glycosylated, ANGPTL4 levels [56]. Finally, some studies conducted on and oligomerized isoforms. In humans, the truncated form is human myofibroblasts, using genome-wide transcriptional secreted from liver, whereas the full-length form is released profiling technology, revealed that human ANGPTL4 ex- from adipose tissue [61, 62]. Furthermore, ANGPTL4 has pression might be synergistically induced by the functional beenshowntobindtoheparinsulfateproteoglycansand interactions of TGF-𝛽 and PPAR 𝛽/𝛿 signaling [79]. interact with ECM (extracellular matrix) proteins, by inhibit- Since ANGPTL4 expression was found mainly in liver ing endothelial cell adhesion and migration and altering actin and adipose tissue, this molecule was classified, at first, cytoskeleton [68–70]. as an adipokine exclusively involved in lipid metabolism. 4 PPAR Research

286–315 439–452

N N N N SSN SS

ANG1 92 122 154 243 295 280–309 432–455

N N S S SS

ANGPTL1 160 188 ANGPTL2

ANGPTL3

LIPASIN 188–216 341–354

N S S S S

ANGPTL4 177 ANGPTL5

ANGPTL6

ANGPTL7

Signal peptide Coiled-coil domain SE1 Fibrinogen-like domain

Figure 2: Structural organization and homology between ANGPTL family members. Signal peptide domain is shown in green, specific epitope 1 (SE1) (region present in ANGPTL3 and ANGPTL4 important for binding LPL and inhibiting its activity in vitro and in vivo) in purple red, coiled-coil domain in blue, and N-terminal fibrinogen-like domains in orange. Glycosylation sites (N) are shown at positions 92, 122, 154, 243, and 295 for ANG1; positions 160 and 188 for ANGPTL1; and position 177 for ANGPTL4. Disulfide bonds (SS) are shown at positions 286–315 and 439–452 for ANG1; 280–309 and 432–455 for ANGPTL1; 188–216 and 341–354 for ANGPTL4.

Afterwards, thanks to a large number of studies, this protein on LPL activity in fatty acid (FA) uptake, whereas the has been shown to have a highly multifaceted role, since it is effect of PPAR𝛿 activation on 𝛽-oxidation is independent of involved in several nonmetabolic and metabolic conditions, ANGPTL4 [83]. Interestingly, the authors investigated also both physiological and pathological, including angiogenesis the role played by ANGPTL4 in regulating LPL activity, not and vascular permeability, cell differentiation, tumorigenesis, only at the level of the surface of capillaries, highlighting the glucose homoeostasis, lipid metabolism, energy homeostasis, intracellular lipase degradation [83]. Other studies tried to wound healing, inflammation, and redox regulation [80]. better assess the cellular localization and molecular mechanisms underlying ANGPTL4 role in lipid metabolism, as 3. ANGPTL4: A Regulative Role in reported by Dijk et al. [84]. These authors performed ex vivo and in vivo studies on adipocytes and adipose tissue from Glucose and Lipid Metabolism −/− wild-type and ANGPTL4 mice, showing that ANGPTL4 As previously introduced, among the “pleiotropic” roles of stimulates LPL processing in the endoplasmic reticulum (ER) ANGPTL4, greater attention was focused on its involvement leading to its intracellular degradation [84]. Another study in glucose and lipid metabolism regulation [81]. A positive proposed that LPL regulation by ANGPTL4 occurs at cell correlation between increased ANGPTL4 and NEFAs levels surface [85]. in plasma of healthy subjects after dietary regimens has The homeostasis of lipid metabolism is promoted through been shown. Conversely, the negative energy balance caused the intervention of lipases, enzymes that counterbalance LPL by fasting increases the hydrolysis of intracellular TG in activity. Indeed, they hydrolyze stored TG, allowing adipo- adipocytes and other peripheral tissues. This leads to an cytes to release FA. Starting from these evidences, an inter- increase in plasma NEFA levels [82]. esting work by Koliwad et al. [78] showed that ANGPTL4 is a Another recent work by Robciuc et al. showed the indirect glucocorticoid receptor (GR) target and is involved in volvement of ANGPTL4 in mediating the PPAR𝛿 effects GR-dependent TG metabolism. Indeed, ANGPTL4-null mice PPAR Research 5 showed lower plasma TG levels and increased ability to gain the hypothesis that they are active during different metabolic weight compared to mice overexpressing the gene, suggesting states. a role of ANGPTL4 in modulating TG homeostasis by A recent work by Zhang proposed an ANGPTL3-4-8 regulation of its expression [78]. molecular model to explain TG trafficking specifically in Lichtenstein et al. [73] have well explained the correlation cardiac and skeletal muscles [95]. The model suggests that between ANGPTL4 role and inhibition of LPL-mediated feeding can induce ANGPTL8, resulting in the activation of plasma TG lipolysis. Thanks to studies derived from trans- the ANGPTL3-8 pathway. This causes LPL inhibition and genic mice, the molecular mechanisms underlying mouse increase in plasma levels of TG which can be stored in WAT. blood TG were deeply revealed. Normally, LPL monomer is In this district, decreases in ANGPTL4 concentration allow associated with the N-terminal domain of ANGPTL4 protein, anincreasedLPLactivity.Theoppositescenarioispresented thus shifting the balance between LPL dimers and monomers during fasting. The impact of the ANGPTLs on plasma lipid towards the latter, causing LPL inhibition and, consequently, levels has led to considering them as therapeutic targets for determining the alteration of TG clearance from the plasma dyslipidemia [96]. and FFA uptake decrease into the peripheral tissues [73]. Decreased plasma levels of this angiopoietin-like protein Another mechanism by which ANGPTL4 inhibits LPL have been detected also after insulin induction. A recent was proposed by Chi et al. These authors showed that study focused on the evaluation of the systemic effect of ANGPTL4 can bind and inactivate LPL complexed to GPI- insulinonLPLanditsregulativemachineryinsubjectswith HBP1. Therefore, the ANGPTL4-mediated LPL inactivation a different tolerance degree to insulin and showed a decrease greatly reduces the affinity of LPL for GPIHBP1 [86] (Fig- in the adipose tissue ANGPTL4 expression in type 2 diabetes ure 3). mellitus patients and healthy subjects [97]. Considering the discussed role about ANGPTL4 in lipid Other evidences concerning the ANGPTL4 role in glu- metabolism, a possible link of this angiopoietin-like protein cose metabolism regulation were reported in different studies with obesity was investigated. Different murine models were on transgenic mice, where the decrease of blood glucose, proposed, highlighting a significant role of this protein in improvement of glucose tolerance, and induction of hyper- central regulation of energy metabolism [87]. More recently, lipidemia and hepatic steatosis have been linked to the Robciucetal.carriedoutaninterestingstudyonhomozygous protein [44, 98]. More recently, other studies carried out on twins, in which a positive correlation between ANGPTL4 humans and animal models suggested the involvement of expression levels, adipose tissue hormone-sensitive lipase ANGPTL4 in nephrotic syndrome, revealing that ANGPTL4 (LIPE), and CGI-58 gene was shown, supporting the hypoth- acts by linking proteinuria and hypertriglyceridemia through esis of the ANGPTLs’ involvement in promoting lipolysis of negative feedback loops [99]. adipocytes [88]. As previously reported, ANGPTL4 is mainly expressed in tissues such as WAT, liver, and skeletal muscle. A recent 4. Role of ANGPTL4 in Cancer work by Alex et al. [89] highlighted protein expression also in The analysis of the different components of the tumor human colon adenocarcinoma cells. The authors investigated microenvironment and their cross talk have been the focus the role of short-chain fatty acids (SCFA) in HT29 and T48 of the research of many molecular laboratories, especially cell lines and showed an induction of ANGPTL4 synthesis by 𝛾 since a lot of studies showed the key role played by tumor SCFA through PPAR receptor activation [89]. Furthermore, microenvironment in cancer development and progression. long-chain fatty acids have been shown to induce ANGPTL4 𝛿 In vivo and in vitro models affirmed the ability of different production and secretion by PPAR activation in skeletal molecular factors belonging to the tumor microenvironment muscle cells [90]. in regulating cell–cell and cell–matrix communications, cell As many scientific papers showed, several proteins, migration, invasion, and metastatic dissemination, but also including ANGPTL4, inhibit LPL. A recent study focused in conditioning drastically the efficacy of antitumor therapies the attention on the regulation mechanisms of another lipase, [100–102]. that is, the pancreatic lipase (PL). Interestingly, Mattijssen et Currently, we know well that the malignance grade of a al. suggested the involvement of ANGPTL4 also in endoge- tumor is related to several factors, including genomic instabil- nous inhibition of dietary lipids, through knockout mice ity, heterogeneity (cells types such as fibroblasts, endothelial experiments [91] (Figure 4). cells, pericytes, and immune cells), and composition of the However, ANGPTL4 is not the only angiopoietin-like microenvironment, which has been shown to change in family member involved in maintaining energy metabolism different cancer types and also among different patients homeostasis. Other two members, such as ANGPTL3 and harbouringthesametumorhistotype. ANGPTL8, can inhibit LPL activity by affecting plasma TG To date, the role of ANGPTL4 in cancer progression is levels. Indeed, different works showed overexpression of not well defined, and there is still some controversy in the these proteins in mice and humans with increased plasma TG literature indicating the need of more studies addressing this levels. Conversely, mutant mice carrying a loss-of-function interesting topic. of ANGPTLs exhibited low plasma TG levels [32, 92–94], Several studies identified the presence of ANGPTL4 in although all three proteins show different tissue expression various solid tumors, such as breast cancer, colorectal cancer, patterns and are regulated by different stimuli. This has led to prostate cancer, hepatocarcinoma, and renal cell carcinoma 6 PPAR Research

VLDL IDL/LDL CE TG Angptl4

Angptl4 Inactive LPL-dimer GPIHBP1

FFA

Oxidative PPAR stress/lipotoxicity

Angptl4

Figure 3: Molecular mechanism governing ANGPTL4-mediated TG hydrolysis. LPL monomer is linked to nANGPTL4 protein fraction, thus shifting the balance between LPL dimers and monomers towards the latter. As a consequence, LPL is inhibited and this causes the alteration of TG clearance from the plasma and uptake decrease of FFA into the peripheral tissues. The intervention of ANGPTL4 on LPL causes also the reduction of LPL affinity for GPIHBP1 protein.

[103–107], suggesting its important role in cancer growth SRC kinase and mitogen-activated protein kinase (MAPK) and progression, anoikis resistance, altered redox regulation, signaling pathways favoring cancer cell growth and survival angiogenesis, and metastasis [80, 104, 108]. One potential [113]. link between ANGPTL4 and tumorigenesis is provided by As regards tumor angiogenesis, there are discordant data hypoxia conditions, which represent a prominent feature of about the proangiogenic or antiangiogenic role of ANGPTL4. tumor microenvironment. Indeed, hypoxia induces overex- Several experiments showed that the ANGPTL4 increase by pression of cyclooxygenase-2 (COX-2) by hypoxia-inducible HIF-𝛼 stimulates the secretion of multiple proangiogenic fac- factor-1 (HIF-1), an oxygen-sensitive transcriptional reg- tors regardless of vascular endothelial growth factor (VEGF). ulator [109–111], leading to the synthesis of prostanoids, In this regard, one of the first evidences regards Kaposi’s sar- especially prostaglandins PGE2. COX-2 is upregulated in coma, which is characterized by a deregulated angiogenesis recruited macrophages to trigger activation of other immune process promoted by the release of proangiogenic molecules. cells involved in antitumor response. Increased levels of Several in vitro and in vivo studies detected a significant PGE2 stimulate an intracellular signaling cascade leading ANGPTL4 upregulation in endothelial cells expressing a to the induction of the ANGPTL4 expression and cANG- deregulated herpesvirus-8- (HHV-8- or KSHV-) encoded PTL4 secretion [112]. Although most of studies have not G protein-coupled receptor (vGPCR), which is considered explained the specific role played by ANGPTL4 as entire a key factor in Kaposi’s sarcoma tumorigenesis. ANGPTL4 molecule or generated fragments, currently, several evidences inhibition has been associated with a significant decrease of suggested a prevalent activity of cANGPTL4. This fragment neovascularization and vascular leakage in vitro and vGPCR- seems to be involved in “anoikis resistance,” which is a mediated tumorigenesis in vivo [114–116]. However, other peculiar feature of metastatic cells acquiring ability to escape studies have reported that ANGPTL4 exhibits an antian- programmed cell death. cANGPTL4 interacts with beta- giogenic role, inhibiting the proliferation, chemotaxis, and integrins to maintain an elevated ROS rate, inducing a redox- tubule formation of endothelial cells. One of such studies based survival mechanism that involves the activation of the explored the effects of ANGPTL4 on the mouse epidermis PPAR Research 7

Skeletal muscle Heart Lipid overload regulation PPAR𝛿 Fatty acid-induced oxidative stress control Liver Regulation of local uptake of PPAR𝛾 plasma triglyceride-derived 𝛼 PPARδ PPAR fatty acids GR

Lipolysis regulation ANGPTL4 HDL and triglyceride level regulation ANGPTL4 Glucose production modulation White adipose tissue Inflammation response GPIHBP1 LPL-dimer

PPAR𝛾 HIF1𝛼 GR

Intestine ↑ Lipolysis PPAR ↑ ANGPTL4 Triglyceride metabolism modulation Macrophages Homeostasis of energy metabolism Glucose metabolism regulation Pancreatic PPAR𝛿 lipase

↑ ANGPTL4 Fat storage regulation ANGPTL4 Inflammation response

Inflammation response

Figure 4: Regulation of expression and role of ANGPTL4 in lipid metabolism. In WAT, fasting induces ANGPTL4 expression through the action of different molecules such as PPARs, HIF-1𝛼, and GR. The protein stimulates TG degradation via LPL inhibition. In liver, PPAR isoforms and GR stimulate ANGPTL4 expression. In this district, ANGPTL4 acts in part on hepatic LP and, in part, is released into the bloodstream, acting on LPL of peripheral tissues. In skeletal muscle, heart, and macrophages, FAs induce ANGPTL4 by PPAR𝛿 activation. Also in intestine, FAs stimulate ANGPTL4 expression via one of the PPARs. ANGPTL4 produced by enterocytes is thus released towards the lumen and inhibits pancreatic LP.

throughaninvivoneovascularizationassay,revealingthat microenvironment. A recent study carried out on patients ANGPTL4 decreased only VEGF-induced neovasculariza- withuvealmelanoma(UM)showedthatANGPTL4secretion tion, whereas it was not able to influence VEGF-independent is regulated by HIF-1 and cooperates with VEGF in the neovascularization [27]. angiogenesis promotion, supporting the potential benefit Finally, all these studies suggested that both proangio- of a combined VEGF-ANGPTL4 inhibition to increase the genic and antiangiogenic effects of ANGPTL4 are reliable and efficacy of antiangiogenic treatments [119–121]. In addition, strongly dependent on the related tumor microenvironment. a recent work by Xin et al. [122] showed that HIF-1-induced Several evidences suggested that tumor microenvironment upregulation of ANGPTL4 may promote vessel permeability playsacrucialroleinmultiplestepsoftumordevelop- in ischemic retinopathies, such as diabetic eye. All these ment and progression, including drug resistance, immune- evidences emphasize the need for further investigations escaping, distant metastasis, and angiogenesis [117]. In par- about the posttranslational modifications that ANGPTL4 ticular, stromal cells are able to secrete multiple factors, can undergo, to better understand how generated fragments including ANGPTL4, to enhance vasculature permeability in could modulate pro- or anti-angiogenic events. both lung and brain cancers [118]. Some studies suggested that metastatic process seems Furthermore, as mentioned before, both the secretion and to be pushed by the activation of human ANGPTL4 via proangiogenic activities of ANGPTL4 are highly dependent TGF-𝛽. This protein is an essential multifunctional cytokine on the vGPCR expression by the Kaposi Sarcoma tumor involved in embryo development and tissue homeostasis but 8 PPAR Research is secreted also in response to hypoxia and/or inflammation. thiazolidinediones, aimed at contrasting pathological condi- Inparticular,ithasbeenshowntoinduceanincreasein tions, including the dyslipidemic state (hypertriglyceridemia) human ANGPTL4 levels in breast cancer cells, by acti- and diabetes mellitus [134, 135]. vating SMAD transcription factors, ultimately favoring the An interesting work by Sethi et al. [136] showed that LDLs transendothelial migration of tumor cells through disrup- oxidation in endothelial cells causes their activation by PPAR tion of endothelial cell junctions [104, 123]. In hepatocellu- agonists. Other PPAR agonists, such as bezafibrate, have been lar carcinoma cells, ANGPTL4 also favors transendothelial shown to directly improve insulin sensitivity through the migration and metastasis, through upregulation of vascular activation of PPAR𝛾 isoform [137]. cell adhesion molecule-1 (VCAM-1) on endothelial cells, As previously reported, the different PPAR isoforms are and stimulates the VCAM-1/integrin 𝛽1signalingpathway, involved in lipid metabolism with different mechanisms of facilitating the cancer cell transendothelial extravasation to action, depending also on the tissue context in which they develop distant metastasis [124]. Another study concerning are [4, 83, 138–142]. Their deregulation can be evidenced the role of ANGPTL4 in colorectal cancer patients posi- in various tissue contexts. For example, an increase in tively correlated ANGPTL4 expression and venous invasion, PPAR𝛽/𝛿 expression was associated with a decreased lipid which is considered the first step of metastatic process. accumulation during a fat-rich diet in cardiac cells, whereas However, the biological mechanism remains elusive [104]. its overexpression in intestine was linked to colon cancer Finally, a study only showed that ANGPTL4 may prevent development [142]. Indeed, Sertznig et al. [4] showed that tumor invasiveness and metastasis through modulation of colon cancer cell activation depends on the stimulus induced both endothelial and tumor cell cytoskeleton organization by arachidonic acid, which leads to COX-2 upregulation [116]. Taken together, all these data suggest a potential and overproduction of prostaglandin PGE2. As previously prometastatic role for ANGPTL4, which of course needs to reported, increased levels of PGE2 stimulate an intracellular be deeply investigated in further studies, in order to elucidate signaling cascade, leading to the induction of ANGPTL4 the biological mechanisms underlying these processes. Since expression and cANGPTL4 secretion [112]. Furthermore, several studies suggested the involvement of ANGPTL4 macrophage PPAR𝛿 induced by Th2 cytokines released by in vascular permeability, angiogenesis, and inflammatory adipocytes has been shown to modulate the polarization of processes, ANGPTL4-modulating agents, such as PPARs, adipose tissue-resident macrophages, causing activation of an fatty acids, and specific drugs, could be useful for treatment anti-inflammatory phenotype and consequently improving of associated diseases [125]. insulin sensitivity [143]. Also PPAR𝛾 is involved in lipid metabolism. Similar to 𝛽 𝛿 5. ANGPTL4 as Potential Modulator of the PPAR / isoform, it regulates the activity of proteins like LPL [139, 140]. Due to this evidence, probably ANGPTL4, Cross Talk between Metabolism and Cancer as well as other adipokines, is also a target of PPAR𝛾, acting as a mediator of lipid metabolism. Moreover, PPAR𝛾 Last decade has progressively evidenced two diseases, such as deregulation was detected not only in peripheral tissues diabetes mellitus and obesity, as contributing factors to cancer linked to lipid metabolism, but also in inflammation and onset and development. The first can favor tumor growth cancer [144]. Several evidences suggested that PPAR𝛾 ligands by increasing the availability of nutrients (e.g., glucose and may be potent inhibitors of angiogenesis mechanisms useful FFA) or through alteration of the normal insulin signaling for anticancer therapy [145, 146]. Trombetta et al. [147] machinery, that causes an increase in blood lipid concen- showed that fatty acids, such as docosahexaenoic acid (DHA), trations [126, 127]. Moreover, FFAs themselves can favor activate PPAR𝛾 in cancer cells, leading to the inhibition the instauration of oxidative stress through the formation of tumor development. Moreover, studies carried out on of stress molecules such as reactive oxygen species (ROS), WAT, using long-chain monounsaturated fatty acids (LC- contributing to inflammation and tumor growth [128, 129]. MUFAs), revealed a PPAR𝛾 overexpression and a decrease of The obesity could also be linked to cancer development inflammatory markers in diabetes syndrome [148]. through regulative mechanisms linked to adipokines and The increasing recognition of the dynamic entity of inflammatory cytokines [130, 131]. Indeed, obesity is char- ANGPTL4 and its multifunctional role in different metabolic acterized by accumulation of visceral adipose tissue that and nonmetabolic pathways, the expression network linking produces high quantities of inflammatory cytokines, mainly PPARs isoform to this angiopoietin-like protein, together 𝛽 𝛼 leptin, but also IL-1 ,TNF- , IL-8, and IL-6 [74, 132]. In with the recent evidences of involvement of PPAR in can- 𝛾 adipocytes, PPAR activation has been associated with the cer,ledtodosomesignificantspeculationsonthepoten- upregulation of IRS-2 and CAP components of insulin path- tial molecular cross talk between these molecules, lipid way and hence to increased insulin sensitivity [133]. In this metabolism, and cancer [149]. complex scenario, we speculated on the potential role played As previously reported, the ANGPTL4 expression de- by some “pleiotropic” molecules, such as PPARs isoforms and pends on different stimuli, such as hypoxia and fasting and, ANGPTL4, in connecting lipid and glucose metabolism with finally, PPAR𝛽/𝛿 isoform induction [80]. Interestingly, recent cancer. evidences showed that ANGPTL4 expression is activated by As previously reported, PPARs activation depends on the PPAR𝛽/𝛿, not only in adipose tissue but also in response to binding of different ligands. Among the PPAR ligands, there inflammation during wound healing [70]. Indeed, ANGPTL4 are natural and synthetic compounds, such as fibrates and was defined as novel matricellular protein interacting with PPAR Research 9

PC cleavage site

ANGPTL4 CCD FLD

Oligomerization Secretion and cleavage

CCD ∼ 26 kDa FLD ∼ 47 kDa nANGPTL4 cANGPTL4 (oligomer) (monomer)

Angiogenesis inhibition Inhibition of LPL Tumor cell survival Tumor cell proliferation

Inhibition of endothelial cell adhesion, Increased wound migration, and tubule formation closure efficiency

Figure 5: Different functional domains of ANGPTL4. Native full-length ANGPTL4 (flANGPTL4) is present as dimeric or tetrameric complexes. It can be processed to generate the N-terminal coiled-coil fragment (nANGPTL4) and COOH terminal fibrinogen-like domain (cANGPTL4), respectively. These protein fractions seem to have distinct roles depending on the tissue context.

specific ECM proteins and integrins to facilitate cell migra- the tumor progression. Indeed, ANGPTL4 mRNA has been tion during this event [54, 69]. This specific function could found to be upregulated in the perinecrotic areas of different be linked to the C-terminal domain. Indeed, cANGPTL4 tumor types [154, 155]. can activate 𝛽1and𝛽5 integrins, through their binding, A deeper investigation needs to be conducted on the in order to regulate cell migration via the focal adhesion roles played by different functional domains of ANGPTL4. kinase (FAK)/p21-activated kinase- (PAK-) signaling cascade As previously described, together with the full-length protein, [69]. Huang et al. [150] demonstrated that cANGPTL4 may nANGPTL4 and cANGPTL4 fragments can also be detected induce vascular disruption through a direct and sequential in plasma. Many scientific papers showed a different func- association with integrin 𝛼5𝛽1, VE-cadherin, and claudin-5, tional role for each of these protein domains. nANGPTL favoring metastasis. Other evidences showed that this protein fragment seems to be mainly responsible for the regulation of fragment can bind to specific matrix proteins and delay their lipid metabolism by inhibiting LPL activity, whereas a weaker proteolytic degradation through the intervention of metallo- effect in modulating triglyceride availability seems to be proteinases [70]. Also, the endothelial ANGPTL4 secretion attributable also to flANGPTL4. Conversely, the cANGPTL4 inducedbytumor-releasedsemaphorin4D(SEMA4D)has fragment is involved in tumor cell growth, anoikis resistance, been shown to modulate vascular permeability [151]. The angiogenesis inhibition, and wound healing, depending on its absence of ANGPTL4 in macrophages has been shown to interacting molecules [54] (Figure 5). promote atherosclerosis, inducing foam cell formation and Interestingly, the different domains of ANGPTL4 and, vascular inflammation [152]. Conversely, Georgiadi et al. consequently, their different roles are correlated to the tissue [153] showed that ANGPTL4 overexpression reduces uptake context in which they act. Obviously, this dependence is of oxidized low-density lipoprotein (oxLDL) by macrophages linked also to the different partners interacting with the and inhibits foam cell formation in murine models, conse- various protein fragments. These evidences taken together quently by counteracting the atherosclerosis development. led to speculate on the evolutionary benefit of having one More recently, Goh et al. [70] reported that ANGPTL4 up- protein with many functions correlated to distinct structural regulation after inflammatory stimulus determines the regu- domains. lation of transcription factors involved in epidermal differentiation, such as protein kinase C (PKC) and activator protein- 6. Conclusions 1 (AP-1). Considering the similarities between the wound healing Abundant evidences opened the way to speculate a potential and cancer microenvironment, it becomes clear how the synergic role of PPARs and ANGPTL4 as key players in matricellularroleofANGPTL4anditsup-ordownregulation the cross talk between metabolic syndromes and cancer canbetranslatedintheneoplasticcellularcontext.As (Figure 6). evidence of its role in cancer development, several scientific Indeed, as shown, (1) diabetes and obesity are impor- works showed a mRNA deregulation pattern associated with tant cancer comorbidity factors; (2) PPARs are involved 10 PPAR Research

LDL oxidation by PPARs ligands PPARs agonists as pioglitazone (endothelial cells) (PPAR𝛾 ligand) LPL TG ↑ PPARs activation Inflammation and LIPID tumor growth METABOLISM/ Dysregulated adipokines and inflammatory cytokines production: leptin, IL-1𝛽, TNF-𝛼, Hypoxia Angiogenesis Obesity IL-8, IL-6, ANGPTL3, PAI-1) deregulation HIF-1𝛼

NO Cox-2 overexpression

PGE2 synthesis ANGPTL4 Cancer metabolism Interaction with 𝛽1 and 𝛽5 integrins

Elevated ROS rate Src/ERK/PKB𝛼-MAPK GLUCOSE pathway activation METABOLISM/ Insulin, IGF-1 Anoikis resistance Diabetes Inflammation and Insulin resistance tumor growth (obese mutant Tumor cell survival knockout mice) ↑↑↑ Blood FFA and ROS PPARs activation ↑↑↑ Availability of nutrients (eg., glucose, FFA)

Figure 6: Potential cross talk between lipid/glucidic metabolism and cancer. Molecular pathways involved in communication between lipid/glucidic metabolism and cancer highlighting the key roles of PPAR and ANGPTL4. Abbreviations: ANGPTL3, angiopoietin-like 3; ANGPTL4, angiopoietin-like 4; Cox-2, cyclooxygenase-2; ERK, extracellular signal-regulated kinases; FFA, free fatty acids; HIF-1𝛼,hypoxia- inducible factor-1 alpha; IGF-1, insulin-like growth factor-1; IL-1𝛽, interleukin-1 beta; IL-6, interleukin-6; IL-8, interleukin-8; LDL, low-density lipoprotein; LPL, lipoprotein lipase; MAPK, mitogen-activated protein kinase; PAI-1, plasminogen activator inhibitor-1; PGE2, prostaglandin E2; PKB𝛼, protein kinase B alpha; PPARs, peroxisome proliferator-activated receptors; ROS, reactive oxygen species; Src, V-SRC Avian Sarcoma (Schmidt-Ruppin A-2) Viral Oncogene; TG, triglycerides; TNF-𝛼, tumor necrosis factor-alpha.

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Review Article MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers

Dorothea Portius, Cyril Sobolewski, and Michelangelo Foti

Department of Cell Physiology and Metabolism and Diabetes Center, Faculty of Medicine, University of Geneva, Geneva, Switzerland

Correspondence should be addressed to Michelangelo Foti; [email protected]

Received 24 August 2016; Accepted 5 December 2016; Published 12 January 2017

Academic Editor: Valeria Amodeo

Copyright © 2017 Dorothea Portius et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-dependent nuclear receptors, which control the transcription of genes involved in energy homeostasis and inflammation and cell proliferation/differentiation. Alterations of PPARs’ expression and/or activity are commonly associated with metabolic disorders occurring with obesity, type 2 diabetes, and fatty liver disease, as well as with inflammation and cancer. Emerging evidence now indicates that microRNAs (miRNAs), a family of small noncoding RNAs, which fine-tune gene expression, play a significant role in the pathophysiological mechanisms regulating the expression and activity of PPARs. Herein, the regulation of PPARs by miRNAs is reviewed in the context of metabolic disorders, inflammation, and cancer. The reciprocal control of miRNAs expression by PPARs, as well as the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNA, is also discussed.

1. Introduction proteins, for example, NCOR (nuclear receptor corepressor) or SMRT (silencing mediator for retinoid and thyroid Peroxisome proliferator-activated receptors (PPARs) are a hormone receptor), which hamper PPARs interactions with family of nuclear receptors involved in various biological PPRE [3]. functions but with a prominent role in metabolic homeostasis Through complex regulatory mechanisms, PPARs exert of carbohydrates and lipids [1]. The three PPAR isoforms, a tight control on energy homeostasis by modulating the PPAR𝛼 (NR1C1), PPAR𝛽/𝛿 (NR1C2), and PPAR𝛾 (NR1C3), expression of key genes involved in lipid metabolism [5, 6], share 60% to 80% of structural homology [2, 3] and exhibit adipocytes differentiation [5], and carbohydrate metabolism a distinct tissue expression pattern but can exert similar or [5, 6]. The implication of PPARs in inflammatory processes different physiological functions [3]. In the canonical model, and specific cancers is further suggested by recent studies PPARs are activated in the cytoplasm by specific ligands (reviewed in [3, 8, 9]). These key and pleiotropic roles of [1–6] and then translocate into the nucleus, where they PPARs in cellular processes have led to the development of form a complex predominantly with the nuclear receptor pharmacologic agonists, for example, thiazolidinediones and Retinoid-X-Receptor (RXR), to transactivate gene expression fibrates [10, 11], to treat metabolic disorders or other diseases bybindingtoPPARresponseelements(PPREs)ongene such as atherosclerosis [2, 5, 12]. However, long-term treat- promoters [6, 7]. In contrast, noncanonical PPAR activity ment with PPARs agonists triggers uncontrolled side effects suppresses gene transcription through direct protein-protein in patients (e.g., oedema, weight gain, heart failure, and bone interactions with other transcription factors, for example, fractures)andinsomecasestheymayevenpromotetumori- the nuclear factor-kB (NFkB) or activated protein-1 (AP-1) genesis [6, 8, 13]. Alternative therapeutic options to control [1, 3]. PPARs activity is also tightly dependent on the binding distinct PPARs activities in specific tissues are therefore of other cofactors such as PGC1𝛼 (peroxisome proliferator- desirable but require that we deepen our understanding activated receptor coactivator-1𝛼) and p300 or CREB binding of the molecular mechanisms controlling PPARs expres- protein—or on the contrary on the binding of corepressor sion/activity in diseases. 2 PPAR Research

Recently, a wealth of studies has suggested that epige- although it frequently appears to be degraded and devoid of netic mechanisms, for example, DNA methylation, histone any functions [19, 21, 23, 24]. modifications, or small noncoding RNA (i.e., microRNAs), More than 2000 miRNAs have been identified and it importantly affect physiological or pathological mechanisms is considered that 60% of human genes are regulated by involved in a wide variety of diseases and cancers. In the miRNAs with around 45 000 miRNA targets within the tran- case of PPARs, methylation of their promoters [14, 15], or scriptome [21–23, 25]. miRNAs act within an intricate histone acetylation [16], has been reported to affect PPARs regulatory network, where one specific miRNA can control expression and physiological processes under their control. the expression of several hundred mRNAs and conversely one More recently, other epigenetic alterations, in particular those mRNA can be targeted by several miRNAs [19, 23]. Through leading to abnormal microRNAs (miRNAs) expression, have their wide action, miRNAs are involved in the control of also been implicated in the regulation of PPARs expression or almost all cellular functions and alterations of their expres- activity [17]. Indeed several miRNAs were reported to either sion/activity are observed in various pathological conditions directly target PPARs mRNA or to indirectly affect their including metabolic diseases and associated cancers [2, 21, expression/activities by targeting PPARs-associated cofactors 22, 25–29]. The bulk of the studies investigating PPARs and and repressors, thus providing a further level of complexity in associated cofactors/repressors (e.g., RXR, NCOR) regulation these regulatory mechanisms [18–20]. by miRNAs has been performed in the frame of metabolic In this review, we discuss the current knowledge about diseases, where the role of PPARs is the best characterized. miRNAs-dependent regulation of PPARs and their cofactors Indeed, bioinformatics analyses using the miRWalk 2.0 platin physiological and pathological processes. Most of available form (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/ studies dealing with this topic are restrained to metabolic index.html), which integrates different prediction software diseases (e.g., diabetes, fatty liver diseases, and cardiovascular programs for miRNA-mRNA interactions, point to multiple diseases) and associated cancers (e.g., liver cancers) in tissues candidate miRNAs potentially targeting directly PPAR iso- where the role of PPARs is well characterized (e.g., liver, forms. However, only a restricted number of these candidates adiposetissue,muscles,andheart).Otherrarestudiesinvesti- have been validated by experimental approaches (see Table 1). gating PPARs regulation by miRNAs in different tissues (e.g., Combining MetaCore6 and miRWalk 2.0 based analyses in bone marrow, neurons, and cartilage) or type of cancers (e.g., human studies exclusively revealed that 606 miRNAs were neuroblastoma, prostate cancer), unrelated to metabolic dis- implicated in human cancers, and among those 34 are in orders, are also considered. Finally, the reciprocal regulation metabolic disorders (e.g., obesity, diabetes, hepatomegaly, of specific miRNAs by PPARs, as well as potential miRNA- fatty liver diseases, hypertension, dyslipidemia, and other based pharmacological approaches to therapeutically modu- diabetic complications). Among the 606 cancer-related late PPARs expression and/or activity, was also examined. miRNAs, eight were targeting PPAR𝛼,fourweretargeting PPAR𝛽/𝛿, and eight were targeting PPAR𝛾. Interestingly, two miRNAs targeting PPAR𝛼 (miR-21 and miR-519d) and 2. miRNAs two miRNAs targeting PPAR𝛾 (miR-27 and miR-20) were MicroRNAs (miRNAs) are endogenous small noncoding also previously associated with metabolic diseases (Figure 1). RNAs of approximately 16–22 nucleotides, which bind to Although such predictive analyses using available software 󸀠 complementary sequences (seed sequences) in the 3 UTR of programs are subject to multiple biases and should be consid- target mRNAs and mediate either their decay or translation ered with extreme caution, they suggest that fine-tuning of PPARs signaling by miRNAs may sit at the crossroad inhibition [21, 22]. miRNAs are encoded within intronic, between metabolic diseases and cancers in human. intergenic regions or in polycistronic clusters [19, 23], and their biogenesis starts with a RNA polymerase II-dependent 3. miRNAs-Dependent Regulation of PPARs in transcription of a primary transcript (pri-miRNA), which is then maturated by a nuclear microprocessor complex (RNase Metabolic Diseases and Cancer III Drosha and its mammalian double-stranded RNA- 3.1. miRNAs-Dependent Regulation of PPAR𝛼. PPAR𝛼 is a binding partner DGCR8). This leads to the release ofa nutritional sensor adapting metabolic homeostasis to energy pre-miRNA, which is then exported into the cytoplasm by deprivation [3]. It is mostly expressed in the liver, where it Exportin-5, where the RNase III Dicer1, together with its regulates lipid catabolism (i.e., 𝛽-oxidation) and critical genes binding partner TARP2 (T-cell receptor gamma-chain con- (e.g., fatty acid transport protein 1, CD36, Acyl-CoA oxidase stant region), removes the pre-miRNA hairpin loop and gen- 1, and Carnitine palmitoyltransferase 1) involved in fatty acid erates a miRNA duplex of mature miRNA (guide strand) and ∗ transport [68] and in ketogenesis (e.g., Hmgcs2, Hmgcl) of a complementary strand (passenger strand or miRNA ). 𝛼 ∗ [68]. PPAR exerts also an anti-inflammatory function, as The guide strand and the miRNA arethenassociatedwith evidenced in mouse models of acute inflammation [68]. Argonaute proteins and incorporated into the RNA-induced This effect results from an attenuation of proinflammatory silencing complex (RISC). A second maturation step is initi- cytokines (e.g., Il-6, Il-1𝛽) expression as well as an upregula- ated within the RISC to separate both strands and the mature tion of anti-inflammatory factors such as Il-1ra (Il-1 receptor 󸀠 miRNA binds to the 3 UTR of target mRNAs. Recent evi- antagonist) or I𝜅B𝛼 [68]. PPAR𝛼 is also expressed in other dence also indicates a pathophysiological role of the passen- organs such as adipose tissues, heart, skeletal muscles, and ∗ ger strand of miRNA (miRNA ) in specific conditions, kidneys, where it controls also some aspects of the glucose PPAR Research 3

Table 1: Experimentally validated miRNAs targeting PPAR isoforms in specific tissues and pathophysiological processes. (a) PPAR𝛼

miRNA Biological process Cell/tissue Reference Human studies Cancer cell invasion and HCC tissue Drakaki et al., 2015 miR-9 proliferation Hepatic cell lines [30] HCC tissue Lipid metabolism Cui et al., 2015 [31] Hepatic cell lines Zheng et al., 2010 miR-10b Hepaticsteatosis Hepaticcelllines [32] Zhou et al., 2011 Vascular inflammation Endothelial cell lines [33] miR-21 Liver cell injury Liver tissue Loyer et al., 2015 Inflammation Primary biliary and hepatic [34] Fibrosis inflammatory cells miR-33 Liver fibrosis Hepatic stellate cell line Li et al., 2014 [35] miR-141-3p HBV replication Hepatic cell line Hu et al., 2012 [36] Liver tissue miR-199a-5p Hepatic steatosis Li et al., 2014 [37] Hepatic cell lines Tong et al., 2011 miR-506 Drug resistance Colon cancer cell line [38] White adipose tissue Martinelli et al., miR-519d Adipocyte differentiation Primary preadipocytes 2010 [39] Rodent studies Liver cell injury Liver tissue Loyer et al., 2015 miR-21 Inflammation Primary biliary and hepatic [34] Fibrosis inflammatory cells Heart tissue Cardiac hypertrophy Gurha et al., 2013 miR-22 Primary neonatal Cardiac contractility [40] cardiomyocytes Brown/white adipose tissue, primary adipose derived stromal miR-27 Adipocyte differentiation Sun et al., 2014 [41] cells, brown preadipocyte cell line Baek et al., 2008 miR-124-3p Protein secretion Isolated neutrophils [42] Liver tissue miR-199a-5p Hepatic steatosis Li et al., 2014 [37] Hepatic cell lines (b) PPAR𝛽/𝛿

miRNA Biological process Cell/tissue Reference Human studies miR-199a Myocardium El Azzouzi et al., 2013 Mitochondrial metabolism miR-214 Primary cardiomyocytes [43] miR-9 Inflammation Isolated monocytes Thulin et al., 2013 [44] Skin tissue Wound healing, miR-138 Hypertrophic scar Xiao et al., 2015 [45] Proliferation, migration fibroblasts Rodent studies miR-199a Myocardium El Azzouzi et al., 2013 Mitochondrial metabolism miR-214 Primary cardiomyocytes [43] (c) PPAR𝛾

miRNA Biological process Cell/tissue Reference Human studies Bone marrow derived Zhang et al., 2011 miR-20 Osteogenic differentiation stromal cell line [46] 4 PPAR Research

miRNA Biological process Cell/tissue Reference HCC tissue Proliferation Li et al., 2015 [47] Hepatic cell lines miR-27a Lung tissue Kang et al., 2013 Proliferation Pulmonary endothelial [48] cell lines Isolated monocytes Jennewein et al., Inflammation Monocyte cell line 2010 [49] Adipose derived stromal Karbiener et al., Adipocyte differentiation miR-27b cell line 2009 [50] Tumor growth and Neuroblastoma cell line Lee et al., 2012 [51] progression Primary hepatic stellate miR-34a Liver fibrosis cells Li et al., 2015 [52] miR-34c Hepatic stellate cell line Primary hepatic stellate Povero et al., 2015 miR-128-3p Liver fibrosis cells [53] Hepatic stellate cell line Adipocyte differentiation Primary preadipocytes Lee et al., 2011 [54] Epithelial-mesenchymal miR-130 transition HCC tissue Tu et al., 2014 [55] Cancer cell migration and Hepatic cell lines invasion White adipose tissue miR-130a Type 2 diabetes mellitus Jiao et al., 2015 [56] Adipocyte cell line Primary adipose derived Yang et al., 2011 miR-138 Adipocyte differentiation stromal cells [57] Bone marrow derived miR-548d-5p Adipocyte differentiation Sun et al., 2014 [58] stromal cells Rodent studies White adipose tissue, Primary white adipocytes Kim et al., 2010 Adipocyte differentiation Primary adipose derived [59] stromal cells Preadipocyte cell line miR-27 Brown/white adipose tissue Primary adipose derived Adipocyte differentiation Sun et al., 2014 [41] stromal cells Brown pre-adipocyte cell line Lung tissue Kang et al., 2013 Proliferation Pulmonary endothelial [48] cell lines miR-27a Kidney tissue Hou et al., 2016 Renal fibrosis Kidney tubular epithelial [60] cells Cardiac hypertrophy Myocardium Wang et al., 2012 miR-27b Heart failure Primary cardiomyocytes [61] Primary hepatic stellate miR-34a Liver fibrosis cells Li et al., 2015 [52] miR-34c Hepatic stellate cell line Primary hepatic stellate Povero et al., 2015 miR-128-3p Liver fibrosis cells [53] Hepatic stellate cell line PPAR Research 5

miRNA Biological process Cell/tissue Reference Adipocyte inflammation Preadipocyte cell line Kim et al., 2013 [62] miR-130 Primary hepatic stellate Liver fibrosis cells Lu et al., 2015 [63] Hepatic stellate cell line White adipose tissue miR-130a Type 2 diabetes mellitus Jiao et al., 2015 [56] Adipocyte cell line Primary bone marrow miR-210 Osteoporosis Liu et al., 2015 [64] derived stromal cells White adipose tissue miR-301a Adipocyte inflammation Li et al., 2016 [65] Preadipocyte cell line White adipose tissue miR-302a Adipocyte differentiation Jeongetal.,2014[66] Pre-adipocyte cell line Primary adipose derived miR-540 Adipocyte differentiation Chen et al., 2015 [67] stromal cells

Metabolic Metabolic Metabolic diseases diseases diseases 34 34 34 Cancer Cancer Cancer 606 606 mmiR-21R 21 606 miR-27miiR 227 mmiR-519diR 519dd miR-20miR 20

PPAR𝛽/𝛿 PPAR𝛼 8 PPAR𝛾 8 4

(a) (b) (c)

Figure 1: Human miRNAs targeting PPAR isoforms and involved in metabolic diseases and cancer. MetaCore pathway analysis software from Thomson Reuters was used to identify experimentally the number of validated human miRNAs involved in cancer (grey circle). Among those, the numbers of miRNAs involved in metabolic diseases, also identified by MetaCore pathway analysis, are indicated in blue circles. In red circles are the number of miRNAs identified using miRWalk 2.0 atlas and targeting PPAR𝛼 (Panel (a)), PPAR𝛽/𝛿 (Panel (b)), and PPAR𝛾 (Panel (c)). The identities of miRNAs targeting specific PPAR isoforms and involved in both cancer and metabolic diseases are indicated in violet. miRWalk 2.0 atlas is a software integrating 12 different prediction algorithms (miRWalk 2.0, MicroT4, miRanda, miRBridge, miRDB, miRMap, miRNAMap, PICTAR2, PITA, RNA22, RNAhybrid, and TargetScan) for identification of miRNAs target mRNAs.

and lipid homeostasis (i.e., 𝛽-oxidation)[6,68,69].PPAR𝛼 metabolically active tissues, for example, skeletal muscles or is usually activated through the binding of specific ligands, in pancreas, remains to be established. particular unsaturated fatty acids (𝜔-3 fatty acids), eicosanoid derivatives (e.g., 8-hydroxy-eicosatetraenoic acid, prostacy- 3.1.1. miRNAs-Dependent Regulation of PPAR𝛼 in the Liver. clin), or metabolized fatty acids (e.g., oxidized fatty acids) [6]. Intheliver,PPAR𝛼 is implicated in the lipid catabolism and Alterations of PPAR𝛼 expression or activity were associated inflammatory processes [68]. miRNAs-dependent alterations with a variety of human pathologies such as obesity, liver of PPAR𝛼 signaling are reported by numerous studies to diseases, inflammation, and cancers [3, 68, 69]. It is now contribute to the onset of liver diseases such as nonalcoholic clear that deregulations of specific miRNA can significantly fatty liver disease (NAFLD) [19, 21, 29], chronic diseases contribute to PPAR𝛼 abnormal signaling in these pathophys- associated with viral infections (HBV, HCV) [70], or hepatic iological conditions (see experimentally validated miRNAs cancers [30, 71]. targeting PPAR𝛼 in Table 1 and Figure 2). Such alterations have been investigated only in specific tissues, such as the Hepatic Steatosis. Two miRNAs were shown to alter PPAR𝛼 liver or adipose tissue, as well as in inflammatory cells and expression in hepatocytes and to lead to steatosis develop- cartilage and specific tumors (e.g., in the colon). Whether ment (Table 1) [19, 69]. Upregulation of miR-199a-5p was PPAR𝛼 expression/activity is affected by miRNAs in other observed in various in vivo mouse models of obesity (ob/ob 6 PPAR Research

Liver Endothelial tissue Monocytes miR-9 miR-21 miR-9 miR-10b miR-21 Neutrophils Skin miR-33 miR-124-3p miR-138 miR-141-3p miR-199a-5p

𝛽 𝛿 PPAR𝛼 PPAR / Heart miR-22 Heart Adipose tissue miR-199a Colon miR-27 miR-214 miR-506 miR-519d

(a) (b)

Liver Adipose tissue miR-27a Kidney miR-27 miR-34a/c miR-27a miR-27b miR-128-3p miR-130 miR-130 miR-138 miR-301a miR-302a 𝛾 miR-540 PPAR miR-548d-5p Neuroblasts miR-27b Lung Monocytes miR-27a miR-27b Bone miRNA-20 Heart miR-210 miR-27b

(c)

Figure 2: miRNAs targeting PPAR isoforms in specific tissues. miRNAs (also referred to in Table 1) that have been experimentally demonstrated to specifically target PPAR𝛼 (Panel (a)), PPAR𝛽/𝛿 (Panel (b)), and PPAR𝛾 (Panel (c)) in different tissues are illustrated. miRNAs identified in human studies are in blue, those identified in mouse/rat studies are in green, and those identified in both human androdents studies are in red.

and db/db mice, mice fed a high-fat diet), as well as in liver in these cells. Interestingly, in mice knockout, specifically for samples from patients with NAFLD. In vitro analyses of hep- miR-21 in hepatocytes, PPAR𝛼 expression was not altered, atoma cell lines (HepG2 and murine AML12 cells) exposed even when mice were challenged with an obesogenic diet, to fatty acids as a surrogate model of steatosis further confirm therefore suggesting that, in different cell types, miR-21 an upregulation of miR-199a-5p, which in turn downregulates may have different activities and/or cellular targets [34, 72]. PPAR𝛼 and caveolin-1 thereby promoting abnormal cellular In hepatic stellate cells (HSCs), which are the main non- redoxequilibriumandfattyacidsintracellularaccumulation parenchymal liver cells contributing to the abnormal extra- [37]. In human hepatic LO2 cells, Zheng et al. uncover cellular matrix deposition in liver fibrosis, miR-33 and miR- another miRNA, miR-10b, upregulated following exposure to 27a/-27b were also found upregulated and to target PPAR𝛼 fatty acids and having a unique binding site in the PPAR𝛼 and the PPAR𝛼 cofactor RXR, respectively. Inhibition of these 󸀠 3 UTR sequence [32]. However, the relevance of miR-10b miRNAs with synthetic nucleotides in rat primary and alterations in human liver metabolic disorders was not immortalized human HSCs (LX-2 cells) increased PPAR𝛼 evaluated. expression concomitantly with a decreased activation of the cells, thus suggesting a tight link between HSC activation and Hepatic Inflammation and Fibrosis. PPAR𝛼 downregulation PPAR𝛼 expression [35, 73]. by miRNAs was recently suggested to trigger hepatic inflammation and fibrosis. Indeed, Loyer et al. [34] reported an Hepatic Carcinogenesis. The role of PPAR𝛼 in cancer is still upregulation of miR-21 in biliary and inflammatory cells of debated but few studies suggested that miRNAs-dependent mice and patients with nonalcoholic steatohepatitis (NASH). alterations of PPAR𝛼 expression/activity are relevant for the They further discover that miR-21 was promoting hepatic development of hepatocellular carcinoma (HCC). In par- inflammation and fibrosis by suppressing PPAR𝛼 expression ticular, high-throughput screening of human HCC samples PPAR Research 7 revealed 28 miRNAs differentially expressed with top hits from a study performed in a drug-resistant colon cancer cell for miR-9, miR-21, and miR-224 [30]. In addition to miR- line (SW1116) showing that miR-506 overexpression in this 21, which is discussed in the previous section, prediction cancer cell model directly affects PPAR𝛼 expression [38]. software programs identified conserved miR-9 binding sites In another study, the growth inhibitory properties of 1.25- 󸀠 within the 3 UTR of PPAR𝛼.miR-9upregulationcorrelated dihydroxyvitamin D3 in human prostate adenocarcinoma with tumor invasiveness, cell growth, and tumor stage, but cells (LNCaP) were associated with an increased expression whether this was related to a decreased PPAR𝛼 expression of the miR-17/92 cluster, which correlated with PPAR𝛼 down- remains unclear. Indeed, whereas the direct regulation of regulation, but whether miR-17/92 directly target PPAR𝛼 was PPAR𝛼 by miR-9 in human HCC cells was confirmed by not investigated [77]. luciferase reporter assay [31], molecular analysis of human Snu-449 and HepG2 cancer cell lines indicated an indirect 3.2. miRNAs-Dependent Regulation of PPAR𝛽/𝛿. PPAR𝛽/𝛿 is role for miR-9 overexpression in PPAR𝛼 downregulation ubiquitously expressed with the highest levels in liver, intes- [30]. tine, kidneys, and skeletal muscles [6, 78]. Major PPAR𝛽/𝛿 activators are natural ligands such as polyunsaturated fatty 3.1.2. miRNAs-Dependent Regulation of PPAR𝛼 in the Adi- acids, prostaglandin derivatives (e.g., prostacyclin), or com- pose Tissue. PPAR𝛼 assumes important functions in brown ponents of VLDL (Very Low Density Lipoproteins) particles adipose tissue and adaptive thermogenesis and browning of [6]. This PPAR isoform regulates multiple cellular pro- white adipose tissue [41]. Although experimental evidence cesses including developmental aspects, the lipid metabolism, in human showing miRNAs-dependent PPAR𝛼 regulation in insulin sensitivity, vascular function, and anti-inflammatory brown/whiteadiposetissueisscarce,severalanimalmodels responses [4, 6, 44, 79]. The best-characterized role of have suggested such regulatory mechanisms. For example, PPAR𝛽/𝛿 has been described in metabolically active tissues. miR-27a and miR-27b, which are downregulated in mouse Intheliver,PPAR𝛽/𝛿 appears to increase glucose utilization brown/white adipose tissue after cold exposure, directly mod- through the pentose-phosphate pathway and to promote lipo- ulate components of the adipocyte transcriptional network genesis [80]. However, in mice fed an obesogenic diet, acti- including PPAR𝛼 [41]. MiR-519d, which is increased in sub- vation of PPAR𝛽/𝛿 surprisingly prevents the development of cutaneous white adipose tissue of obese subjects as compared steatosis [81]. In muscles and white adipose tissue, PPAR𝛽/𝛿 to nonobese individuals, decreases fatty acid catabolism, exerts an adaptive response to fasting and exercise by favoring and increases intracellular lipid accumulation by directly fatty acids oxidation [82], through the direct induction of repressing PPAR𝛼 [39]. Finally, other miRNAs upregulated key genes involved in this process (e.g., mitochondrial CPT-1 inbrownadiposetissueand/orwhiteadiposetissueofdiet- (Carnitine palmitoyltransferase-1) and FoxO1 (Forkhead box induced obese mice, or during human white and beige protein O1)) [82, 83]. In brown adipose tissue, PPAR𝛽/𝛿 con- adipose differentiation, for example, miR-106b/miR-93 [74, tributes to adaptive thermogenesis by inducing the expres- 75] and miR-26a and miR-26b [50], have been correlated with sion of UCP-1 and UCP-3 [81, 82] and to 𝛽-oxidation, by alterations of PPAR𝛼 expression. However, whether PPAR𝛼 is upregulating several genes involved in this process (e.g., long a direct target of these miRNAs was not investigated. chain acyl-CoA synthetase, Acyl-CoA oxidase). In addition to these well established roles of PPAR𝛽/𝛿,thisisoformwas 3.1.3. miRNAs-Dependent Regulation of PPAR𝛼 in further implicated in the regulation of multiple other cellular Other Cell Types/Organs processes including developmental aspects, vascular function, and anti-inflammatory responses [4, 6, 44, 79]. Finally, Inflammatory Cells and Cartilage. Functional miRNAs- in cancer, the role of PPAR𝛽/𝛿 is controversial with evidence dependent PPAR𝛼 alteration in inflammatory processes was pointing at PPAR𝛽/𝛿 as an oncogene (e.g., in breast and suggested by two studies. First, miR-21, which is upregulated prostate tumors) [84] or as a tumor suppressor (e.g., in colon in cultured human endothelial cells from umbilical vein cancer) [4, 6, 85]. Despite the key functions of PPAR𝛽/𝛿, exposed to oscillatory shear stress, was shown to directly solid experimental evidence indicating miRNAs-dependent inhibit PPAR𝛼 translation [33]. The decreased expression regulation of this isoform is very limited and restricted to of PPAR𝛼 in turn promotes AP1-dependent upregulation studies described below (see Table 1 and Figure 2). of VCAM-1 (vascular cell adhesion molecule-1) and MCP-1 (monocyte chemotactic protein-1), which favor the adhesion 3.2.1. miRNAs-Dependent Regulation of PPAR𝛽/𝛿 in the Liver. of inflammatory cells [33]. In a second study, bioinformatics Based on Affymetrix microarrays, in vivo inhibition of miR- and molecular analyses combined with clinical data identified 122 by antisense oligonucleotides (ASO) in mice affected hun- an increased expression of miR-22 in osteoarthritic cartilage, dreds of hepatic mRNAs including PPAR𝛽/𝛿 [86]. Whether which was correlated with PPAR𝛼 downregulation and an miR-122 directly targets PPAR𝛽/𝛿 is still undetermined; increased body-mass-index (BMI) of patients [76]. However, however its downregulation following injection of miR-122 this study did not provide any direct molecular link between inhibitors in mice was suggested to affect circadian clock- miR-22 upregulation and PPAR𝛼 downregulation. dependent energy homeostasis and in particular regulation of lipid transport and catabolism [86]. Nonhepatic Cancers. The only evidence that PPAR𝛼 may potentially behave as a tumor suppressor downregulated by 3.2.2. miRNAs-Dependent Regulation of PPAR𝛽/𝛿 in the aberrantly expressed miRNAs in transformed cells comes Heart. By stimulating fatty acid utilization in the myocar- 8 PPAR Research dium, PPAR𝛽/𝛿 exerts a protective vascular function. In a 88]. As illustrated in Table 1 and Figure 2, posttranscriptional mouse model of heart failure, an impaired fatty acid oxidation regulation of PPAR𝛾 by miRNAs has been reported in many and a metabolic switch towards glycolysis were attributed to pathophysiological situations. the direct repression of PPAR𝛽/𝛿 by two miRNAs, miR-199a and miR-214, which are upregulated following aortic pressure 3.3.1. miRNAs-Dependent Regulation of PPAR𝛾 in the Liver overload and subsequent heart failure [43]. Hepatic Fibrosis. PPAR𝛾 is a negative regulator of hepatic 3.2.3. miRNAs-Dependent Regulation of PPAR𝛽/𝛿 in Mono- stellate cells (HSCs) activation [89]. The induction of various cytes/Macrophages. PPAR𝛽/𝛿 exerts an anti-inflammatory miRNAs expressed in nonalcoholic steatohepatitis (NASH) function, by promoting the switch from proinflammatory and fibrosis correlated with PPAR𝛾 downregulation and M1 macrophages to the anti-inflammatory M2 macrophages overexpression of profibrogenic markers like 𝛼-SMA. Among intheliverandinadiposetissue[44].Bioinformaticsand those miRNAs, upregulation of miR-34a/-34c [52], miR-128- luciferase reporter assays revealed the presence of a func- 3p [53], and miR-130a/miR-130b [63] in activated human 󸀠 󸀠 tional miR-9 binding site within PPAR𝛽/𝛿 3 UTR in human or rat HSCs was reported to directly bind the 3 UTR of monocytes. Downregulation of PPAR𝛽/𝛿 and its targets genes PPAR𝛾 and to repress its expression. miRNAs-dependent was further observed in proinflammatory M1 macrophages PPAR𝛾 downregulation in hepatic fibrosis can also occur treatedwithlipopolysaccharide(LPS)andcorrelatedwith through indirect mechanisms. For example, in HSCs from an upregulation of miR-9 in these cells, thus suggesting mice treated with CCL4 to induce fibrosis, miR-132 is down- a potential functional regulation of PPAR𝛽/𝛿 by miR-9 in regulated thereby leading to an increase of one of its targets, monocytes and macrophages [44]. MeCP2 (Methyl CpG binding protein 2), and repression of PPAR𝛾 transcription via different epigenetic mechanisms 3.2.4. miRNAs-Dependent Regulation of PPAR𝛽/𝛿 in Other [90]. Cell Types/Organs. MiRNAs-dependent PPAR𝛽/𝛿 regulation was finally reported in hypertrophic scar formation, where Hepatitis C Virus (HCV) Infection. Infection of Huh-7.5 hep- PPAR𝛽/𝛿 promotes proliferation of fibroblasts. A decrease atoma cells with a HCV-derived JFH1 strain induces expres- in miR-138 expression was noted in scar tissue as compared sion of miR-27a. This miRNA directly targets PPAR𝛾 thereby to paired normal skin tissues and inversely correlated with reducing lipid synthesis and increasing lipid secretion [91], the level of PPAR𝛽/𝛿. Further analyses using luciferase two processes likely promoting HCV replication and virions reporter assays and synthetic miR-138 mimics and inhibitor egress. nucleotides in human hypertrophic scar fibroblasts (hHSFs) 𝛾 confirmed that PPAR𝛽/𝛿 isadirecttargetofmiR-138andthe Hepatic Carcinogenesis. PPAR has a tumor suppressive func- 𝛾 functional relevance of this regulatory mechanism in hHSFs tion in hepatocarcinogenesis [7, 51, 92–95]. PPAR down- proliferation [45]. regulation in HCC correlated with upregulation of specific miRNAs [3, 93, 96], among which the best characterized ones 3.3. miRNAs-Dependent Regulation of PPAR𝛾. There are two are miR-130b and miR-27a. These two miRNAs directly target 𝛾 PPAR𝛾 isoforms (PPAR𝛾1andPPAR𝛾2). PPAR𝛾1isbroadly PPAR and decrease its expression thus promoting cancer expressed in adipose tissue, liver, intestine, kidneys, small cells growth and aggressiveness [47, 55]. intestine, immune cells, and endothelium, while PPAR𝛾2 is predominantly expressed in the adipose tissue [2, 3, 6]. 3.3.2. miRNAs-Dependent Regulation of 𝛾 Activation of PPAR is induced mostly by unsaturated fatty PPAR𝛾 in Adipose Tissue acids and endogenous arachidonic acid-derived metabolites (e.g., leukotriene B4 and eicosatetraenoic acid). The best- Adipocyte Differentiation. Regulation of PPAR𝛾 activity/ described functions of PPAR𝛾 are to transcriptionally pro- expression by miRNAs represents an important posttran- mote adipocyte differentiation and lipogenesis as well as de scriptional mechanism controlling adipocyte differentiation. novo lipogenesis in the liver [5, 87]. In addition, PPAR𝛾 con- Several miRNAs in murine and human preadipocytes, trols also the expression of various adipocyte genes involved including miR-540, miR-302a, miR-138, miR-548d-5p, miR- 󸀠 in glucose homeostasis (e.g., Glut4 expression) and endocrine 130, and miR-27, were described to bind the 3 UTR of PPAR𝛾 signaling (e.g., adiponectin, resistin, and TNF𝛼) affecting and to decrease its expression thus preventing differentiation insulin sensitivity in other peripheral organs such as liver and towards mature adipocytes [54, 57–59, 67, 97, 98]. In particu- muscles [2, 3, 5, 12]. Finally, several other cellular processes lar,miR-130wasreportedtobedownregulatedinmicefedan including cholesterol transport, kidney function, food intake, obesogenic diet and in adipocytes of obese and type 2 diabetic and inflammation have been suggested to be modulated patients, who also have high levels of PPAR𝛾 in adipose by PPAR𝛾 isoforms[2,3,5,12].Consistentwiththerole tissues and a low abundance of preadipocytes [54, 56, 62]. of PPAR𝛾 inglucoseandlipidhomeostasis,anabnormal Further in vitro analyses using embryonic fibroblasts-derived activity of PPAR𝛾 is often associated with the development preadipocytes (3T3-L1) indicated that synthetic nucleotides of metabolic disorders (e.g., obesity, type 2 diabetes, and mimicking or inhibiting miR-130a were able to modulate fatty liver disease) [5]. In contrast, in cancer, increasing PPAR𝛾 expression and its downstream target genes involved evidence indicates a beneficial tumor suppressive role for in glucose and lipid metabolism [56]. miR-27a and miR-27b PPAR𝛾 (e.g., gastric, pancreatic, and hepatic cancers) [2, 6, are other key miRNAs regulating adipocyte differentiation, PPAR Research 9 and both are downregulated during adipocyte differentiation, targeting of PPAR𝛾 was also highlighted in several other tis- thus leading to an induction of PPAR𝛾 [59]. Consistent with sues (inflammatory cells, renal tubular cells, and pulmonary this role, expression of miR-27a/-27b is lower in obese ob/ob endothelial cells as well as neuroblastoma). mice as compared to lean animals and decreases during adipogenic differentiation of 3T3-L1 cells and mouse bone mar- Inflammatory Cells. PPAR𝛾 is a potent inhibitor of M1 row derived mesenchymal stem cells (OP9 cell line). In the macrophage activation (Th1 proinflammatory macrophages) same study, miR-27a/miR-27b mimic nucleotides decreased and promoter of M2 macrophage activation (Th2 anti- PPAR𝛾 expression and prevented adipocyte differentiation. inflammatory macrophages) [102]. Upregulation of miR-27b However, experimental evidence indicated that the mecha- in human macrophages upon LPS exposure was demon- nisms by which miR-27 affect PPAR𝛾 expression are indirect strated to directly target PPAR𝛾 andtoelicitaTh1differen- [98]. tiation [49]. Although these findings suggest that miR-27b- dependent downregulation of PPAR𝛾 may represent a key Inflammation. The role of PPAR𝛾 inadiposetissueinflamma- process in macrophage polarization, whether miR-27b con- tion is still poorly characterized, but downregulation of miR- trols M2 macrophage activation via PPAR𝛾 was however not 301a, which directly targets PPAR𝛾, was correlated with the investigated. production of proinflammatory cytokines in obese mice and in 3T3L1 preadipocytes [65]. Kidneys. Upregulation of miR-27a occurs in glucose- stimulated rat renal proximal tubular cell line (NRK-52E 3.3.3. miRNAs-Dependent Regulation of PPAR𝛾 in Bone Mar- cells) and renal tubular epithelial cells of streptozotocin- induced diabetic rats. In these cellular contexts, the increased row. The commitment of mesenchymal stem cells (MSCs) in 𝛾 the bone marrow towards osteogenic or adipogenic differen- miR-27a expression was shown to trigger PPAR downregu- tiation might be also tightly dependent on PPAR𝛾 regulation lation, which in turn promoted renal fibrosis [60]. by miRNAs. Indeed, miR-548d-5p, which is downregulated Lung. In mice and human pulmonary artery endothelial during adipogenic differentiation of human bone marrow 󸀠 cells (HPAECs), hypoxia upregulates miR-27a expression derived MSCs, targets the 3 UTR of PPAR𝛾. Overexpression and decreases PPAR𝛾 expression [103]. Given the important of this miRNA abrogates adipogenic differentiation and antiproliferative, and antithrombotic and vasodilatory effects increases the osteogenic potential of MSCs by downregulat- of PPAR𝛾 on the lung vasculature [104], upregulation of ing PPAR𝛾 and C/EBP𝛼 [58]. Induction of other miRNAs miR-27a may thus represent an important contributor to the such as miR-20 during osteogenic differentiation leads also development of pulmonary hypertension. to a direct downregulation of PPAR𝛾 [46]. In addition, miR- 17-5p and miR-106a also promote adipogenesis and inhibit Neuroblastoma Cells. Although miR-27b-dependent down- osteogenic differentiation in human adipose derived MSCs 𝛾 𝛼 𝛾 regulation of PPAR promotes cell proliferation in HCC, by indirect mechanisms, which increase C/EBP and PPAR itmayleadtooppositeeffectsinothercancers[7,51,92– expressions [99]. Finally, alterations of the osteogenic/adipo- 95].ThisisthecaseintheSK-N-ASneuroblastomacells genic differentiation balance is an important component of and derived mouse xenografts, where miR-27b was shown to specificosteogenic-relateddisorderssuchasosteoporosisand 𝛾 𝛾 repress PPAR expression resulting in a decreased inflamma- deregulation of the expression of miRNAs targeting PPAR , tory response and tumor growth [51, 105, 106]. for example, miR-210, have been involved in these diseases [64, 100]. 4. Indirect Regulation of PPARs by miRNAs 3.3.4. miRNAs-Dependent Regulation of PPAR𝛾 in the Heart. UpregulationofmiR-27bexpressionwasshowninheart- The activity of PPARs is tightly linked to the binding of transcriptional partners (i.e., RXR, Prdm16), cofactors/repressors specific smad4 knockout mice, which develop cardiac hyper- 𝛼 trophy [61]. Overexpression of this miRNA specifically in car- (e.g., PGC1 , NCOR), or other regulators (e.g., Sirt1) [3]. diomyocytes using transgenic mice was sufficient to induce Most of the PPARs binding partners and cofactors are also cardiac hypertrophy through PPAR𝛾 downregulation [61]. finely tuned by specific miRNAs, which thereby indirectly Conversely, treatment of a mouse model of heart failure with regulate the expression/activity of PPARs isoforms [18, 107– miR-27b inhibitors (antagomirs) improved cardiovascular 112]. A brief overview of miRNAs targeting PPARs binding functions by increasing PPAR𝛾 expression [61]. Similarly, partners and cofactors is provided in the next section (see in vivo inhibition of miR-128 by antagomirs protected Figure 3). cardiomyocytes from apoptosis in a model of myocardial ischemia/reperfusion injury and increased PPAR𝛾 expression 4.1. miRNAs-Dependent Regulation of in neonatal rat ventricular myocytes (NRVM) [101]. However, PPARs Binding/Heterodimerization Partners whether miR-128 modulates PPAR𝛾 through direct or indirect mechanisms was not assessed in this study. 4.1.1. miRNAs-Dependent Regulation of RXRs. RXR isoforms (RXR𝛼/𝛽/𝛾) are the obligate binding partners for PPARs. 3.3.5. miRNAs-Dependent Regulation of PPAR𝛾 in Other Together they form heterodimeric complexes and induce Cell Types/Organs. In addition to its role in hepatocytes, gene transactivation by binding to PPAR response elements adipocytes, and cardiomyocytes, the relevance of miR-27b (PPREs) [3]. As illustrated in Figure 3, several miRNAs have 10 PPAR Research

miR-29 miR-34a Sirt1 miR-217

v miR-181a PPAR miR-34a miR-27a/-27b miR-574-3p miR-133 miR-16 miR-128-2 miR-100 miR-696 miR-130a

NCOR2 Pgc1𝛼 Pgc1𝛼

PPARv RXR PPAR RXR PPAR Prdm16

PPRE PPRE PPRE

Figure 3: miRNAs targeting PPAR transcriptional partners, cofactors/repressors, and other regulators. miRNAs that have been experimentally demonstrated to specifically target PPAR transcriptional partners (RXR and Prdm16), PPAR cofactors (Pgc1𝛼), PPAR repressors (NCOR2), and other PPAR regulators (Sirt1) are illustrated. miRNAs identified in human studies are in blue, those identified in mouse/rat studies are in green, and those identified in both human and rodents studies are in red. PPAR: peroxisome proliferator-activated receptor 𝛼, 𝛽/𝛿,or𝛾; RXR: Retinoid-X-Receptor; Prdm16: PR domain-containing 16; Sirt1: Sirtuin-1; NCOR2: nuclear receptor corepressor 2 (SMRT); Pgc1𝛼: peroxisome proliferator-activated receptor gamma coactivator 1; PPRE: peroxisome proliferator response element.

been reported to directly target RXR isoforms thus affecting instead of RXR𝛼 heterodimerizes with PPAR𝛾2 and mediates indirectly PPARs activities [108, 109, 113, 114]. For example, brown adipocyte differentiation [115]. MiR-133a was demon- miR-128-2 was shown to suppress cholesterol efflux in HepG2 strated to regulate directly Prdm16 expression in immortal- cells and in the liver of diet-induced obese mice by binding to ized brown preadipocytes [18] and inhibition of miR-133a 󸀠 the 3 UTR of RXR𝛼 and of ATP-binding cassette transporters andmiR-133bledtoanincreasedexpressionofadipogenic (ABCA1 and ABCG1) and repressing their expressions [114]. markers including PPAR𝛾 as well as differentiation towards Chondrogenesis, which is inhibited by RXR𝛼,wasalso mature brown adipocytes [18, 116, 117]. promoted in mesenchymal stem cells by miR-574-3p, which downregulates specifically RXR𝛼 expression [113]. Interest- 4.1.3. miRNAs-Dependent Regulation of PGC1𝛼. PGC1𝛼 is ingly, specific miRNAs targeting PPAR𝛾,thatis,miR-34aand a critical transcriptional coactivator of PPAR𝛾 in brown miR-27a/b, also control RXR𝛼 expression in liver cells [73, 98, preadipocytes and of PPAR𝛼 in white preadipocytes (3T3-L1 109]. Indeed upregulation of miR-34a, which was correlated cells) [118]. To date only two miRNAs have been described with fibrosis development, downregulates RXR𝛼 by binding in hepatocytes to directly target PGC1𝛼 mRNA: (i) miR-696, 󸀠 within the coding region and not the 3 UTR of this isoform which is upregulated with obesity, decreases PGC1𝛼 expres- in hepatocytes [109]. In the case of miR-27a and miR-27b, sion in the liver of ob/ob mice [119] and (ii) miR-130a, which these two miRNAs were upregulated in rat activated HSCs is downregulated in HBV-infected human hepatocytes, 󸀠 anddecreaseRXR𝛼 expression through 3 UTR-dependent increases PGC1𝛼 and PPAR𝛾 expression thus favoring HBV mechanisms [73, 98]. It thus appears that abnormal miRNAs- replication [120]. dependent inhibition of RXR𝛼 in distinct liver cells contributes to the development of hepatic fibrogenesis. Finally, 4.1.4. miRNAs-Dependent Regulation of NCOR. In the inhibition of RXR𝛼 by upregulation of miR-27a was also absence of PPARs ligands, the transcriptional activity of reported in aggressive rhabdomyosarcoma (RMS) [108]. PPARsisinhibitedbythebindingofcorepressorssuchNCOR Altogether, these studies suggest that particular miRNAs, proteins [12]. miRNAs-dependent regulation of NCOR such as miR-34a and the miR-27 family, may affect PPARs proteins is supported by two studies showing that (i) miR-16 signaling by simultaneously targeting different key players in in LPS-activated human monocytes (U937) and biliary this pathway. epithelial cells (H69) targets SMRT (NCOR2), which leads to NF-𝜅B-mediated transactivation of the IL-8 gene [107], and 4.1.2. miRNAs-Dependent Regulation of Prdm16. During (ii) miR-100 targets SMRT (NCOR2) in glioblastoma cells brown adipogenesis, Prdm16 (PR domain-containing 16) thereby inhibiting their proliferation [112]. PPAR Research 11

Macrophages Fibroblasts miR-223 miR-145 Liver Adipose tissue Let-7 Endothelial cells miR-98 miR-339-5p miR-200c miR-378 Ovarian Cofactor Cofactor miR-125b Cerebral cortex RXR 𝛼 RXR PPAR𝛾 miR-145 Colon PPAR Prdm16 Prdm16 miR-329 miR-145

PPRE pri-miRNA PPRE pri-miRNA

(a) (b)

Figure 4: miRNAs expression induced by PPAR𝛼 and PPAR𝛾. Induction of miRNAs by PPAR𝛼 (Panel (a)) and PPAR𝛾 (Panel (b)) binding to PPRE in pri-miRNA promoters in specific tissues is indicated. miRNAs identified in human studies are in blue, those identified in mouse/rat studies are in green, and those identified in both human and rodents studies are in red. RXR: Retinoid-X-Receptor; Prdm16: PR domain- containing 16; PPRE: peroxisome proliferator response element.

4.1.5. miRNAs-Dependent Regulation of Sirtuin-1. The NAD- that is, LRH-1 (liver receptor homolog-1), was proposed also dependent deacetylase Sirtuin-1 (Sirt1) is a critical regulator to drive miR-200c transcription through a direct binding to of PPAR signaling and of energy homeostasis [121]. Posttran- its promoter [132]. Although the role of miR-200c in HCC scriptional control of SIRT1 and other sirtuins by miRNAs was not investigated in this study, miR-200 was previously represents important regulatory mechanism for this protein shown to have tumor suppressive activities by inhibiting cell family and has been extensively reviewed elsewhere [111]. migration [135]. Regarding PPAR𝛽/𝛿,treatmentofHUVEC Among the various miRNAs directly targeting SIRT1, miR- endothelial cells with a PPAR𝛽/𝛿 agonist (GW501516) led to 217 [122], miR-181a [123], miR-29 [124], and miR-34a [125] an increase of miR-100 expression, which improved lipidemia in particular were shown to affect hepatic lipid metabolism, and vascular function [136]. However, as for the study using 𝛼 𝛽 𝛿 insulin sensitivity, or carcinogenesis by modulating SIRT1 PPAR agonists, a direct binding of PPAR / to the miR-100 expression. Of note, miR-34a is also a direct regulator of promoter was not investigated and additional experiments 𝛾 𝛼 are required to confirm these data and exclude off-target PPAR and RXR expression [111, 125–128] therefore sup- 𝛽 𝛿 porting again the biological relevance of fine-tuning PPARs effects of pharmacological agonists of PPAR / . signaling by modulating several factors involved in this 𝛾 transcriptional pathway. 5.2. PPAR -Dependent Regulation of miRNAs Expression. In contrast to the other PPAR isoforms, PPAR𝛾 was reported to regulate several miRNAs in distinct pathophysiological 5. miRNAs Regulated by PPARs processes (see Table 2). Recent evidence indicates that the expression of particular Endothelial Functions. miR-98, which is reduced in endothe- miRNAs can also be under the transcriptional control of lial cells of patients suffering from idiopathic pulmonary PPARs [129] (see Figure 4). Most of the studies reviewed here hypertension (IPAH) and of mouse models of this disease, rely on the identification of PPAR response elements (PPREs) directly targets endothelin-1 (ET1). PPAR𝛾 was shown to within the promoter of genes encoding pri-miRNAs or on the exert a beneficial role in pulmonary hypertension (PH) effects of PPARs agonists [17, 96, 130, 131]. miRNAs described by attenuating, likely through activation of miR-98, ET1 to date to be regulated by PPARs are short-listed in Table 2. expression. In support of this hypothesis, activation of PPAR𝛾 with specific agonists (e.g., rosiglitazone) restores miR-98 5.1. PPAR𝛼-andPPAR𝛽/𝛿-Dependent Regulation of miRNAs expression in hypoxic mouse and in primary human pul- Expression. Limited information is available on PPAR𝛼- monary artery endothelial cells (PAECs); however whether and PPAR𝛽/𝛿-dependent regulation of miRNAs expression. PPAR𝛾 is a direct regulator of miR-98 was not assessed [103]. PPAR𝛼 was suggested to promote the expression of let-7 and miR-200c in hepatic cancer cells. Indeed, expression of let- Adipocytes Differentiation and Function. PPAR𝛾 agonists 7, which targets c-myc in hepatocytes, was decreased in mice (rosiglitazone and pioglitazone) modulated the expression of treated with the PPAR𝛼 agonist Wy-14,643, which in turn fos- 27 different miRNAs in human subcutaneous and visceral tered myc-dependent liver oncogenesis [71]. In Huh-7 hep- adipocytes. Among those, miR-329, miR-145, and miR-339- atoma cells, PPAR𝛼 insynergywithanothernuclearreceptor, 5p are involved, based on predictive bioinformatics analyses, 12 PPAR Research

Table 2: PPAR𝛼-andPPAR𝛾-dependent miRNAs induction in specific tissues and pathophysiological processes. (a) PPAR𝛼

miRNA Biological process Organism Cell/tissue Reference Liver tissue, Shah et al., 2007 Let-7 Proliferation Mouse HCC cell line [71] Zhang et al., 2011 miR-200c Migration Human HCC cell line [132] (b) PPAR𝛾

miRNA Biological process Organism Cell/tissue Reference Endothelial dysfunction Mouse Primary pulmonary artery Kang et al., 2016 miR-98 Pulmonary hypertension Human endothelial cells [103] Proliferation Ovarian cancer tissue, Luo et al., 2015 miR-125b Human Apoptosis Ovarian cancer cell lines [130] Inflammation Cerebral cortex, Dharap et al., Rat Oxidative stress Pheochromocytoma cell line 2015 [17] Zhu et al., 2015 Collagen synthesis Human Hypertrophic scar fibroblasts miR-145 [131] Cell cycle Colorectal cancer tissue and Panza et al., Invasion Human colorectal cancer cell lines 2014 [96] Differentiation Bone marrow derived macrophages Ying et al., 2015 miR-223 Inflammation Mouse Adipocyte-derived stromal cells [133] Primary adipocytes Inflammation Cerebral cortex, Dharap et al., miR-329 Rat Oxidative stress Pheochromocytoma cell line 2015 [17] Adipocyte White adipose tissue Yu et al. 2014 miR-339-5p Human differentiation Isolated preadipocytes [134] White adipose tissue Adipocyte Yu et al., 2014 miR-378 Human Primary adipose derived stromal differentiation [134] cells

in metabolic (e.g., insulin signaling) and proliferative (e.g., further enhanced by a PPAR𝛾 agonist (i.e., pioglitazone) Wnt/𝛽-catenin signaling) pathways [17, 134]. Interestingly, and inhibited by a PPAR𝛾 antagonist (i.e., GW9662). Since miR-329 and miR-145 contain a PPRE in their promoters and 𝛾 󸀠 PPAR -dependent M2 activation is inhibited in BMDMs both miRNAs also bind to PPAR𝛾 3 UTR, thus suggesting the from miR-223 knockout mice, these data suggest that miR- existence of positive feedback loop mechanisms regulating 223 and its target genes (e.g., Rasa1 and genuine)arekey expressions of these miRNAs and PPAR𝛾 [17]. In human effectors of macrophages polarization [133]. subcutaneous adipocytes and bovine preadipocytes, PPAR𝛾 also induces the expression of miR-378, which is located in Fibrosis. Whether PPAR𝛾 may control fibrotic processes the first intron of PPAR𝛾 coactivator-1𝛽 (PGC1𝛽)[98,134, through miRNAs-dependent mechanisms is not well estab- 137]. Finally, a list of potential miRNAs, directly regulated lished, but one study supports this concept. Indeed, Dharap by PPAR𝛾 and involved in 3T3-L1 adipose differentiation, et al., reported that miR-145, which contains a PPAR was identified by crossing datasets of miRNAs containing response element in its promoter [17, 96], was increased in putative PPAR𝛾 binding site with datasets of miRNAs altered rosiglitazone-treated hypertrophic scar fibroblasts (HSFDs), during 3T3-L1 differentiation. Authors of this study identified thus leading to a direct decrease of SMAD3 expression and miR-103-1, miR-182/miR-96/miR-183, miR-205, and miR-378 collagen synthesis [131]. as potential PPAR𝛾-regulated miRNAs, whose expression was further induced in 3T3-L1 cells treated by rosiglitazone. Carcinogenesis. AdirecteffectofPPAR𝛾 on the expression Chip analyses also revealed that these miRNAs are directly of specific miRNAs through binding of PPRE in their pro- regulated by PPAR𝛾 through PPRE present in their host genes moters was demonstrated for three different types of cancer (PanK3 and PGC1𝛽)[138]. cell lines. In hepatoma HepG2 cells, miR-122 was strongly induced in cells treated with DNA methylation or histone Inflammation. Exposure of bone marrow derived macropha- deacetylase inhibitors via a direct binding of PPAR𝛾/RXR𝛼 ges (BMDMs) to Th2 stimuli (i.e., IL-4) triggers the expres- in the pre-miR-122 promoter [139]. In ovarian cancer cells sion of miR-223 through a direct binding of PPAR𝛾 in (i.e., Ovcar3, CaOv3, and Skov3 cells), PPAR𝛾 also directly PPREs within the promoter of pre-miR-223. This effect was regulates the transcription of miR-125b and silencing of PPAR Research 13 miR-125 impaired the growth inhibitory capacity of PPAR𝛾 (e.g., miR-103/107 for type 2 diabetes and obesity) [26]. agonists [130]. Finally, miR-145, which is downregulated in However, only few of them, for example, miR-122 inhibitors colorectal cancer cells (Caco2, Sw480, HCT116, and HT29) (Miravirsen) to treat HCV infection, are currently being and colorectal tumor tissues, is induced by the PPAR𝛾 agonist tested in human clinical trials [26, 147–149]. Unfortunately, (i.e., rosiglitazone) via direct binding of PPAR𝛾 to PPRE in none of the miRNAs known to potentially target PPAR the promoter encoding pre-miR-145 [96, 131]. isoforms are under clinical trials in human. Only preclinical studies were performed for miR-33 and miR-21 [26], 6. miRNAs-Based Therapies to Target which targets directly PPAR𝛼 (Figures 2-3). In African green PPARs Expression/Activity monkeys, inhibition of mir-33a/b with specific antagomiRs increased the hepatic expression of ABCA1, thus leading to Targeting tissue-specific miRNAs with pharmacological an increase of HDL (high density lipoprotein) and a decrease compounds may represent novel and valuable alternative in VLDL (very low density lipoproteins) and triglycerides therapeutic approaches to PPAR agonists or antagonists [10– plasma levels [150]. Inhibition of miR-21 in mice with an anti- 13]. Different methods have been developed to modulate sense oligonucleotide prevented hepatic lipid accumulation miRNA expressions in vivo. Of particular interest are chemi- in animals fed an obesogenic diet [151]. cally modified synthetic oligonucleotides inhibiting or mim- The (pre)clinical use of synthetic nucleotides mimicking icking endogenous miRNAs that display increased affinity endogenous miRNAs is less developed compared to miRNAs for their targets and great stability in the serum. Currently, inhibitors and currently only miR-34 mimics nucleotides are 󸀠 󸀠 these oligonucleotides bear modifications on the 2 -or3- tested to treat some cancers [26, 152]. MRX34, a liposome- position of the nucleic acid ribose backbone. For example, formulated miR-34 mimic-based drug is currently in phase 󸀠 󸀠 antagomiRs (3 -cholesterol-conjugated, 2 -O-Me oligonu- I study for melanoma patients. This miR-34 mimic achieved cleotides with terminal phosphorothioate modifications), positive outcomes as a monotherapeutic agent in patients 󸀠 antisense modified oligonucleotides (AMO) (2 -O-methoxy- with renal cell carcinoma, acral melanoma, and HCC (http:// ethyl phosphorothioate modified antisense oligonucleotide www.mirnatherapeutics.com/pipeline/mirna-pipeline.html) 󸀠 or 2 -fluoro-modified antisense oligonucleotides), or locked [26]. However, other in vivo studies indicated that miR-34a/c nucleic acids (LNA) represent potent inhibitors of miRNAs couldalsoactivatehepaticstellatecellsandpromotefibrogen- expression/activity. These synthetic nucleotides are usually esis by targeting PPARy and repressing RXR𝛼 and Sirt1 [52, administered by intravenous injection and hopefully soon 109]. Other miRNAs, such as miR-27 or miR-9, are also able orally with a good efficiency [140, 141]. When administered to regulate the expression/activities of different PPARs iso- by intravenous injection they can broadly reach every tissue forms in distinct tissues. Therefore, although miRNAs-based but tends to accumulate in particular organs, such as the therapies are promising, the potential pleiotropic effects of liver or the kidneys [26]. Special formulations such as systemic administration of pharmacological miRNAs inhibi- liposomes or polyethylenimine-formulated nanoparticles as tors or mimics call also for cautiousness in their therapeutic miRNA nanocarriers have been developed to improve tissue- usesincetheycanlikelylead,asinthecaseofPPARsagonists/ specific distribution, circulation time, and clearance of the antagonists, to conflicting and unwanted side effects. miRNAs-likecompounds[26,142,143].Ofparticularinterest, microvesicles (MVs) were shown to represent efficient and 7. Conclusion functional miRNA delivery tools as it was demonstrated in animals and in the case of miR-130b [144, 145]. Other alter- ThepivotalroleofabnormalPPARssignalinginthedevel- native methods to target specific tissues have been also development and the progression of various pathologies including oped such as inclusion of oligonucleotides into liposomal metabolic diseases, inflammation, and cancer is now well or oleic-based nanoparticles, which target preferentially the established. However, the mechanisms and extent to which liver [142]. Finally, viruses with specific tropisms, for example, miRNAs contribute to alterations of PPARs expressions adenoassociated viruses (AAV), have been used in animal and/or activities in physiopathological conditions are cur- models to robustly express or inhibit specific miRNAs in rently still poorly understood and represent an important particular tissues and may represent an interesting alternative developing field of research. Conversely, the fact that PPARs to chemically modified nucleotides [26]. Importantly, abnor- can drive the expression of specific miRNAs, which may mal levels of circulating miRNAs stimulate toll-like receptors target in turn hundreds of different mRNAs, opens also a therefore promoting inflammation and favoring the develop- new dimension in our understanding of the physiological mentofchronicdiseasessuchasmetabolicandcardiovascu- and pathological roles of PPARs isoforms. Given the tissue- lar disorders as well as cancers. Interestingly, chemically mod- specific and pleiotropic action of PPARs in various cellular ified synthetic oligonucleotides, in particular those modified processes described herein, it is likely that posttranscriptional 󸀠 in the 2 position of the ribose, have the ability to reduce, but regulation of PPARs and related cofactors by miRNAs is not completely prevent, such unintended immune responses tissue- and process-specific. In addition, the simplistic view [26, 146]. that only changes in the intracellular levels of miRNAs Several miRNA inhibitors have been tested in preclinical impact the expression of target genes is likely incorrect. studies with rodents or primates in the context of various Indeed increasing evidence indicates that the activity and pathologies (e.g., miR-155 in inflammatory diseases, miR- bioavailability of miRNAs are also key factors to consider 208 in cardiac remodeling) including metabolic diseases in these regulatory mechanisms. This concept is further 14 PPAR Research supported by the emerging role of long noncoding RNAs [31] T2DM: Type 2 diabetes mellitus and RNA-binding proteins, which could interfere with the TARP2: T-cell receptor gamma-chain constant region activity/expression of specific miRNAs [153, 154] and regula- TNF-𝛼: Tumor necrosis factor-𝛼 tion of their target genes. Further studies are thus required to TZD: Thiazolidinedione deepen our knowledge of miRNAs-based posttranscriptional UCP-1: Uncoupling protein-1 regulatory mechanisms controlling PPARs expressions and UTR: Untranslated region activities. VCAM: Vascular cell adhesion protein WAT: White adipose tissue. Abbreviations Competing Interests AP-1: Activator protein-1 ApoA: Apolipoprotein-A The authors declare that they have no competing interests. BAT: Brown adipose tissue CD36: Cluster of differentiation 36 FAT: Fattyacidtranslocase Authors’ Contributions C/EBP𝛼: CCAAT/enhancer-binding protein 𝛼 C. elegans: Caenorhabditis elegans Dorothea Portius and Cyril Sobolewski have equal contribu- COX-2: Cyclooxygenase-2 tions. 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Review Article Peroxisome Proliferator-Activated Receptor Modulation during Metabolic Diseases and Cancers: Master and Minions

Salvatore Giovanni Vitale,1 Antonio Simone Laganà,1 Angela Nigro,2 Valentina Lucia La Rosa,3 Paola Rossetti,2 Agnese Maria Chiara Rapisarda,4 Sandro La Vignera,5 Rosita Angela Condorelli,5 Francesco Corrado,1 Massimo Buscema,2 and Rosario D’Anna1

1 Unit of Gynecology and Obstetrics, Department of Human Pathology in Adulthood and Childhood “G. Barresi”, University of Messina, Messina, Italy 2Unit of Diabetology and Endocrino-Metabolic Diseases, Hospital for Emergency Cannizzaro, Catania, Italy 3Unit of Psychodiagnostics and Clinical Psychology, University of Catania, Catania, Italy 4Department of General Surgery and Medical Surgical Specialties, University of Catania, Catania, Italy 5Department of Clinical and Experimental Medicine-CRAMD (Research Centre of Motor Activity and Metabolic Rehabilitation in Diabetes), University of Catania, Catania, Italy

Correspondence should be addressed to Salvatore Giovanni Vitale; [email protected]

Received 19 October 2016; Accepted 12 December 2016

Academic Editor: Daniele Fanale

Copyright © 2016 Salvatore Giovanni Vitale et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The prevalence of obesity and metabolic diseases (such as type 2 diabetes mellitus, dyslipidaemia, and cardiovascular diseases) has increased in the last decade, in both industrialized and developing countries. This also coincided with our observation ofa similar increase in the prevalence of cancers. The aetiology of these diseases is very complex and involves genetic, nutritional, and environmental factors. Much evidence indicates the central role undertaken by peroxisome proliferator-activated receptors (PPARs) in the development of these disorders. Due to the fact that their ligands could become crucial in future target-therapies, PPARs have therefore become the focal point of much research. Based on this evidence, this narrative review was written with the purpose of outlining the effects of PPARs, their actions, and their prospective uses in metabolic diseases and cancers.

1. Introduction includes the receptors for thyroid hormones, retinoids, 1,25- dihydroxyvitamin D3, and steroid hormones [3]. After bind- The prevalence of obesity and metabolic diseases (for exam- ing with their agonists (natural or synthetic) in cytoplasm, ple, type 2 diabetes mellitus (T2DM), dyslipidaemia, and PPARs heterodimerize with the retinoid acid receptor (RNR cardiovascular diseases) has increased in the last decade, in or NR2B) and translocate to the nucleus, subsequently bind- both industrialized and developing countries. At the same ing to specific DNA regions termed peroxisome proliferator time,wehaveobservedsimilarincreaseintheprevalence response elements (PPREs). Here they activate the transcrip- of cancers. The aetiology of these disorders is very com- tion of numerous genes that play a role in mechanisms plex and involves genetic, nutritional, and environmental associated with glucose and lipid metabolism, body energy factors. There is much evidence that peroxisome proliferator- production, inflammation, cell cycle arrest, apoptosis, and activated receptors (PPARs) play a significant part in the DNA damage response [4, 5]. progression of these diseases [1, 2]. Currently, we know of three different types of PPARs Peroxisome proliferator-activated receptors (PPARs) are (PPAR𝛼,PPAR𝛽/𝛿,andPPAR𝛾), which present many differ- a group of ligand-activated nuclear hormone receptors (NRs), ent features, such as tissue distribution, ligand specificities, existing within the steroid receptor superfamily, which and effects. The principal differences among PPARs aredue 2 PPAR Research to their structure; despite the DNA-binding domains being These data were confirmed when liver and whole-body fatty 80% identical, the ligand-binding domains are different. acid homeostasis impairment was recently demonstrated The biological effects of PPARs depend on their different in a hepatocyte-specific PPAR𝛼 knockout mouse model. ligand and the presence of several proteins that operate as Results included hepatic lipid accumulation (nonalcoholic coactivators or corepressors and whose presence may alter fatty liver disease, NAFLD) and hypercholesterolemia dur- the expression of genes [6]. About this point, recent evidence ing ageing [16]. In addition, mice conditionally expressing suggests that the E6-associated protein (E6-AP) is an E3 human PPAR𝛿 demonstrated pronounced weight loss and ubiquitin ligase that affects the activity of other NRs: in promoted hepatic steatosis when treated with GW501516 particular, E6-AP is able to inhibit the ligand-independent (PPAR𝛿-agonist)whencomparedtowildtypemice[17]. transcriptional activity of PPAR𝛼 and PPAR𝛽,withmarginal Fibrates are weak PPAR𝛼 ligands; they reduce triglyceride effects on PPAR𝛾,anddecreasedbasalmRNAlevelsof (30–50%) and very low-density lipoprotein (VLDL) levels PPAR𝛼 target genes [7]. Similarly, Murine Double Minute 2 through an increased rate of lipid uptake, lipoprotein lipase- (MDM2), an E3 ubiquitin ligase, was identified as a PPAR𝛼- mediated lipolysis, and 𝛽- oxidation; in addition, fibrates also interacting protein that regulates the transcriptional activity induce a modest increase in HDL cholesterol levels (5–20%), of PPAR𝛼 and PPAR𝛽/𝛿,butnotPPAR𝛾 [8]. secondary to the transcriptional induction of apolipoprotein A-I/A-II synthesis found in the liver [18]. In this way, they 2. PPAR𝛼 Role in Metabolic Diseases decrease the systemic availability of fatty acids as well as fatty acid uptake in muscles [19], consequently leading to PPAR𝛼 is expressed in large amounts in the liver, skeletal fibrates reducing arteriosclerosis progression and cardiovas- muscles, heart, intestinal mucosa, and brown adipose tis- cular events. They also increase insulin sensitization and sue, where it undertakes an important role in fatty acid reduce plasma glucose levels. metabolism, as well as glucose and lipid metabolism [9] PPAR𝛼 activation by omega-3 fatty acids results in an PPAR𝛼 activation induces the expression of genes involved anti-inflammatory effect, caused in all probability by the in lipid and lipoprotein metabolism (apolipoprotein genes inhibition of their own oxidation due to the activation of A1, A2, and A5), in fatty acid oxidation (acyl-coenzyme the nuclear factor kappa-light-chain-enhancers of activated A oxidase and carnitine palmitoyltransferases I and II), in Bcells(NF-𝜅B). PPAR𝛼 alsoplaysakeyroleinthemediation the desaturation of fatty acyl-CoA (delta-6-desaturase), in of the anti-inflammatory actions of palmitoylethanolamide, High Density Lipoprotein (HDL) metabolism (Phospholipid the natural amide of palmitic acid, and ethanolamide [20]. Transfer Protein), and in ketone synthesis (3-Hydroxy-3- Recently, a PPAR𝛼 agonist (K-877) displaying high levels Methylglutaryl-CoA Synthase 2) [10]. Activated PPAR𝛼 also of potency and selectivity demonstrated optimal effects stimulates the expression of the fibroblast growth factor on atherogenic dyslipidemia [21]. In addition, a recent gene 21 (FGF21) and the angiopoietin-like protein gene 4 study indicated that statins, which are normally employed (ANGPLT4). In response to PPAR𝛼 activation, production as cholesterol-lowering drugs, induce an increase in neu- of FGF21 in the liver begins, activating white adipose tissue rotrophin expression in the brain, as a result of their binding lipolysisinordertoprovidenonadiposetissuewithfatty to a specific PPAR𝛼 domain independent of the mevalonate acidsaswellascontrollingketogenesisintheliverwiththe pathway. In a mouse model with Alzheimer’s disease, the use purpose of procuring energy from fatty acids [11]. In partial of Simvastatin led to an increase in neutrophin expression, as agreement with these data, it was found that increased FGF21 well as an improvement in memory and learning [22]. expression was observed in the livers of PPAR𝛽/𝛿-null mice and in mouse primary hepatocytes when this receptor was 3.TheRoleofPPAR𝛽/𝛿 in Metabolic Diseases knocked down by small interfering RNA (siRNA) and that this increase was associated with enhanced protein levels in PPAR𝛽/𝛿 is expressed ubiquitously, particularly in tissue the heme-regulated eukaryotic translation initiation factor which is metabolically active, such as the liver, skeletal 2𝛼 (eIF2𝛼) kinase (HRI) [12]. Recent studies indicate that and cardiac muscle, adipose tissue, and macrophages. Its the physiological fluctuations in lipoproteins lipase (LPL) involvement in the oxidation of fatty acids is crucial, and it activity are mediated by ANGPLT4 as well as the decrease improves lipid and cholesterol profiles. It plays a central role in adipose LPL activity observed during intervals of fasting in the oxidation of fatty acids as well as improving lipid and [13]. The natural and pharmacological ligands for PPAR𝛼 are, cholesterol profiles, which reduces adiposity and prevents respectively, omega-3 fatty acids resulting from diet (such as the development of obesity [23, 24]. It also regulates glucose linolenic, 𝛼-linolenic, 𝛾-linolenic, and arachidonic acids) and blood levels. In several animal studies, PPAR𝛽/𝛿 acted as fibrate, normally used as potent hypolipidemic agents [14]. regulator of fat consumption; the deficiency of this receptor In the liver, PPAR𝛼 plays the role of lipid sensor, normally leads to obesity, while the activation of PPAR𝛽/𝛿 conversely undergoing activation due to fatty acids and resulting in the results in resistance to this condition [25]. In the heart, increased burning of energy, the reduction of fat storage, in the presence of high-level dietary fat, PPAR𝛽/𝛿 lowers and the prevention of steatosis; conversely, when PPAR𝛼 lipid accumulation and increases glucose metabolism and sensing is not efficient or when fatty acid concentration consequently seems to be useful in diabetic cardiomyopathy, is decreased (for genetic, toxic, or metabolic causes), this as it protects the heart against ischemia-reperfusion injury causes a reduction in energy burning and the resulting lipo- [26]. For all these reasons, PPAR𝛽/𝛿 agonists (GW501516, toxicity promotes hepatic steatosis and steatohepatitis [15]. GW0742,andL-165041)couldbecomeapotentialtarget PPAR Research 3 in the treatment of metabolic disorders. However, adverse (VEGF) (121), VEGF(165), and VEGF(189) expression in effects, particularly for PPAR𝛾 agonists, are also observed HPV (Human Papillomavirus) positive HeLa cells: consider- with the use of investigational PPAR agonists and even some ing the intrinsic connection between HPV-related cancer of approved drugs [27]. uterine cervix and VEGF levels, it is possible that PPAR𝛽- Recently,itwasfoundthatGW501516significantly mediated pathway may play a key role in the development increased fatty acid oxidation and reduced the triglyceride of this type of cancer [35]. Confirming these data, L-165041 amount in VLDL-loaded foam cells, suggesting a key role was found to inhibit VEGF-stimulated angiogenesis by sup- of PPAR𝛽/𝛿 in modulating macrophage lipid overload [28]. pressing the cell cycle progression independently of PPAR𝛿: Intriguingly, PPAR-𝛿 agonist GW501516 decreases uptake inparticular,itreducesthenumberofendothelialcellsin of VLDL and expression of VLDL receptor at mRNA and theSphaseandtheexpressionlevelsofcellcycleregulatory protein levels through the regulation of miRNA-100 in proteins such as cyclin A, cyclin E, cyclin-dependent kinase Human Umbilical Vein Endothelial Cells [29]. Confirming (CDK) 2, and CDK4 [36]. Furthermore, a recent in vitro the human findings, clear data from mouse model showed study found that L-165041 significantly inhibits high glucose- that PPAR𝛽/𝛿-deficient mice fed with fructose exacerbated induced interleukin-6 and TNF-𝛼 production, receptor for glucose intolerance and this led to macrophage infiltration, advanced glycation end products expression, and NF-𝜅B inflammation, enhanced mRNA and protein levels of CD36, translocation in human embryonic kidney 293 (HEK) cells; and activation of the c-Jun N-terminal kinase pathway in in addition, it increases superoxide dismutase expression and white adipose tissue; fascinatingly, these effects were partially attenuates apoptosis in HEK and mesangial cells [37]. prevented by the PPAR𝛽/𝛿 activator GW501516 [30]. In addition, topical application of polymer-encapsulated GW501516 4. PPAR𝛼/𝛿 Role in Metabolic Diseases was found to have therapeutic wound healing activity, through stimulation of glutathione peroxidase 1 (GPx1) and The dual PPAR𝛼/𝛿 agonist (GFT-505-Elafibranor) seems to catalaseexpressioninfibroblasts:indeed,GPx1andcata- have potentially beneficial effects in the treatment of NAFLD. lase are known to scavenge excessive H2O2 accumulation In 2013, Staels et al. [38] showed in a mouse model that in diabetic wound beds, preventing H2O2-induced extra- GFT505 protects liver from steatosis, inflammation, and cellular matrix modification and facilitating keratinocyte fibrosis. This agonist also improves liver markers, decreases migration [31]. Furthermore, PPAR𝛿 plays pivotal roles in hepatic lipid accumulation, and inhibits proinflammatory wound healing by promoting fibroblast-to-myofibroblast dif- (IL-1, TNF𝛼) and profibrotic (transforming growth factor ferentiation via transforming growth factor (TGF)-𝛽/Smad3 beta, tissue inhibitor of metalloproteinase 2, collagen type I, signalling: according to recent findings [32], GW501516- alpha 1, and collagen type I, alpha 2) gene expression with activated PPAR𝛿 increases the migration and contractile aPPAR𝛼 dependent and independent mechanism [38]. In properties of human dermal fibroblasts and upregulates the partial agreement with these data, it was recently found that expression of myofibroblast markers such as collagen I and Biliverdin reductase A protects against hepatic steatosis by fibronectin, with a concomitant reduction in expression of inhibiting glycogen synthase kinase 3𝛽 (GSK3𝛽)byenhanc- the epithelial marker E-cadherin. ing serine 9 phosphorylation, which inhibits its activity: in Regarding GW0742, it was recently demonstrated that it particular, GSK3𝛽 phosphorylates serine 73 of the PPAR𝛼, canreversethelungtissuedamageinducedbyelastasein which in turn increased ubiquitination and protein turnover, emphysema-model mice and improves respiratory function as well as decreasing activity [39]. in mouse model: in particular, GW0742 increases the in In phase 2a trials (duration 8 weeks) involving twenty- vivo expression of surfactant proteins A and D, which are two obese males with dyslipidemia, prediabetes, or T2DM, known alveolar type II epithelial cell markers, reduces the GFT505 reduced fasting plasma triglycerides, low-density average distance between alveolar walls in the lungs, and lipoprotein (LDL) cholesterol, and liver enzyme concentra- improves tissue elastance, as well as the ratio of the forced tions improving peripheral insulin sensitivity and hepatic expiratory volume in the first 0.05 s to the forced vital capacity insulin sensitivity [40]. The liver-specific action of GFT505 [33]. In addition, recent evidence suggests that GW0742 wassuggestedbythefactthatneitherPPAR𝛼 nor PPAR𝛿 administration to mice fed in high-fat diet prevented the target genes were induced in skeletal muscle. Recently, it gain of body weight, heart and kidney hypertrophy, and fat was demonstrated that Elafibranor was capable of improving accumulation:namely,itpreventstheincreaseofinplasma the histological features of severe and moderate nonalcoholic levels of fasting glucose, glucose tolerance test, homeostatic steatohepatitis (NASH) and presents a favorable safety profile model assessment of insulin resistance, and triglyceride; from [41]. the molecular point of view, it increases both protein kinase B (Akt) and endothelial nitric oxide synthase phosphorylation 5. PPAR𝛾 Role in Metabolic Diseases and inhibits the increase in caveolin-1/endothelial nitric oxide synthase interaction, ethidium fluorescence, nicoti- PPAR𝛾 was the first to be cloned and studied in depth, due namide adenine dinucleotide phosphate (NADPH) oxidase 1, toitsbeingthetargetofaclassofantidiabeticdrugscalled Toll-like receptor 4, tumor necrosis factor-𝛼,andinterleukin- thiazolidinediones (TZD). Currently, we know of three iso- 6 expression, and I𝜅B𝛼 phosphorylation [34]. forms of PPAR𝛾:PPAR𝛾1 and PPAR𝛾3, which are expressed Regarding the PPAR𝛽 agonist L-165041, it was demon- in the liver, intestine, and spleen; PPAR𝛾2 is present only in strated that it induces vascular endothelial growth factor white and brown adipose tissue. Activated PPAR𝛾 induces the 4 PPAR Research expression of many genes, essential for adipogenesis, energy blocks only the phosphorylation of serine 273 by CDK5. In a balance, insulin sensitivity, lipid and glucose metabolism, recent study, CDK5 deficient mice (CDK5 KO) demonstrated and inflammation [42]. In adipocytes, PPAR𝛾 is necessary a paradoxical augmentation of PPAR𝛾 phosphorylation at in order for adipose tissue to develop. PPAR𝛾2 is a potent serine 273 by a protein kinase (extracellular signal-regulated transcription activator and is triggered as a response to kinase, ERK), normally suppressed by CDK5 [53], suggesting nutrient intake and obesity [43]. Indeed, mice deprived of a key role in the modulation of the abovementioned path- PPAR𝛾2 (obese POKO mice) presented higher levels of fat ways. Finally, as extensively summarized elsewhere [54, 55], accumulation in adipocytes in comparison with normally it was showed that natural PPAR𝛾 ligands have different obese mice fed an identical diet [44]. According to these data, binding modalities to the receptor with respect to the full PPAR𝛾2 is essential for preventing lipotoxicity by promoting TZD agonists and can activate also PPAR𝛼 (asitoccursfor the expansion of adipose tissue and an increased lipid- genistein, biochanin A, sargaquinoic acid, sargahydroquinoic buffering capacity in liver, muscle, and pancreatic beta cells. acid, resveratrol, and amorphastilbol) or the PPAR𝛾-dimer A proliferative response of 𝛽-cells to insulin resistance is also partner retinoid X receptor (RXR; as it occurs for the promoted by PPAR𝛾 [45]. In adipocytes, activated PPAR𝛾 neolignans, magnolol, and honokiol). causes a both balanced and adequate adipocytokine secretion (adiponectin and leptin), which regulates the behavior of 6. PPAR𝛼 and Tumorigenesis insulin when introduced to peripheral tissues (such as the liver, skeletal muscle). As a consequence, PPAR𝛾 leads to To date, many studies have analysed the role played by improved insulin sensitivity in the entire body, additionally PPARs in the complex mechanism of tumorigenesis. Not protecting the nonadipose tissue against excessive lipid levels all data are clear and PPARs seem to possess both positive [46]. Activated PPAR𝛾 induces the expression of genes that and negative effects, depending on the type of tumor. In regulate the release, transport, and storage of fatty acid, particular, it leads to negatively regulated colonic inflam- suchasthegeneofLPLandfattyacidtransporterCD36 mation and proliferation. In an animal model of IL-10 [44]. PPAR𝛾 is also found in endothelial cells and vascular −/− mice, the inhibition of colitis was mediated by fenofi- smooth muscle cells, where it seems to be an important factor brate, increasing the PPAR𝛼 expression of lymphocytes, in inflammation and atherosclerosis [47]. Polyunsaturated macrophages, and colonic epithelial cells and resulting in fatty acids are the natural ligand of PPAR𝛾;theyincrease proinflammatory cytokine production, such as interleukin- glucose uptake and insulin sensitivity, but they do not have 17, interferon-𝛾, and chemokine (C-C motif) ligand 20 many effects on adipocytes differentiation [48]. As already (CCL20), being inhibited [56]. In partial agreement with mentioned, TZDs (pioglitazone, rosiglitazone) are synthetic these results in the mouse model, it was recently found that agonists of PPAR𝛾 and are widely used for the treatment of activation of PPAR𝛼 through fenofibrate suppressed migra- type 2 diabetes. TZDs are also described as insulin sensitizing, tion of oral cancer cells: in particular, differential protein as they indirectly induce a higher insulin-stimulated glucose profiling demonstrated that expressions of genes related to uptake in adipocytes, hepatocytes, and skeletal muscle; they mitochondrial energy metabolism were either upregulated also reduce free fatty acids levels and increase lipid storage (Atp5g3, Cyc1, Ndufa5, Ndufa10, and Sdhd) or downregulated in adipocytes. In the liver, TZDs decrease fasting plasma (Cox5b, Ndufa1, Ndufb7, and Uqcrh), conforming the key glucose levels through the increase of insulin sensitivity and role of PPAR𝛼 activation and response in mitochondrial the inhibition of gluconeogenesis [34]. In the muscles, TZDs energy metabolism [57]. In addition, recent data suggests that reduces postprandial glucose levels [49]. A typical effect of the selective activation of PPAR𝛼 by palmitoylethanolamide TZDs is weight gain, due (at least in part) to fat being inhibits colitis-associated angiogenesis, decreasing VEGF redistributed from visceral depot to subcutaneous depot [49]. release and new vessels formation, via the phosphatidylinos- In diabetic patients, the two principal types of TZDs itol 3-kinase/Akt/mammalian-target-of-rapamycin (mTOR) (rosiglitazone e pioglitazone) have different effects on car- signalling pathway [58]. diovascular outcomes. According to the data of a PROactive In breast cancer, the data are still not clear. In some study, pioglitazone reduced 16% of cardiovascular complica- studies, PPAR𝛼 inhibits breast cancer progression, promoting tions compared to a placebo [50]. Conversely, rosiglitazone apoptosis of cancer cells through NF𝜅B signalling. Recently, it was linked with a significantly increased death rate due to was demonstrated that clofibrate presents a high chemosen- cardiovascular causes; consequently, in 2010 the European sitivity towards breast cancer cells, in all likelihood through Medicines Agency withdrew the usage of this molecule [51]. the inhibition of NF-𝜅B and ERK1/2 activation, which lowers These existing differences between pio- and rosiglitazone cyclin D1, cyclin A, and cyclin E and induces proapoptotic are most likely due to their differing effects on lipid levels; P21 levels [59]. In contrast, in other studies PPAR𝛼 promoted in fact, pioglitazone leads to an increased level of HDL breast cancer progression by releasing leukotriene B4 that cholesterol whilst lowering levels of triglycerides and fasting activates PPAR𝛼 in B cells, inducing the differentiation of B fatty acids. Rosiglitazone increases total and HDL cholesterol cells and metastasis [60]. but also LDL cholesterol, which is negatively associated with Despite the fact that PPAR𝛼 clearly acts in a tumor- cardiovascular diseases [52]. dependent fashion [61], recent evidence suggests that its over- Recently, a new synthetic antidiabetic drug (SR1664) was expression enhances cancer cell chemotherapy sensitivity, proposed: when compared to TZDs, it does not induce weight whereas silenced PPAR𝛼 decreased this event. In this regard, gain. SR1664, with respect to the classic agonist of PPAR𝛾, it is possible that it induces cell apoptosis by destructing PPAR Research 5

B-cell lymphoma 2 (Bcl2): as summarized elsewhere [62], secretion, and that 7,12-dimethylbenz[a]anthracene (DMBA) PPAR𝛼 serves as an E3 ubiquitin ligase to govern Bcl2 plus Rosiglitazone is able to reduce average mammary protein stability; PPAR𝛼 binds to BH3 domain of Bcl2 and, tumor volumes by 50% [72]. Conversely, heterozygous or subsequently, transfers K48-linked polyubiquitin to lysine-22 homozygous intestinal-specific PPAR𝛾 deficiency enhanced site of Bcl2 resulting in its ubiquitination and proteasome- small intestine and colon tumorigenesis in Apc(Min/+) mice dependent degradation. Confirming these results, it was [73].Lastbutnotleast,robustdatafrommyeloid-specific found that ectopic expression of PPAR𝛼 in hepatocarcinoma bitransgenic mouse model allow us to hypothesize that anti- cells significantly suppressed cell proliferation and induced inflammatory PPAR𝛾 in myeloid-lineage cells plays a key role apoptosis by inhibition of NF-𝜅Bpromoteractivity,diminu- in controlling proinflammatory cytokine synthesis, myeloid- tion of phosphor-p65, phosphor-p50, and BCL2 levels, and derived suppressor cell expansion, immunosuppression, and enhancing IkB𝛼 protein [63]. the development of cancer [74]. Finally, recent evidence suggests that PPAR𝛾 is able to induce apoptosis in lung 7. PPAR𝛾 and Tumorigenesis cancer, although it can be inhibited by NR0B1, an orphan nuclear receptor whose knockdown reduces tumorigenic and PPAR𝛾 or dual PPAR 𝛼/𝛾 agonists, in rodent carcinogenicity antiapoptotic potential [75]. studies, were frequently associated with the development of hemangioma or hemangiosarcoma, fibrosarcoma, bladder, 8. PPAR𝛿 and Tumorigenesis and hepatic tumors [64], suggesting that these types of cancer are drug specific [65]. The role of PPAR𝛿 in carcinogenesis is uncertain and seems Recently, a hypothetical mechanism was proposed that to be context-dependent. In particular, PPAR𝛿,throughits could clarify the induction of liposarcoma by differing PPAR anti-inflammatory effects, seems to prevent cancer before its agonists:inthismodel,thefirststageoftumordevelopmentis development; conversely, after the development of cancer, initiation, during which DNA damage ensues independent of the activation of PPAR𝛿 promotes angiogenesis and can- PPAR activation. The second step, promotion, relies on PPAR cer growth [76]. Clinical data suggest a strong association and is defined by tumor cell recruitment, proliferation, and between PPAR𝛿 and aggressive cancer; in particular, inverse differentiation[44].Amultitudeofinvitroandinvivostudies correlation of PPAR𝛿 expression with survival in gastroin- have demonstrated much evidence for the antitumor effects testinal cancer has been noted [77]. In addition, PPAR𝛿 of natural and synthetic PPAR𝛾,sinceitseemstobeupreg- is required for chronic colonic inflammation and colitis- ulated in several human cancer lines. Indeed, recent data associated carcinogenesis: specifically, the cyclooxygenase suggest that PPAR𝛾 ligands have an antitumorigenic effect (COX)-2-derived prostaglandin E2 (PGE2) signalling medi- in prostate cancer as a result of antiproliferative and prodif- ates crosstalk between tumor epithelial cells and macrophages ferentiation effects [66]. It would appear that TZDs possess to promote chronic inflammation and colitis-associated protective effects in the development of pancreatic ductal tumor genesis [78]. In agreement with what reported in the adenocarcinoma, through improvement in insulin sensitivity previous chapters, high-fat diet is associated with increased and inflammation [67]. Interestingly, it was recently found colorectal cancer incidence, probably because many of its that PPAR𝛽/𝛿 plays a role in regulating pancreatic cancer cell effects on stem and progenitor cell compartment are driven invasion through regulation of genes via ligand-dependent by a robust PPAR-𝛿 program and contribute to the early release of B-cell lymphoma-6 and that activation of the steps of intestinal tumorigenesis [79]. In addition, recent receptor may provide an alternative therapeutic method for evidence suggests that high-fat diet modifies the PPAR𝛾 controlling migration and metastasis [68]. pathway leading to disruption of microbial and physiological Despite the fact that data are still elusive, recent evi- ecosystem in murine small intestine [80]. dence from human follicular thyroid carcinoma seems to Recently, the expression of PPAR𝛿 in breast cancer has underlie a key role for paired box gene 8 (PAX8)/PPAR𝛾 been negatively linked with patient survival. In 2016, Wang fusion protein in enhancing in vivo angiogenesis through et al. [81] showed that PPAR𝛿 upregulation increases the VEGF expression [69]. PPAR𝛾 haspositiveeffectsonbreast expression of catalase and Akt in breast cancer cells and cell cancer: it downregulates the expression of the C-X-C in this way cells are able to survive in harsh conditions chemokine receptor type 4 (CXCR-4) gene, which is crucial (including in the presence of chemotherapies), promoting in the growth and progression of cancer, as well as in the progression and metastasis. Not surprisingly, both the proin- development of metastasis. This mechanism seems to be flammatory PGE2 and the BRCA1 tumor-suppressor gene PPAR𝛾 dependent, because it could be reversed by GW9662, were found to regulate aromatase expression [82] and, fur- that is, a PPAR𝛾 antagonist [70]. In partial agreement with thermore, pioglitazone is able to inhibit aromatase expression these results, it was recently found that 𝛾-tocopherol-rich by inhibition of PGE2 signalling and upregulation of BRCA tocopherol decreased tumor volume and multiplicity in [83]. Finally, recent evidence suggests that PPAR𝛿 modulates estrogen-induced breast cancer female rats, increasing the the migration and invasion of melanoma cells by upregulating expression of PPAR𝛾 and its downstream genes, phosphatase Snail expression: in an elegant in vitro study, it was found that and tensin homolog (PTEN), and p27 [71]. In addition, activation of PPAR𝛿 by GW501516 significantly increased the it was found that in vivo PPAR𝛾 expression in mammary migration and invasion of highly metastatic A375SM cells, stromal adipocytes attenuates breast tumorigenesis through but not that of low metastatic A375P cells, by upregulating breast cancer 1 (BRCA1) upregulation and decreased leptin Snail expression [84]. Despite the promising results, further 6 PPAR Research studies are necessary in order to clarify the role of PPAR [10] A. Shah, D. J. Rader, and J. S. 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Review Article PPARs and Mitochondrial Metabolism: From NAFLD to HCC

Tommaso Mello,1 Maria Materozzi,1 and Andrea Galli1,2

1 Clinical Gastroenterology Unit, Department of Biomedical Clinical and Experimental Sciences “Mario Serio”, University of Florence, Viale Pieraccini 6, 50129 Florence, Italy 2Careggi University Hospital, Florence, Italy

Correspondence should be addressed to Tommaso Mello; [email protected]

Received 22 July 2016; Revised 8 November 2016; Accepted 10 November 2016

Academic Editor: Daniele Fanale

Copyright © 2016 Tommaso Mello et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Metabolic related diseases, such as type 2 diabetes, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD), are widespread threats which bring about a significant burden of deaths worldwide, mainly due to cardiovascular events and cancer. The pathogenesis of these diseases is extremely complex, multifactorial, and only partially understood. As the main metabolic organ, the liver is central to maintain whole body energetic homeostasis. At the cellular level, mitochondria are the metabolic hub connecting and integrating all the main biochemical, hormonal, and inflammatory signaling pathways to fulfill the energetic and biosynthetic demand of the cell. In the liver, mitochondria metabolism needs to cope with the energetic regulation of the whole body. The nuclear receptors PPARs orchestrate lipid and glucose metabolism and are involved in a variety of diseases, from metabolic disorders to cancer. In this review, focus is placed on the roles of PPARs in the regulation of liver mitochondrial metabolism in physiology and pathology, from NAFLD to HCC.

1. Introduction histologically classified into nonalcoholic fatty liver (NALF), defined as the presence of steatosis in the absence of Liver cancer is a major challenge in contemporary medicine. causes for secondary hepatic fat accumulation (i.e., alcohol It represents the fifth most common cancer in men, the consumption, steatogenic drugs, or genetic disorders), and ninth in women, and the second most frequent cause of nonalcoholic steatohepatitis (NASH), in which steatosis is mortality among oncological patients. It was responsible for complicated by inflammation and hepatocellular damage nearly 746,000 deaths in 2012, with an estimated incidence of (ballooning hepatocytes), with or without fibrosis [6]. A over 780,000 new cases yearly worldwide [1]. The prognosis relatively small portion of NAFL patients evolve into NASH, a for liver cancer is extremely poor (overall ratio of mortality progressive type of liver disease with the potential of evolving to incidence of 0.95), reflecting the absence of effective into cirrhosis and HCC. The cumulative incidence of HCC in treatments.Themostfrequenttypeofprimarylivercanceris NASH cirrhosis ranges from 2.4% to 12.8%, and although it hepatocellular carcinoma (HCC), accounting for up to 85% is lower than in HCV cirrhotic patients, the absolute burden of total cancers [2]. of NASH related HCC is higher due to the epidemic spread Major risk factors include HBV or HCV infection, alco- of NAFLD [7]. Moreover, NAFLD greatly increases the risk holic liver disease, and most likely nonalcoholic liver disease of HCC from other aetiologies, especially HCV and HBV. (NAFLD) [2]. These and other chronic liver diseases lead to While the vast majority of HCC arise in cirrhotic livers, cirrhosis, which is found in 80–90% of HCC patients [2]. it can also occur in noncirrhotic patients [2]. Of notice, a NAFLD is now the most common liver disease worldwide significant amount of new HCC cases is diagnosed in patients [3], with a global prevalence of about 25%. NAFLD is closely with noncirrhotic NASH [4, 8]. The global incidence of HCC associated with other metabolic disorders such as obesity, among NAFLD patients was recently estimated to be 0.44 metabolic syndrome, and type 2 diabetes [3]. Indeed, obesity per 1,000 person-year [3], which combined with the epidemic and diabetes are now definitively recognized as independent spread of metabolic disorders results in an enormous burden. risk factors for the development of HCC [4, 5]. NAFLD is The recent meta-analysis by Younossi et al. raised the question 2 PPAR Research whether NAFLD could even precede the onset of metabolic 2. PPARs and Mitochondrial Metabolism syndrome rather than just being the hepatic manifestation of in the Liver it [3]. The mechanisms that promote HCC development in 2.1. PPAR𝛼. Peroxisome proliferator activated receptor 𝛼 NASH/NAFLD patients are complex and still poorly under- (PPAR𝛼) is the main PPAR isotype expressed in the liver stood. A number of molecular mechanisms have been linked and plays a major role in energy homeostasis, by regulating to obesity and related metabolic disorders that may accelerate lipid metabolism and ketone body formation [12]. In mice 𝛼 the development of HCC, such as adipose-derived inflam- but not in humans, PPAR also controls the glycolysis- 𝛼 mation, lipotoxicity, and insulin resistance. These and other gluconeogenesis pathway [13]. PPAR natural ligands are pathological events in obesity have complex interactions endogenous lipids such as fatty acids (FA) and their deriva- while their relative contribution to hepatocarcinogenesis in tives (eicosanoids, oxidized phospholipids) [14], while syn- various stages of NAFLD progression remains to be deter- thetic ligands include the class of hypolipidemic drugs mined. fibrates, xenobiotic agents, and plasticizers. Despite the fact that FA and derivatives can bind and Mitochondria can be seen as the energetic hub of the 𝛼 cell. As such, beyond their role in energy production, they activate PPAR in the liver, not all FA are created equal. play a central role in coordinating the cell anabolic and Indeed, it has been now recognized that FA released in the bloodstreambytheadiposetissue(i.e.,duringfastingor catabolic processes, in balancing the cell energetic demands intense physical exercise) have little role as PPAR𝛼 agonist in response to internal and external stimuli, and in the [15], while preferentially activating PPAR𝛽/𝛿,whereasfatty regulation of several cell signaling pathways. Deregulation of acids derived from dietary intake or de novo lipogenesis mitochondrial activity is a common trait to several human are efficient PPAR𝛼 activators [15–18]. However, PPAR𝛼 is diseases, including cancer. Since Warburg, it has long been absolutely required for the metabolic adaptation to fasting, known that cancer cells undergo a radical metabolic shift 𝛼−/− toward glycolysis, irrespective of the oxygen availability since PPAR mice, either full body [19] or liver-specific [20], develop steatosis with prolonged fasting. Moreover, (aerobic glycolysis) [9]. However, the actual significance of 𝛼 this metabolic remodeling, its consequences on cancer cell the time course activation of PPAR in the liver mimics biology, and its plasticity have begun to be grasped only in the kinetics of circulating FFA during fasting, and liver transcriptomic profiling revealed that the fasted state (ver- recent years. The initial perception of the Warburg effects 𝛼 was that cancer cells rely primarily on glycolysis for energy sus fed or refed) triggered the broader PPAR -dependent response, strengthening the functional link between hepatic production due to a defective mitochondrial respiration [10]. 𝛼 On the contrary, it is now clear that cancer cells hijack PPAR and adipose tissue-FA disposal [20]. Since activation of hepatic PPAR𝛼 requires de novo lipogenesis [15, 21], the their mitochondria metabolism toward massive anabolic pro- 𝛼 cesses, in order to cope with the cell fast-growing rates [11]. In mechanisms that fine-tune PPAR activation in different metabolic conditions are still unclear and possibly involve this line of view, exacerbate biosynthesis, in particular lipid 𝛼 biosynthesis, rather than glycolysis dependence, emerges as separate pools of PPAR that can be activated in a context- cancer metabolic hallmark. dependent manner. Peroxisome proliferator activated receptors (PPARs) are Moreover, the adipose tissue cross-talk with the hepatic master regulators of whole body and liver metabolism. PPARs can occur via adiponectin-induced FAO, which is 𝛼 Despite a similar structure, the three PPAR isotypes 𝛼, 𝛽/𝛿, dependent upon AdipoR2 subtype and requires PPAR and 𝛾 vary greatly in tissue distribution, pharmacological induction [22], and via FGF21, produced mainly in the liver 𝛼 and endogenous ligands, and biological effects. In the past in a PPAR -dependent manner [20], which promotes both decades PPARs have been the focus of massive research glucose uptake and lipolysis in the adipocytes [23], as well as hepatic lipid disposal [24]. effort that helped uncovering their contribution to can- 𝛼 cer, metabolic, and cardiovascular diseases. The different In hepatocytes, PPAR promotes the expression of sev- PPAR isotypes regulate lipid metabolism by a number of eral genes involved in FA uptake, activation to acyl-CoA, andtransporttothemitochondriaorperoxisomesand mechanisms: (i) controlling the rate of FA disposal through 𝛽 𝜔 mitochondrial and peroxysomal 𝛽-oxidation (FAO), (ii) subsequent -or -oxidation, ketogenesis, and lipoprotein regulating lipid biosynthesis via de novo lipogenesis, (iii) trafficking [25, 26] (Figure 1). 𝛼 regulating FA uptake in peripheral tissue and in the liver, (iv) Many of the PPAR regulated genes directly modulate regulating whole body lipid trafficking through apolipopro- mitochondrial metabolism. Interestingly, among the many teins, (v) interacting in complex regulatory network with PPAR𝛼-regulated genes in hepatocytes, those involved in other nuclear receptors (LXR, FXR), coactivators (PGC-1𝛼 mitochondrial metabolic functions, especially in fatty acid and 𝛽, SREBP), or corepressors (NCOR) involved in the oxidation, are consistently dependent upon PPAR𝛼 regard- metabolic homeostasis. As liver is primarily a metabolic less of the nutritional condition [20]. PPAR𝛼 target genes organ, PPARs-regulated processes are involved virtually in are also carnitine palmitoyl transferase 1 (CPT-1) and carni- any liver disease. tine palmitoyltransferase 2 (CPT-2) [19, 25], which mediate Thisreviewsummarizescurrentnotionsontherolesof transport of long-chain fatty acids through the outer and PPARs in the regulation of liver mitochondrial metabolism inner mitochondrial membrane, respectively, to initiate their in physiology and pathology, from NAFLD to HCC. degradation in the 𝛽-oxidative pathway (Figure 1). The PPAR Research 3

FABP CD36 LPL APOE APOA1 LDLR

LCFA LCFA-CoA MUFA SCD1 SFA MC/SC-FA CPT1 ACS ACS FASN Glycolysis

LCFA-carnitine Malonyl-CoA Pyruvate PDK CoA CPT2 PDH L-carnitine MCFA ACAC LCFA-CoA SCFA MCD

𝛽-oxidation Acetyl-CoA Acetyl-CoA

CS Citrate TCA ACL cycle CiC Citrate 1𝛽

Cytoplasm PGC- Oxaloacetate PGC-1𝛼 De novo

Mitochondrial matrix lipogenesis Gluconeogenesis + NADH NAD SIRT3 + Clock/Bmal FADH FAD

− + ROS e O2 + H PPAR𝛼 PPAR𝛿 − H2O e MRC I II IVIV PPAR𝛾 II III C

Figure 1: Role of hepatic PPARs in mitochondrial metabolism: fatty acid oxidation, circadian control of NAD+ dependent SIRT activity, de novo lipogenesis, and gluconeogenesis. Color-coding depicts PPAR isotypes-dependency of target genes.

𝛽-oxidation cycle consists of four reactions, catalyzed by stream and, after conversion to citrate, fuel the TCA cycle in acyl-CoA dehydrogenases (ACADs), enoyl-CoA hydratases, peripheral tissues (mostly heart, muscle, and brain). L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA FAO is functionally and physically linked to OXPHOS: thiolase, that sequentially remove two carbons—one acetyl- the reducing equivalents produced by FAO are directly CoA molecule, until the acyl-CoA is completely converted used by the electron transport chain (ETC); moreover, the to acetyl-CoA. The initial step of the 𝛽-oxidation cycle is two pathways are likely happening in large mitochondrial catalyzed by length specific acyl-CoA dehydrogenases (such supercomplexes containing both FAO and ETC complexes as ACADM, ACADS, and ACADVL), all of which are PPAR𝛼 [28]. Therefore, an unbalance in FAO or ETC directly affects target genes [26]. The last three steps are carried on by the the other pathway. mitochondrial trifunctional protein (MTP), a large complex PPAR𝛼,aswellas𝛽/𝛿 and 𝛾, also induces the expression of four 𝛼 and four 𝛽 subunits. The expression of both subunits of all uncoupling protein (UCP-1, UCP-2, and UCP-3), of (encoded by genes HADHA, HADHB) as well as the MTP which UCP-2 is the main type expressed in liver [29, 30]. protein 3-ketoacyl-CoA thiolase (encoded by ACAA2) is Uncoupling proteins allow protons to reenter the mitochon- regulated by PPAR𝛼 [26]. drial matrix without production of adenosine triphosphate, Theacetyl-CoAproducedduringFAOisthenusedto thus promoting energy expenditure and FA oxidation. produce ketone bodies (acetoacetate and 𝛽-hydroxybutyrate) Paralleling its role in promoting energy expenditure viamitochondrialHMG-CoAsynthase,anotherPPAR𝛼 through FA disposal, PPAR𝛼 also inhibits the lipogenic regulated gene [27]. Ketone bodies are released in the blood pathway by induction of the malonyl-CoA decarboxylase 4 PPAR Research which degrades malonyl-CoA, a precursor of FA biosynthesis 2.3. PPAR𝛾. PPAR𝛾 isthemainPPARisotypeexpressedin and inhibitor of the mitochondrial transporter CPT-1 [31] whiteandbrownadiposetissue.Itisthemasterinducerof (Figure 1). adipogenesis and promotes glucose uptake and utilization in the novo lipogenic pathway, therefore regulating whole bodylipidmetabolismandinsulinsensitivity.NaturalPPAR𝛾 2.2. PPAR𝛽/𝛿. PPAR𝛽/𝛿 is ubiquitously expressed, often at ligands are lipid molecules derivates, such as unsaturated FA, higher level than PPAR𝛼 or 𝛾.Overall,PPAR𝛽/𝛿 role in lipid PGJ2, and oxidized LDL [14, 46, 47] while potent synthetic metabolismappearstobelargelyoverlappingwithPPAR𝛼 𝛽 𝛿 ligands include the insulin sensitizer class of drug TZD [48]. in most tissues. Indeed, PPAR / stimulates FAO in muscle PPAR𝛾 induces the expression of genes regulating glucose and heart, the latter organ being extremely dependent on 𝛽 𝛿 sensitivity (GLUT-4, IRS-1, IRS-2, and PI3K), as well as PPAR / function [32]. genes involved in FA uptake and mobilization (FAT/CD36, 𝛼 Several PPAR target genes are thus not surprisingly fatty acids binding proteins aP2, and lipoprotein lipase) 𝛽 𝛿 induced also by PPPAR / (UCP-1, UCP-2, and UCP-3, andtriglyceridesynthesis(acyl-CoAsynthetase,glycerol FABP, FAT/CD36, LPL, ACS, and CPT-1) [33, 34] and loss kinase, and PEPCK) [46, 49] (Figure 1). In the liver, PPAR𝛾 𝛼 𝛽 𝛿 of PPAR in muscle is efficiently compensated by PPAR / is expressed in macrophages, endothelial cells, quiescent 𝛽 𝛿 [33]. Indeed, numerous studies have shown that PPAR / (nonactivated) stellate cells, and hepatocytes. Its complex overexpressionoractivationinmuscledramaticallyimproves actions on liver physiology are mostly mediated by its FA utilization as energy source, reduces hyperlipidemia, anti-inflammatory functions on macrophages and endothe- improves endurance, and decreases insulin secretion from 𝛽- lial cells, antifibrotic function in hepatic stellate cells, and cells [32, 35–37]. metabolic cross-talk between hepatocytes and adipocytes via 𝛽 𝛿 However, in the liver PPAR / seems to play a different FGF family members (FGF21, FGF-1). Mice with selective 𝛼 role than PPAR . Adenoviral-mediated overexpression of deletion of PPAR𝛾 in hepatocytes developed relative fat 𝛽 𝛿 PPAR / in the liver enhanced glucose utilization either to intolerance, increased adiposity, hyperlipidemia, and insulin increase glycogen storage or to promote de novo lipogen- 𝛾 𝛽 𝛿 resistance. Loss of hepatic PPAR increased TG blood esis, rather than inducing FAO [38] (Figure 1). PPAR / level and redistribution to other tissues, aggravating insulin induced the expression of several genes involved in glu- resistance in muscle and adipose tissue [50, 51]. These cose metabolism (GLUT2, GK, and pyruvate kinase) and models highlighted the role of liver PPAR𝛾 in maintaining lipogenesis (FAS, ACC1, ACC2, SCD1, SREBP-1c, and PGC- lipid/glucose homeostasis and insulin sensitivity. 𝛽 1 ) [38]. Conversely, gluconeogenesis genes (PEPCK, HNF- PPAR𝛾 also induces the expression of mitochondrial pro- 𝛽 𝛿 4) were inhibited by PPAR / expression in hepatocytes. teins, common to the other PPARs, such as CPT-1 and UCPs, 𝛼 Importantly, the levels of PPAR and its target (CPT-1, suggesting a possible degree of overlap in mitochondrial acyl-CoA oxidase, and MCAD) were unaffected; therefore metabolism regulation with other PPAR members. Probably 𝛽 𝛿 𝛼 PPAR / seems not to overlap with PPAR function in the themostrelevantfunctionofPPAR𝛾 in mitochondria biology liver [38]. Consistently, whole transcriptome profiling and comes with its interaction with PGC-1 family members. PGC- 𝛼−/− 𝛽 𝛿−/− liver metabolites analysis of PPAR and PPAR / mice 1𝛼 was initially identified as a nuclear PPAR𝛾 coactivator in revealed clearly divergent roles [39]. Very interestingly, liver mitochondrial rich brown adipose tissue-tissue [52]. Since 𝛽 𝛿 𝛼 PPAR / signals to PPAR andactivatesFAOinmusclevia then,ithasbecomeclearthatPGC-1𝛼 and 𝛽 control virtually the lipid molecule PC (18:0/18:1), whose production in the any aspect of mitochondria function and biogenesis [53], liver is PPAR𝛽/𝛿-dependent [40]. 𝛼 𝛽 𝛿 by thoroughly coordinating a plethora of nuclear receptors Different roles for PPAR and PPAR / in mitochondri- (including all three PPARs, EER𝛼) and nonnuclear receptor ogenesis are also beginning to emerge. A transitory upregula- protein [54]. Indeed, PPAR𝛾 can promote the expression tion of PPAR𝛼, and consequent induction of PGC-1𝛼,isnec- of PGC-1𝛼, which in turn potentiates PPAR𝛾 activity [55]. essary to promote mitochondriogenesis in the early steps of Recently,steatogenicFAwereshowntoinducePPAR𝛾 via differentiating embryonic stem cells. A robust upregulation of PGC-1𝛼, suggesting a link between mitochondria biogenesis PPAR𝛽/𝛿 is instead needed to promote mitochondriogenesis andtriglycerideaccumulation[56]. at later stages of cells differentiation and correlates with the expression of mature hepatocytic markers [41]. Functional peroxisome proliferator response elements have been identified in the distal promoter of PGC-1𝛼,pro- 3. Mechanisms of Mitochondrial Oxidative viding the mechanistic basis for PPAR-induced mitochon- Stress Damage drial biogenesis. However, the contribution of the diverse PPAR isotypes to PGC-1𝛼 induction appears to be cell Reactive oxygen species (ROS) are small reactive molecules 𝛼 𝛼 generated by the normal cell metabolism, involved in homeo- context-dependent. PGC-1 is activated by PPAR in brown − 𝛾 stasis and signaling. ROS such as superoxide anion (O2 ), adipose tissue [42] and by PPAR in both white and brown ∙ adipose tissue [42]. In skeletal muscle, PPAR𝛽/𝛿 but not hydrogen peroxide (H2O2), and hydroxyl radical (HO ) PPAR𝛼 induce PGC-1𝛼 expression [43, 44]. consistofradicalandnonradicaloxygenspeciesformed In liver, PCG-1𝛼 is induced by fasting, paralleling PPAR𝛼 by the partial reduction of oxygen. Cellular ROS levels are activation, and promotes gluconeogenesis, a process medi- controlled by antioxidant systems such as reduced/oxidized ated by PPAR𝛽/𝛿 [45]. glutathione (GSH/GSSG), reduced/oxidized cysteine PPAR Research 5

(Cys/CySS), tioredoxin (Trx), peroxiredoxin (Prx), super- pathways and inducing the expression of proteins, such as oxide dismutase (SOD), and catalase. NF-𝜅B, Akt, MAPK, JNK, and PPARs. Lipid peroxidation An imbalance of the generation/neutralization of ROS, occurs through a radical reaction; it is therefore extremely driven by an overproduction of ROS or a depletion of the harmful to biological membranes where the damage can antioxidant defenses, leads to a prooxidant state defined as rapidly spread. “oxidative stress.” Oxidative stress can directly damage pro- The depletion of mitochondrial ROS scavenger is a key teins, lipids, and DNA, leading to damaged macromolecules step in the pathogenesis of ROS-related liver disease. and organelles, but also deranges the redox circuits that In NASH animal model, depletion of mGSH occurs due regulate many signaling pathways [57]. In fact, while exces- to cholesterol accumulation in the mitochondrial membrane sively high levels of oxidative stress lead the cell to apoptosis, [67] that disrupts the functionality of GSH transport from a controlled increase of ROS serves as critical signaling cytosol to mitochondria. Depletion of mGSH and other molecules in cell proliferation and survival [58]. ROS can be antioxidant systems are documented in NASH patients [68]. generated by growth factor signaling through activation of ROS can also act as second messengers in cellular sig- the NADPH oxidase NOX1 or through the mitochondria. In naling oxidizing proteins on cysteine residues, resulting in turn, they can induce cellular signaling cascades by oxidation protein activation or inhibition. High levels of ROS can there- of phosphatases such as PTEN or PTP or kinases such as fore activate pathways in a signal-independent manner and Src. This leads to the activation of several pathways such as self-sustain many proproliferative pathways highly involved a Src/PKD1-dependent NF-𝜅Bactivationmechanism,MAPK in cancer and liver diseases such as NASH/NAFLD. (Erk1/2, p38, and JNK), and the PI3K/Akt signaling. Aberrant For example, it has been demonstrated that ROS can levels of ROS induce a deregulation of these pathways, directly oxidize and activate complexes such as inflam- which are involved in several pathological conditions, such masomes: protein platforms that assemble in the pres- as NAFLD [59], diabetes [60], and cancer [58, 61]. ence of exogenous or endogenous danger signals such Several different sources of ROS exist in mitochondria. as pathogen associated molecular patterns (PAMPs) and ETCcomplexIandcomplexII,aswellasothermitochondrial damage-associated molecular patterns (DAMPs) to activate enzymes such as 𝛼-ketoglutarate dehydrogenase, pyruvate and amplify inflammatory pathways [69]. Typically, inflam- phosphate dehydrogenase, fatty acyl-CoA dehydrogenase, masomes consist of a sensor (NLRs, ALRs, and TLRs), an ∙− and glycerol 3-phosphate dehydrogenase, can produce O2 adaptor (ASC), and the effector molecule caspase-1 [70]. as byproduct, releasing it within the mitochondrial matrix. Once caspase-1 is recruited and activated through autocat- Moreover, H2O2 is produced by the monoamine oxidases alytic cleavage by the inflammasome, it can proteolytically (MAOs) located in the outer mitochondrial membrane process the inflammatory cytokines IL-1𝛽 and IL-18 that lead [62, 63]. Therefore, mitochondria can produce a significant to a specialized form of cell death called pyroptosis. Pyropto- amountofROSduringOXPHOSandFAO,especiallyinthe sis causes the release of IL-1𝛽 and amplify the inflammatory context of reduced antioxidant defense such as depletion of response downstream of inflammasome activation [70]. In the mitochondrial glutathione pool [64]. the liver, inflammasomes are expressed in hepatocytes as Four main alterations are the direct result of ROS for- well as in immune cells and can also be activated by fatty mation: lipid, protein and DNA oxidation, and depletion of acids through a mechanism involving mitochondrial ROS, antioxidant molecules. decreased autophagy, and IL-1𝛽 secretion. Inflammasomes Mitochondrial DNA (mtDNA) is particularly suscep- are found overexpressed in NAFLD and NASH and their tibletooxidativedamageduetotheabsenceofprotec- silencing reduced hepatic injury, steatosis, and fibrosis [69]. tivehistones,incompleteDNArepairmechanisms,andthe Interestingly, agonists of PPAR𝛽/𝛿 were shown to reduce close proximity to ROS production site, which increase the fatty acid induced inflammation and steatosis by inhibiting risk of double-strand breaks and somatic mutations with inflammasomes [69, 71]. increased ROS production [65]. Since the ETC proteins are encoded exclusively in mtDNA, oxidative damage leads to LipidoverloadinNAFLDandNASHleadstomitochon- defective mitochondrial respiration and to a second burst drial dysfunction and increased oxidative stress, which results of ROS production that damages mitochondrial membrane from both increased electron flux through the ETC and and eventually results in loss of mitochondrial membrane depletion of the mitochondrial antioxidant defense systems potential and activation of proapoptotic pathways due to [64]. the ROS induced-ROS-release avalanche [64, 65]. Indeed, Reduced levels of GSH, SOD, and catalase as well as depletion and mutation of mtDNA have been described in increased protein oxidation, a hallmark of increased oxidative several type of liver injury, including NASH [66]. stress, are found in NASH patients [68]. Consistently, the Lipid peroxidation is the process under which lipids, mitochondria of NASH patients have altered morphology mainly polyunsaturated fatty acids, are attacked by oxidants [72, 73], reduced or mutated mDNA content [66], and such as ROS. These reactions can form a variety of pri- reduced oxidative phosphorylation capacity [74]. Oxidative mary and secondary products, among which malondialde- stress constitutes one of the key factors driving NAFLD hyde (MDA) appears to be the most mutagenic and 4- progression to NASH [75]. Indeed, histological markers of hydroxynonenal (4-HNE) the most toxic. MDA induced oxidative stress, such as oxidized phosphatidylcholine, local- mutations are involved in cancer and other genetic diseases. ize into steatotic/apoptotic hepatocytes and macrophages and 4-HNE can also act as a signaling molecule modulating many correlate with the degree of steatosis [76]. 6 PPAR Research

Depletion of mtGSH and mitochondria oxidative damage NAFLDinducedbyMCDandshort-termHFD.Interest- ℎ𝑒𝑝−/− are recapitulated also in several animal models of NASH. ingly, PPAR𝛼 mice developed steatosis and hyperc- −/− Interestingly, Llacuna and colleagues highlighted that mito- holesterolemia with aging similarly to whole body PPAR𝛼 chondrial damage in diverse animal models of NASH seemed micebutdidnotbecomeobesenorhyperglycaemic[20], to be dependent more on mitochondria cholesterol accumu- confirming that hepatocytic PPAR𝛼 deletion by itself is a lation (ob/ob mice or HFD administration), rather than only primary cause of liver steatosis. fatty acid/triglyceride overload (choline deficiency model) On the other hand, in leptin deficient (ob/ob) and [67]. Consistently, statins reduced mitochondrial damage in leptin resistant (db/db) mouse models, PPAR𝛼 expression ob/ob mice and HFD models. was found reduced, unchanged, or increased [83]. Rate of Thislineofviewisconfirmedbyarecentreporthigh- FAO also varies greatly depending on the study. While these lighting the crucial role of dietary cholesterol in delivering discrepancies could be generated by different study protocols, the “second hit” for NASH onset, in context of moderate they may be interpreted also in the light of different PPAR𝛼 dietary fat administration (45% of total calories from fat) [77]. pools that can be differentially activated in the metabolism of In this study, addition of a moderate level of cholesterol in dietary, versus adipose tissue-derived fatty acids. HF elicits the onset of hepatocellular damage and inflam- Since FA can bind and activate PPAR𝛼,thuspromoting mation through activation of the inflammasomes response, mitochondrial and peroxysomal FAO, downregulation of while neither dietary cholesterol nor HF alone produced PPAR𝛼 in NASH mice models and patients may be counter- the NASH phenotype. Importantly, addition of cholesterol intuitive. Moreover, high FAO can increase oxidative stress; to HF resulted in blunted adaptation of mitochondrial therefore stimulating PPAR𝛼 activity and FAO is somewhat metabolism to HF and markedly reduced mitochondrial expected to worsen the oxidative damage in hepatocytes. biogenesis, effects paralleled by a decrease in PGC-1𝛼 and However, it should be recalled that although mitochondria TFAM expression levels [77]. Moreover, while hepatic inflam- are potentially a major source of ROS, they are also very well mation recovered after removal of excess dietary cholesterol, equipped with antioxidant defense systems. In fact, whether mitochondrial functions remained hampered alongside ele- significant ROS production occurs in mitochondria in vivo is vated NRLP3 inflammasome protein levels, indicating slow highly debated, and the endoplasmic reticulum is currently recovery dynamics from mitochondrial damage. emerging as the major source or toxic ROS within the cell Excess accumulation of free cholesterol in mitochondrial [64]. The current view is that liver triglycerides accumulation membranes emerges as a hallmark of cellular transformation, per se does not result in inflammation [84, 85]. Rather, accu- potentially fueling the metabolic derangement required for mulation of free fatty acids, in particular saturated fatty acids cancer cell growth and resistance to apoptosis [78]. (SFA), results in marked lipotoxicity, hepatocellular damage, and inflammation [86, 87]. The onset of inflammation drives the progression from NAFLD to NASH and causes PPAR𝛼 4. PPARs and Mitochondrial Dysfunction, downregulation by TNF𝛼 [88]. Moreover, TNF𝛼 also reduces from NAFLD to HCC adiponectin levels. Adiponectin promotes FAO and blunts 𝛼 𝛼 liver gluconeogenesis signaling through AdipoR2 receptor, 4.1. PPAR . AroleforPPAR in NASH pathogenesis in 𝛼 animal models has long been established. which promotes PPAR activity [89] and depends upon −/− 𝛼 PPAR𝛼 micefedaMCDdietdevelopedmoresevere PPAR induction. Thus, inflammation-mediated disruption of the metabolic cross-talk between the adipose tissue and the NASH than WT mice, and Wy-14,643 administration com- liver may account for reduced PPAR𝛼 activity, mitochondrial pletely prevented the development of NASH in WT mice, 𝛼−/− dysfunction, and NASH development (Figure 2). A recent but not in PPAR mice [79]. The protective effect of the report by Ande and coworkers highlights the importance of 𝛼 PPAR agonist Wy-14,643 was unexpected, since the authors the inflammatory cross-talk between adipose tissue and liver, had foreseen a detrimental effect of the oxidative stress proin a sex-dependent manner, in the induction of hepatocytes 𝜔 𝛼 duced by peroxysomal -oxidation after PPAR activation. mitochondrial dysfunction, NASH, and HCC development 𝛼 However, PPAR activation also resulted in increased hepatic [90]. lipid turnover through the 𝛽-oxidative pathway, preventing This line of view is consistent with the emerging role of accumulation of lipoperoxides despite peroxysomal induc- PPAR𝛼 in the control of inflammation [12] and provides 𝛼 tion [79]. The beneficial effects of PPAR activation by Wy- additional rationale for pharmacological induction of PPAR𝛼 14,643werealsoconfirmedinasevereNASHmodelwith in NASH treatment. established fibrosis [80]. Reports on PPAR𝛼 inhumanNAFLDarescarce.Very 𝛼 PPAR deletion in mice results in mild, age and sex- recently a thorough investigation of PPARs expression in dependent, lipid accumulation in the liver [81]. Moreover, NAFLD patients was assessed by Staels’ group. The expression overnight fasting results in severe hypoglycemia, hypoke- of PPAR𝛼,PPAR𝛽/𝛿,andPPAR𝛾 was evaluated on mRNA tonemia, and increased plasma free FA levels, impaired 𝛽- −/− extracted from paired liver biopsies collected 1 year apart oxidation, and ketogenesis in PPAR𝛼 mice [19]. As a result, −/− in 85 patients. They found a significant association between HFD feeding worsens NAFLD in PPAR𝛼 mice [19, 82]. decreased PPAR𝛼 expression and histological severity of −/− More recently, the use of a hepatocytic specific PPAR𝛼 NASH. No correlation was found with PPAR𝛽/𝛿 or PPAR𝛾 mice model confirmed the protective role of PPAR𝛼 in expression [91]. PPAR Research 7

FABP CD36 LPL APOE APOA1 LDLR PPAR𝛼 PPAR𝛿 PPAR𝛾 LCFA LCFA MC/SC-FA LCFA LCFA-CoA MC/SC-FA

CPT1 ACS FASN ACS Malonyl-CoA SFA Glycolysis

Pyruvate PDK ACAC LCFA-carnitine SCD1 CoA PDH CPT2 MCFA L-carnitine MCD Acetyl-CoA SCFA MUFA LCFA-CoA 𝛽-oxidation Acetyl-CoA ACL Citrate CS CiC TCA Citrate TG cycle TG PGC-1𝛽 De novo Cytoplasm lipogenesis TG Oxaloacetate PGC-1𝛼 Lipid Mitochondrial matrix Mitochondrial Gluconeogenesis droplets + NADH NAD SIRT3 + Clock/Bmal FADH FAD ROS ROS ROS Mitochondrial dysfunctions ROS Survival/growth pathways activation − + + Activation of inflammasomes e O2 H ↓ Antioxidants − Cell transformation and e H2O ↑ mtDNA oxidation MRC cancer promotion I II IVIV II III C

Figure 2: Altered mitochondrial metabolism in NASH and HCC: role of PPARs. Altered PPARs expression drives metabolic dysfunctions in the mitochondria leading to suppression of FAO, disruption of circadian rhythms, increased ROS levels, and upregulation of de novo lipogenesis. Color-coding depicts PPAR isotypes-dependency of target genes.

The PPAR𝛼 agonists peroxisome proliferators exhibit cancer cells often have scarce OXPHOS and rely mainly liver cancerogenic activity when chronically administered on glycolysis for ATP production. Activation of PPAR𝛼 in mice. The tumor promoting activity has been related induces pyruvate dehydrogenase kinase 4 (PDK4) [97], to massive proliferation of peroxisomes, with consequent which inhibits the pyruvate dehydrogenase complex, thus oxidative stress, and to inhibition of let-7c, a microRNA preventing pyruvate from glycolysis to enter mitochondria that represses c-myc expression [92]. Long-term HCC devel- for acetyl-CoA synthesis and anaplerosis. The net result is opment was also found to be dependent with sustained the blockage of TCA and fatty acid synthesis, which requires PPAR𝛼 activation in a transgenic model overexpressing the acetyl-CoA, and the slowing-down of glycolytic rate [98]. HCV core protein [93]. However, humans are resistant to Activation of PPAR𝛼 suppresses anaplerosis from glu- peroxisome proliferation and indeed no association between tamine, by repressing the expression of glutaminase and fibrates and increased risk of any cancer has ever been found glutamate dehydrogenase, thus potentially counteracting c- [94, 95]. myc-dependent activation of glutaminolysis in tumor [97]. −/− Recently, PPAR𝛼 micewerefoundtobemoresuscep- Therefore, the transrepression activity of PPAR𝛼 on tible to DEN-induced HCC, and PPAR𝛼 anticancer activity lipid biosynthesis and anaplerosis is just as relevant as was shown to be mediated by NF-kB inhibition [96]. its transactivation activity on FAO genes. The transrepres- Interestingly, PPAR𝛼 regulation of mitochondrial sion activity of PPAR𝛼 indeed impacts on mitochondria metabolism may be exploited for cancer treatment. Many metabolism through SIRT1, by competing with ERR tran- cancer types exhibit highly glycolytic metabolism, and scriptional pathway [99]. Interestingly, Pawlak and colleagues cancer cell’s mitochondria have a strong commitment toward recently showed that the transrepression activity of PPAR𝛼 anabolism and cataplerosis. Since TCA intermediates are also regulates the inflammatory response in liver, preventing used mainly in biosynthetic reactions, mitochondria of transition from NAFLD to NASH and fibrosis, and occurs 8 PPAR Research

−/− independently on PPAR𝛼 DNA binding activity and its lipid Consistently, PPAR𝛽/𝛿 mice were prone to inflammation handling properties [100]. derived liver damage. A very recent report established a direct connection bet- In humans, PPAR𝛽/𝛿 agonists for NASH treatment are ween PPAR𝛼-driven FAO and hepatocyte proliferation. currently under investigation in clinical trials. The first evi- CyclinD1, expressed in proliferating cells and a typical pro- dence in men was obtained with GW501516, which proved to tooncogene, was found to inhibit PPAR𝛼 expression, thereby be equal to the PPAR𝛼 agonistGW590735inreducingplasma 𝛼 reducing 𝛽-oxidation, both in normal hepatocytes and in triglycerides levels and superior to the PPAR agonist in HCC cells lines. This link was confirmed also in liver after reducing cholesterol LDL, apolipoprotein B, liver fat content, 𝛽 𝛿 partial hepatectomy, where induction of CyclinD1 timed with and urinary isoprostane [112]. More recently, the PPAR / a reduction of PPAR𝛼 and its target genes [101]. agonist MBX-8025 was tested in 181 dyslipidemic patients in combination with atorvastatin or alone. MBX-8025 proved 𝛽 𝛿 𝛽 𝛿 effective in reducing apolipoprotein B levels, non-HDL- 4.2. PPAR / . As summarized above, PPAR / functions cholesterol, triglycerides, free fatty acids, and high-sensitive significantly overlap with PPAR𝛼 in peripheral tissues, while 𝛾 C-reactive protein [113]. in the liver its functions are more closely related to PPAR PPAR𝛽/𝛿-driven mitochondriogenesis has been impli- regulated processes. cated in the differentiation of hepatic-like tissue from mouse In genetic mice model of NAFLD (ob/ob), adenoviral of ES cells [41]. At the early phase of differentiation, a transi- 𝛽 𝛿 overexpression of PPAR / reduced the lipogenic program tory upregulation of PPAR𝛼 was observed, which resulted in activated by SREBP-1c, via downregulation of the SREBP- induction of PGC-1𝛼 and mitochondriogenesis. Instead, the 1c activator insig-1, thus ameliorating hepatic steatosis [102]. late phase of differentiation required a robust and sustained Conversely, increased activation of SREBP-1c was found in expression of PPAR𝛽/𝛿,whichwastimelyassociatedwith −/− PPAR𝛽/𝛿 versus WT mice, fed either a control or ethanol albumin expression and acquisition of high mitochondrial liquid diet [103], suggesting that PPAR𝛽/𝛿 may play a role in membrane potential. PPAR𝛽/𝛿 agonists L165041 promoted suppressing the lipogenic pathway trough SREBP-1c. differentiation into hepatic-like tissue that was abolished In another study, adenoviral-mediated overexpression of by PPAR𝛽/𝛿 inhibitor GSK0660 [41]. Therefore, PPAR𝛽/𝛿 PPAR𝛽/𝛿 in hepatocytes improved glucose utilization and may promote terminal hepatocyte differentiation associated hepatic insulin sensitivity. After overnight fasting, PPAR𝛽/𝛿 with acquisition of mature mitochondria metabolism and overexpressing livers had higher triglyceride and glyco- function. 𝛽 𝛿−/− gen content than wild-type mice, while fatty acids and Indeed, PPAR / mice show a delay in liver regener- cholesterol level were similar [38]. Moreover, adenoviral- ation after partial hepatectomy, associated with lack of Akt mediated overexpression in C57/BL6 mice induced SREBP-1c activation, lack of induction of glycolytic and lipogenic genes, and PGC-1𝛽 expression. PPAR𝛽/𝛿 overexpression protected and suppression of E2F transcription factors activation [114]. 𝛽 𝛿 mice liver from fatty acid overload by promoting (i) FA Interestingly, PPAR / was associated with nonprolif- conversion into nontoxic MUFA and (ii) FA storage into lipid erating hepatocytes in a gene signature analysis of nuclear droplets as triglycerides (Figure 2). As a result, activation receptor in proliferating livers and HCC [115]. The authors of inflammatory pathways by FA overload was reduced in analyzed the expression of all 49 members of the nuclear receptor superfamily in regenerating mouse liver and PPAR𝛽/𝛿 overexpressing mice fed HFD although steatosis PPAR𝛽/𝛿 (together with TR𝛼 and FXR𝛽)wasfoundcon- was increased [38]. Treatment of db/db mice with the sistently downregulated throughout the process. PPAR𝛽/𝛿 high affinity PPAR𝛽/𝛿 ligand GW501516 resulted in marked was found significantly reduced in a small series of HCC increase of genes involved in fatty acids synthesis and pentose with respect to the surrounding nontumoral tissue and the phosphate pathways, promoting FA synthesis in the liver (in PPAR𝛽/𝛿 agonist GW501516 suppressed CyclinD1 expression parallel with FA oxidation in muscle) [104]. and cell proliferation in Hepa1-6 cells [115]. However, whether These discrepancies are difficult to reconcile and might PPAR𝛽/𝛿 agonists suppress HCC cells growth is still con- be related to the different mice model used, although in troversial [116, 117]. Both PPAR𝛽/𝛿 and PPAR𝛾 have been 𝛽 𝛿 both genetic and dietary models PPAR / has been shown implicated in mediating beta-catenin-Tcf/lef signaling [118]. to either promote or inhibit liver lipogenesis. Moreover, Recently, PPAR𝛽/𝛿 was identified as a target gene of PPAR𝛽/𝛿 inhibits hepatic FGF21 expression [105], while FHL2, a tumor suppressor gene also involved in hepatocel- PPAR𝛼 is a potent activator of FGF21 [20]. Since FGF21 is lular carcinoma [119, 120]. known to inhibit SREBP-1c and several other lipogenic genes in the liver [106, 107], the potential cross-talk of different 𝛾 PPAR isotypes on FGF21 may contribute to eliciting context- 4.3. PPAR . The effectiveness of the insulin sensitizers TZD dependent effects. in ameliorating the lipidemic profile, inflammation, and Despite these striking differences, activation of PPAR𝛽/𝛿 steatosis in T2DM patients is well established. Several clinical consistently resulted in a beneficial effect on liver damage. trials have explored the potential of TZDs in the treatment of Pharmacological activation of PPAR𝛽/𝛿 has been exp- NASHandhaverecentlybeenreviewed[121,122]. lored in several rodents and human studies. Administration A recent meta-analysis of RCT on TZD and NASH (3 with of PPAR𝛽/𝛿 agonists improved hepatic steatosis and reduced pioglitazone, 1 with rosiglitazone) confirmed the effectiveness insulin resistance and hepatic inflammation [71, 108–111]. of TZD in improving steatosis, necroinflammation, and PPAR Research 9 hepatocyte ballooning [123]. A significant improvement in inducing cell-cycle arrest [145], apoptosis/anoikis [146–148], fibrosis was obtained only when the analysis was restricted to and inhibiting EMT [149, 150], angiogenesis [151], and metas- the pioglitazone studies only. Rosiglitazone failed to improve tasis [152]. necroinflammation, ballooning, and fibrosis in the 1-year However, several lines of evidence also support the FLIRT trial [124] and even when treatment was extended notion that this nuclear receptor may support the growth in for additionally 2 years [125]. Combinatory treatment of several cancer types. Conflicting results have been reported rosiglitazone with metformin or losartan did not improve the in breast cancer model. Recently, Avena et al. showed that histological endpoint versus rosiglitazone alone [126]. A very breast cancer growth was inhibited by PPAR𝛾 overexpression recent report suggests that rosiglitazone administration may epithelial cancer cells but promoted by PPAR𝛾 overexpression exert opposite outcome on liver steatosis depending on liver in cancer associated stroma [153]. The authors identify the PPAR𝛾 expressionlevels:RGZworsensteatosisinPPAR𝛾 tumor promoting role of PPAR𝛾 in the metabolic symbiosis overexpressing mice fed a HFD and protected mice with low between stoma and epithelial cancer cells, where cancer asso- PPAR𝛾 expression level [121, 127]. ciated fibroblasts provided intermediates for mitochondrial PPAR𝛾 is indeed markedly overexpressed in the liver metabolism to cancer cells [153]. Moreover, increased de novo of obese patients with NAFLD and NASH, and its expres- lipogenesis, that is promoted by PPAR𝛾,isnowrecognized sion positively correlates with plasma insulin, HOMA-IR, as a metabolic hallmark of cancer cell [154], including and SREBP1-c mRNA levels and inversely correlates with HCC [155–159] (Figure 2). Indeed, de novo lipogenesis is adiponectin [128]. High PPAR𝛾 levels, in particular of activated downstream of the Akt/mTOR pathway, one of the PPAR𝛾2, promotes de novo lipogenesis and liver steatosis most common signaling pathways altered in cancer. Forced andisassociatedwithHFDfeedinginmice[129–131]. activation of Akt/mTOR induces liver cancer [160, 161], a However as recalled above, induction of PPAR𝛾 by TZD, process mediated at least in part by activation of FASN in particular pioglitazone, ameliorates steatosis and NASH. [155, 156]. Consistently, inactivation of FASN was recently This discrepancy may be interpreted in the light of the shown to completely inhibit Akt-driven HCC in mice double nature of PPAR𝛾 target genes, which comprises both [158]. Importantly, FASN is not oncogenic per se. However, genes of de novo lipid synthesis and mitochondrial genes when the PI3K/Akt/mTOR pathway becomes hyperactive, promoting FAO [132]. Moreover, pioglitazone also binds and the induction of the de novo lipogenesis is a requisite for activates PPAR𝛼 with low potency [133], which could explain supporting cancer cell growth. Importantly, PPAR𝛾 is a direct its better performance than rosiglitazone in ameliorating transcriptional target of mTORC1 [162]. Moreover, in PTEN steatosis. Mechanistically, induction of PPAR𝛾 in steatotic null mice PPAR𝛾 was found to directly induce the expression hepatocytes may serve as a protective mechanism to reduce of key glycolytic gene HK and oncogenic PKM2, inducing liver FFA levels by storing them as less toxic triglycerides hepatocyte steatosis, hypertrophy, and hyperplasia [163]. [134, 135]. Therefore, the prosteatotic action of PPAR𝛾 [136] Therefore, PPAR𝛾 may inhibit or promote HCC devel- may not be entirely detrimental. However, excess triglyceride opment depending on the metabolic context, the cell type accumulation eventually results in hepatocyte ballooning and expressing it, the oncogenic signaling pathways involved, and necroinflammation, promoting transition to NASH. dietary or pharmacological treatment. It is however con- The role of PPAR𝛾 in hepatocellular carcinoma is still ceptually very attractive to explore the therapeutic potential debated. A large body of literature on PPAR𝛾 and cancer was interference with the cancer cell lipid handling capacity, produced using TZD, which eventually were proved to have through modulation of mitochondrial FA, ketogenesis, and several anticancer pleiotropic effects also independently of lipogenesis, as an integrated anticancer approach. PPAR𝛾 [137–140]. WeandothershaveinvestigatedtheroleofPPAR𝛾 on hepatocarcinogenesis in mice harboring a hepatocyte 5. PPARs and Circadian Regulation of ℎ𝑒𝑝−/− specific deletion of PPAR𝛾 gene (PPAR𝛾 mice). Yu and Mitochondria Metabolism colleagues found increased DEN-induced HCC in mice lack- Many processes of our metabolism and physiology are 𝛾 ing one PPAR allele, thus suggesting a tumor-suppression regulated by circadian clocks, endogenous time-tracking function for PPAR𝛾 [141]. Moreover, RGZ reduced HCC +/− systems that coordinate daily rhythms of rest, activity, feeding development in DEN-treated WT mice but not in PPAR𝛾 behavior, energy utilization, and storage. Although circadian mice [141]. Using a transgenic model of HBV-related HCC, rhythms are endogenous they respond to external stimuli, we found that RGZ or PGZ effectively reduced HCC onset which include light, temperature, and redox cycles [164]. [142]. Strikingly, TZD treatment resulted more effective in Circadian regulation is coordinated by the suprachiasmatic ℎ𝑒𝑝−/− PPAR𝛾 mice than in WT mice [142], highlighting that nucleusinthebrain,butmostperipheralorganscontain (i) TZD antitumor activity is independent of PPAR𝛾; (ii) their own independent pacemakers [165]. At a cellular level PPAR𝛾 expression reduced TZD activity; therefore in this these oscillations are driven by transcriptional feedback loops model PPAR𝛾 may support, rather than inhibiting, tumor associated with changes in chromatin remodeling, mRNA growth. processing, protein turnover, and activity [166–169]. Main As the master regulator of adipogenic differentia- factors that control circadian rhythmicity in the cells include tion, PPAR𝛾 has been described to promote differentia- BMAL1 and CLOCK (“activators”) and CRYs and PERs tion programs in a variety of tumor cell types [143, 144], (“inhibitors”). Their effects are tissue-specific and in the liver 10 PPAR Research they control approximately 10% of the transcriptome [170], mice lacking PGC-1𝛽, but this resulted in markedly decreased influencing metabolic pathways by modifying the expression activity during the dark cycle, as opposed to the hyperactive or activity of key enzymes and transporters involved in lipid, PGC-1𝛼 KO mice [190] (Figure 2). glucose, and mitochondrial oxidative metabolism. Recip- The liver-specific deletion of PPAR𝛿 in mice showed that rocally, intracellular metabolites and transcriptional factors it is involved in the temporal regulation of several lipogenic modulate CLOCK activity in response to the energy status. genes, such as fatty acid synthase (FAS) and acetyl-CoA Circadian dysregulation of lipid metabolism, ROS pro- carboxylase 1 and acetyl-CoA carboxylase 2 [40]. BMAL1 also duction, and cell-cycle control is linked to various patho- induces the expression of REV-ERB𝛼,anuclearreceptorthat logical conditions including metabolic syndrome, diabetes, downregulates BMAL1 itself, operating a negative feedback, chronic liver diseases, and cancer [171–173]. and upregulates the expression of a liver-specific microRNA: miR-122 [191]. miR-122 is also involved in lipid metabolism in mouse liver [192] and PPAR𝛿 was proven to be one of 5.1. Clock and Lipid Metabolism: Regulation of PPARs and its targets, suggesting that PPAR𝛿 plays a role in hepatic Mitochondrial Functions. The redox state of the cell also circadian regulation [193]. seems to play an important part in the rhythmicity of The circadian regulation of mitochondrial metabolism is metabolism, especially in the mitochondria. NAD+ levels stillinitsearlydays.UsingaMS-basedproteomicapproach, oscillate and are under direct control of clock transcription + the expression of rate-limiting enzymes and metabolites in factors that upregulate the rate-limiting enzyme in NAD mitochondria was quantitatively evaluated throughout the biosynthesis, NAMPT (nicotinamide phosphoribosyl trans- day [194]. Many key mitochondrial enzymes involved in ferase). In mitochondria NAD+ activates SIRT3, an impor- carbohydrates and lipid metabolism were found to peak in the tantregulatorofintrinsicmitochondrialfunctionincluding early morning period and to be regulated by PER2/3 proteins. FAO. In the cytoplasm NAD+ activates SIRT1 that operates Mitochondrial respiration displayed an oscillatory behavior, asmallfeedbackregulatingClockandBmal.Disruption peaking several times of the day. In mice KO for Per2/3, as of circadian rhythms in mice leads to defects in mtFAO well as in those fed a HFD, period protein oscillation was lost, and decreased OCR mainly through deregulation of NAD+ together with OXPHOS oscillation [194]. dependent SIRT3 activity [174, 175] (Figure 2). Several genes involved in lipid metabolism (such as SREBP, HMGCoAR, and FAS) are modulated by PPAR𝛼 and 5.2. Circadian Disturbances in Liver Disease. It is now clear display circadian fluctuations that are lost in PPAR𝛼-KO mice that circadian rhythms are fundamental in liver physiology [176, 177]. and their disruption is observed in many hepatic pathologic PPAR𝛼 is a direct transcriptional target of BMAL1 and conditions, such as NASH, NAFLD, ALD, and HCC [110, 172, CLOCK [178–180] and in the rodent liver operates a feed- 195–198]. back loop binding BMAL1 and REV-ERB𝛼 gene promoters. InamousemodelofNASHitwasfoundthatHFD BMAL1-KO and CLOCK-mutant mice display abolished induces the susceptibility to develop NASH through desyn- PPAR𝛼 oscillation and decreased expression in the liver, chronized Clock gene expression and altered cellular redox whereas PPAR𝛼-KO mice display altered oscillation of PER3 status, accompanied by reduced sirtuin abundance [197]. and BMAL1 [181]. Moreover, administration of PPAR𝛼 ago- HFD in mice is sufficient to induce the loss of circadian nists fenofibrates upregulates the expression of Bmal1 in fluctuations of insulin secretion [199]. Conversely, BMAL1 mouse liver [180]. whole body-KO mice and Clock-mutant mice display hepatic Fatty acids are known to be PPAR𝛼 activators, binding steatosis, obesity, hypoinsulinemia, and increased glucose directly to the transcriptional factor. Interestingly, hepatic intolerance [200]. fatty acids are also produced in a circadian manner by acyl- The molecular alterations found in the liver of HFD-fed CoA thioesterases (ACOTs) and lipoprotein lipases (LPLs). mice include loss of oscillation or phase advance of rhyth- The expression of both enzyme families displays circadian micity of many genes involved in lipid and mitochondrial rhythmicity; it is regulated by PPAR𝛼 and can in fact metabolism (such as NAMPT, acetyl-coenzyme A synthetase, be induced by WY14643. Moreover, silencing members of and ornithine decarboxylase 1) and gain of oscillation of other ACOTs lead to a downregulation of Cyp4a10 and Cyp4a14, genessuchasPPAR𝛾 and its targets [201]. This transcriptional PPARa targets [182–186]. reprogramming relies on changes in the oscillation and chro- Another clock controlled gene, Nocturnin, binds to matin recruitment of PPAR𝛾 that also induces the oscillation PPAR𝛾 modulating its transcriptional activity [187], and of Cidec (cell death activator CIDE-3) [201], a protein that PPAR𝛾 systemic inactivation in mice leads to impaired rhyth- is substantially elevated in the livers of the obese ob/ob micity of the canonical clock genes in liver and adipose tissues mice [202]. Administration of GW9662, a specific PPAR𝛾 [188]. PGC-1𝛼 is also rhythmically expressed in mouse liver antagonist, into HFD-fed animals produced a decrease in and muscle, upregulates circadian factors BMAL1, CLOCK, PPAR𝛾-induced Cidec expression [201]. The expression of and REV-ERB𝛼 [189], and modulates the length of circadian another known PPAR𝛾 target, pyruvate carboxylase (Pcx), an oscillations by controlling Bmal1 transcription in a REV- important regulator of hepatic gluconeogenesis, was signifi- ERB-dependent manner. Mice lacking PGC-1𝛼 show abnor- cantlyelevatedandrhythmicinliversofHFD-fedmice[201]. mal circadian rhythms and altered expression of metabolic In Nocturnin-KO mice fed with HFD, liver PPAR𝛾 oscillation genes [189]. Interestingly, circadian regulation was lost also in was abolished, accompanied by a reduced expression of many PPAR Research 11 genes related to lipid metabolism and resistance to hepatic Abbreviations steatosis [203]. ACAA2: Acetyl-CoA acyltransferase Accumulating evidence supports the importance of the 2 disruption of circadian rhythms in various types in cancer. ACADM: Medium-chain specific Specifically, in HCC patients, low expression of clock genes acyl-CoA dehydrogenase was observed in the cancerous tissue, but not in the non- ACADs: Acyl-CoA dehydrogenases cancerous liver tissue, and correlated with tumor size and ACADVL: Very long-chain specific tumor grade [204]. A number of mechanisms may explain acyl-CoA dehydrogenase the circadian control on HCC. For example, it was found ACC1 and ACC2: Acetyl-CoA carboxylase 1 thatDENexposureinmiceisassociatedwithcircadian and acetyl-CoA carboxylase disturbance, suggesting that liver clocks are involved in the 2 carcinogenesis [196]. Mutations and polymorphisms of the ACS: Acetyl-coenzyme A clock proteins are being screened to assess their association synthetase with HCC. Interestingly, a functional polymorphism of PER3 AdipoR2: Adiponectin receptor 2 wasrecentlyassociatedwithalowerriskofdeathinHCC MAL1: Aryl hydrocarbon receptor patients treated with TACE [205]. nuclear translocator-like protein 1 6. Perspectives and Conclusions CPT-1 and CPT-2: Carnitine palmitoyl transferase 1 and carnitine It is now clear that expression or activation of nuclear rec- palmitoyl transferase 2 eptors, including PPARs, is not sufficient to predict their DAMPs: Damage-associated biological output. The net effect of a nuclear receptor acti- molecular pattern vation in a given cell actually depends on the context of ERRalpha: Estrogen related receptor coactivators, corepressors, dimerization events, availability alpha of endogenous/synthetic ligands, posttranslational modifica- ETC: Electron transport chain tions, competition, and interactions with other NRs. This led FAO: Fattyacidoxidation to the development of partial agonist selective PPAR modula- FAS: Fattyacidsynthase tors (SPPARMs), a second generation of PPAR agonists able FAT/CD36: Fatty acid translocase to selectively activate a subset of target genes downstream a FHL2: Four and a half LIM domains protein 2 specific PPAR isotype. K-877 is a SPPAR𝛼M currently being tested in dyslipi- FXR: Farnesoid X receptor demic patients that exhibits higher lipid lowering activity GK: Glycerol kinase than fibrates and has a favorable risk profile [206, 207]. GLUT2: Glucose transporter 2 INT-131, SPPAR𝛾M, has potent glucose lowering effects not GLUT-4: Glut transporter 4 associated with TZD side-effects [208]. HADHA and HADHB: Trifunctional enzyme AdifferentapproachtoPPARmodulationistosimultane- subunit alpha and beta ously activate, with different potency, more than one isotype: H-FABP: Heart-type fatty dual PPAR agonist or pan-agonists are currently under acid-binding protein investigation. The dual PPAR𝛼/𝛿 agonist GFT-505 is proving HMGCoAR: 3-hydroxy-3- effective in reducing plasma triglyceride levels, improving methylglutaryl-CoA insulin sensitivity, and increasing HDL-cholesterol in obese reductase patients [209, 210] and showed promising results in mice HNF-4: Hepatocyte nuclear factor model of NASH [211]. Very recently a phase 2 multicenter 4-alpha randomized controlled trial, enrolling 274 subjects with IRS-1, IRS-2: Insulin receptor substrate 1 histologically proven NASH, showed that GFT505 produces and insulin receptor a dose-dependent improvement in histology of patients with substrate 2 NASH [212]. JNK: Jun N-terminal kinase As we gain knowledge of the metabolic circadian regu- LC-FA: Long-chain fatty acids lation and of its disruption in disease, an entire new area LPL: Lipoprotein lipase of intervention begins to emerge. Modulation of amplitude LXR: Liver X receptor and phase of PPARs circadian regulation could be exploited M/S-FA: Medium/short chain fatty to drive complex metabolic remodeling of mitochondrial acids metabolism in NASH and cancer models. Finally, the integra- MAPK: Mitogen-activated protein tion of the above-mentioned approaches with the metabolic kinase 1 and genetic profiling of cancers holds the promise for new MCD: Methionine and choline therapeutic approaches that can selectively target the fuel deficient diet requirements of HCC. MDA: Malondialdehyde 12 PPAR Research

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Review Article PPAR𝛿 as a Metabolic Initiator of Mammary Neoplasia and Immune Tolerance

Robert I. Glazer

Department of Oncology, Georgetown University Medical Center and the Lombardi Comprehensive Cancer Center, 3970 Reservoir Rd, NW, Washington, DC 20007, USA

Correspondence should be addressed to Robert I. Glazer; [email protected]

Received 19 July 2016; Accepted 3 November 2016

Academic Editor: Stefano Caruso

Copyright © 2016 Robert I. Glazer. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PPAR𝛿 is a ligand-activated nuclear receptor that regulates the transcription of genes associated with proliferation, metabolism, inflammation, and immunity. Within this transcription factor family, PPAR𝛿 is unique in that it initiates oncogenesis in a metabolic and tissue-specific context, especially in mammary epithelium, and can regulate autoimmunity in some tissues. This review discusses its role in these processes and how it ultimately impacts breast cancer.

1. Introduction 2. PPAR𝛿 and Tumorigenesis ThePPARnuclearreceptorfamilyconsistsofthePPAR𝛼, The role of PPAR𝛿 in tumorigenesis has been investigated PPAR𝛾,andPPAR𝛿/𝛽 isotypes, which function as het- for almost two decades, and whether it exerts an oncogenic erodimeric partners with RXR with specificity dictated by or antioncogenic role depends in large part on the targeted high-affinity binding of PPAR ligands and coactivators [1]. tissue and the gene targeting strategy utilized [14–16]. In the Similar to other nuclear receptors, PPARs contain an N- context of the mammary gland, however, most animal models terminal transactivation domain, a DNA-binding domain, a confirm that PPAR𝛿 exerts an oncogenic effect. This can be ligand-binding domain, and a C-terminal ligand-dependent envisioned to result in part from competition between the transactivation region [2]. PPARs bind to a DR-1 response tumor promoting effects of PPAR𝛿 and the tumor suppressor element (PPRE) with the consensus sequence AGG(T/A)CA effects of PPAR𝛾.PPAR𝛾 agonists reduce mammary carcino- that is recognized specifically by the PPAR heterodimeric genesis [17–19], which correlates with induction of PTEN partner [3]. Ligand-activated PPARs interact with coactiva- [20, 21] and BRCA1 [22] tumor suppressor activity, as well as tors CEBPA/B and NCOA3 and in the unliganded state with reduction of inflammation via the Cox2/Ptgs2 pathway [23]. corepressor NCOR2 [4–7]. Of the three isotypes, PPAR𝛿 Conversely, PPAR𝛾 haploinsufficiency [23] or expression plays a dominant role in regulating fatty acid 𝛽-oxidation, of a dominant-negative Pax8-PPAR𝛾 transgene [24] and glucose utilization, cholesterol transport, and energy balance direct or indirect inhibition of PPAR𝛾 [21, 25] enhance [8–10]butalsomodulatesthecellcycle,apoptosis,angiogene- DMBA mammary carcinogenesis. In MMTV-Pax8-PPAR𝛾 sis, inflammation, and cell lineage specification [11–14]. These mice, the increased rate of carcinogenesis correlates with multifaceted functions indicate that PPAR𝛿 has a critical enhanced Wnt, Ras/Erk, and PDK1/Akt signaling, reduced homeostatic role in normal physiology and that its aberrant PTEN expression, and a more stem cell-like phenotype [24]. expression can impact the initiation and promotion of onco- The respective Yin/Yang functions of PPAR𝛿 and PPAR𝛾 genesis. This review discusses recent advances pertaining to are consistent with the ability of PPAR𝛿 to enhance sur- the involvement of PPAR𝛿 in these processes primarily as vival through the PI3K and PDK1 pathways in response to they relate to mammary tumorigenesis. wound healing [26, 27], as well as with the proliferative and 2 PPAR Research

PPARd in mammary epithelium + adipose PLA2 PLD LPA microenvironment PC LPC

PTGS2 PLD PI3K S100A8/9, SAA1/2/3 Prostaglandins AA (20 : 4) MMP12, KLK6/11 PDK1 OLAH ACSL4 PA

Inflammation AKT Inflammation FA-CoA Invasion, ECM Malonyl CoA Acylcarnitine RAD001 mTOR PLAC1

S6 AcCoA AcCoA Macrophages, Treg,

MDSC, fibroblasts Citrate Tumor growth

Immune tolerance

Figure 1: Interactions between inflammation, metabolism, and mTOR signaling in the mammary gland of MMTV-PPAR𝛿 mice. PPAR𝛿 activates PPRE-containing genes associated with metabolism (Olah, Ptgs2, Pla2, and Pld), invasion (Mmp12, Klk6), and inflammation (S100a8/9, Saa1/2/3). Arachidonic acid (AA) is a substrate for Ptgs2 and is a constituent of phosphatidylcholine (PC) required for prostaglandin synthesis. Lysophosphatidylcholine (LPC) is generated from PC by phospholipase A2 (Pla2), and lysophosphatidic acid (LPA) and phosphatidic acid (PA) are generated by phospholipase D (Pld). LPA stimulates mTOR through a G protein-coupled receptor, and PA directly activates mTOR. The mTOR inhibitor RAD001 (everolimus) inhibits tumorigenesis in this animal model. The net result is an increase in inflammation, extracellular matrix remodeling, immune suppression, and neoplasia. Adapted from [31]. angiogenic response of breast cancer and endothelial cells to theirreversiblePPAR𝛾 inhibitor, GW9662 [25]. These find- conditional activation of PPAR𝛿 [28]. The induction of PDK1 ings support the notion that PPAR𝛾 and PPAR𝛿,eitherby signaling by the PPAR𝛿 agonist GW501516 in DMBA-treated direct competition [36], cofactor competition [37], and/or wild-type mice [19], the increased expression of PPAR𝛿 in ligand-dependent activation [38] have opposing actions that + GW501516-treated MMTV-PDK1 mice [29], and reduction of affect expansion of the ER lineage tumor subtype. Interest- + mammary tumorigenesis in MMTV-Cox2 mice crossed into ingly, ER tumors also arose in MMTV-NCOA3 mice [39, aPPAR𝛿 null background [30] further support its oncogenic 40], but not in other MMTV-driven transgenic models [41], potential. This outcome was ultimately proven by the gener- suggesting that it is the PPAR𝛿 coactivator complex itself, ation of MMTV-PPAR𝛿 mice, which developed infiltrating rather than the MMTV promoter that drives expansion of the + mammary adenocarcinomas and whose progression was ER lineage.Thisconclusionisalsosupportedbythesimilar- accelerated by, but not dependent on, agonist stimulation [31]. ities between MMTV-NCOA3 and MMTV-PPAR𝛿 mice for From a clinical perspective, this result is concordant with activation of the mTOR signaling axis [39, 40], suggesting its + the increased expression of PPAR𝛿 in invasive breast cancer importance in ER luminal tumor specification. [12, 32] and by manifestation of a PPAR𝛿 signaling network Another intriguing feature of MMTV-PPAR𝛿 mice is the that predicts poor survival in this disease [33]. association between the onset of neoplasia and the upregula A signature feature of MMTV-PPAR𝛿 mice is the devel- tion of Plac1 [31], a microvillous membrane protein expressed + + − opment of ER /PR /ErbB2 tumors resembling the luminal primarily in trophoblasts, but not in most somatic tissues [42] B subtype of breast cancer [31], which is denoted by lower (Figure 1). Plac1 is reexpressed in several malignancies [43– ER expression, higher Ki-67 staining, and a higher histologic 45], and reduction of Plac1 in breast cancer cells inhibits grade [34]. Since ER mRNA is relatively low in these mice proliferation and invasion [43]. These findings suggest that in comparison to immunohistochemical staining, it suggests Plac1 may serve as a diagnostic biomarker as shown by the that PPAR𝛿 may affect ER stability posttranslationally, for more favorable prognosis of colorectal cancer patients ex- example, phosphorylation of ER Ser167 by mTOR/S6K [35], a pressing Plac1 autoantibodies [46]. Analysis of a limited set of pathway activated in this mouse model (Figure 1). The devel- paired breast cancer specimens indicates that Plac1 express- + opment of ER tumors in MMTV-PPAR𝛿 mice is similar to ioniselevatedinthemajorityofbiopsies,butnotinadjacent what was observed in DMBA-treated MMTV-Pax8-PPAR𝛾 normal tissue (Isaacs and Glazer, unpublished results), which mice [24] and DMBA-treated wild-type mice administered is consistent with the presence of circulating Plac1 RNA in PPAR Research 3 themajorityofbreastcancersubjects[43,44].Thehighlevel oxidation that increases unsaturated fatty acid, arachidonic of expression of Plac1 in MMTV-PPAR𝛿 mice also suggests acid, LPA, and PA biosynthesis in MMTV-PDK1 mice [29, that Plac1 may be under the transcriptional control of 31] and is in accordance with the capacity of long chain PPAR𝛿 as demonstrated by its dependence on the PPAR𝛿 unsaturated fatty acids to serve as endogenous PPAR𝛿 ligands coactivators CEBPA and CEBPB [47] and the presence of [50–52]. Additionally, PPAR𝛿 upregulates the fatty acid- PPREs in the promoter regions of mouse and human Plac1 binding protein (FABP) gene family [68], which facilitate fatty (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PLAC1 acid transport and potentiate EGFR- and ErbB2-mediated &keywords=plac1). proliferation [69, 70] and invasion [71]. Lastly, PPAR𝛿 and fatty acid oxidation are required to maintain asymmetric + 3. PPAR𝛿 and Inflammation stem cell division [72], an area that may be linked to ER tumor specification and one unexplored thus far in mammary One of the earliest recognized functions of PPAR𝛿 was tumorigenesis. its antiapoptotic, chemotactic, and inflammatory actions mediated through the Akt and Rho pathways in response to 5. PPARs and Immune Tolerance wound healing in keratinocytes [26, 27, 48]. This was the first indication that PPAR𝛿 might be a contributing factor to One of the primary mechanisms associated with cancer inflammatory disorders, such as psoriasis [49], and tumori- progression is the coopting of immune tolerance to produce genesis. It had been previously shown that inflammatory an immunologically permissive tumor microenvironment molecules, such as eicosanoids, could serve as endogenous [73]. This can occur through several mechanisms associated PPAR𝛿 ligands [50–52]. In colon tumorigenesis and colitis, with adaptive immunity, including expansion of tumor infil- Ptgs2 and prostaglandin synthesis are dependent on PPAR𝛿 trating regulatory T cells (Tregs), myeloid-derived suppressor [53, 54], whereas inhibition of tumorigenesis by NSAIDs cells (MDSC), and tumor-associated macrophages (TAM) results from induction of the endogenous PPAR𝛿 antagonist, [74, 75] (Figure 2). Tregs contribute to immune escape by 13-S-hydroxyoctadecadienoic acid [55]. Of note is that a sim- activation of the programmed cell death protein-1 (PD-1) ilar Ptgs2/prostaglandin phenotype is expressed in MMTV- receptor through immune and tumor cell expression of its PPAR𝛿 mice (Figure 1) [31], which is consistent with the ligand, PD-L1 (not shown), which results in suppression of + induction of mammary tumorigenesis in MMTV-Ptgs2 mice effector T cell function mediated by CD4 helper T cells + [56], but not in PPAR𝛿-null mice [30]. These findings suggest and CD8 cytotoxic T cells. MDSC also differentiate into a feed-forward mechanism, whereby transactivation of the TAM with similar T cell inhibitory properties [76], a process prostaglandin E2 receptor, Ptger4, by PPAR𝛿 [57], coupled driven by inflammatory Th2 cytokines, which ultimately with the generation of arachidonic acid by phospholipase leads to tumor progression. Although there are numerous A2 [58] and the biosynthesis of prostaglandin E2 (PGE2)via studies of these pathways in immune tolerance, the role of Pges2, elicits a self-sustaining inflammatory response. PPAR𝛿 in this process has not been examined in mammary In addition to activation of the prostaglandin axis, PPAR𝛿 tumor models. Nevertheless, a clue as to its functional increases expression of the acute phase proteins Saa1, Saa2, role in adaptive immunity may be gleaned from studies in S100a8,andS100a9,aswellasseveralmembersofthe diabetic obese mice. In liver and adipose tissue, PPAR𝛿 is + kallikrein gene family [31], all of which are elevated in ER required to maintain insulin sensitivity via Th2 cytokines, breast cancer [59, 60] and whose promoter regions contain which promote M2 macrophage polarization [77, 78] that PPREs. S100a8 and S100a9 are ligands for Ager (advanced have the characteristics of TAMs, and promotes tolerance glycation end-product receptor), another PPAR-dependent to “self” recognition [79] to prevent diabetes. This suggests gene that mediates acute and chronic inflammation, tumor that PPAR𝛿 may play a similar role in tumorigenesis, but development, and metastasis in several types of cancer and with a decidedly different outcome. As discussed in Section 2, proliferative disorders [61, 62], including gastric carcinogen- PPAR𝛿 regulates the inflammatory Saa1/2/3 and S100a8/9 esis [63] and psoriasis [49]. Thus, there is strong evidence to pathways, which in tumor-bearing mice are associated with implicate PPAR𝛿 in driving multiple inflammatory pathways MDSC expansion [80] and metastasis [81]. Immune tolerance implicated in tumorigenesis. mediated by Tregs, MDSC, and TAM are dependent on PGE2 synthesis, reactive oxygen species generated by NADPH 4. PPAR𝛿 and Metabolism oxidase (NOX1), and tryptophan depletion by indoleamine 2,3 dioxygenase (IDO) [74] (Figure 2), all of which are PPAR𝛿 is one of the primary regulators of intermediary under the transcriptional control of PPAR𝛿.MDSCand metabolism, including fatty acid synthesis and 𝛽-oxidation, Treg infiltration of mammary tumors is dependent on PGE2 + particularly in adipose and muscle tissue [13, 64]. In synthesis and IDO activation [82], and inhibition of CD8 MMTV-PPAR𝛿 mice, PPAR𝛿 functions as an integrator T cell activation via the PD-1/PD-L1 axis is dependent on of metabolism and tumorigenesis via the biosynthesis of mTOR activation [83], a pathway that is activated in MMTV- lysophosphatidic acid (LPA), a metabolite that promotes PPAR𝛿 mice [31]. Since the transcriptions of ARG1, IDO2, mammary tumorigenesis [65, 66], and phosphatidic acid inducible nitric oxide synthetase (NOS2), Ptgs2, Ptger4, and (PA), a metabolite that directly activates mTOR [67] (Fig- NOX1 are all regulated by the coactivators CEBPA/B, which ure 1). The LPA/PA signaling pathway is also coupled to also function in this capacity with PPAR𝛿,thissuggestsa expression of Pdk4, a PPAR𝛿-regulated inhibitor of pyruvate mechanism whereby PPAR𝛿 may control adaptive immunity 4 PPAR Research

Effector T cell Treg

MDSC

Tumor

TAM

Figure 2: Metabolic interactions between tumor, stromal, and immune cells in the tumor microenvironment. Tumor and stromal cells express ARG, IDO, Cox2/Ptgs2, and iNOS/NOS2, which produce reactive oxygen species (ROS), chemokines, and Th2 cytokines that recruit Tregs, MDSC, and tumor-associated macrophages (TAM) to block effector T cell activation. Adapted from [84].

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Review Article PPAR Gamma in Neuroblastoma: The Translational Perspectives of Hypoglycemic Drugs

Serena Vella,1 Pier Giulio Conaldi,1,2 Tullio Florio,3 and Aldo Pagano4,5

1 DepartmentofLaboratoryMedicineandAdvancedBiotechnologies,IRCCS-ISMETT(IstitutoMediterraneoperiTrapiantie TerapieadAltaSpecializzazione),Palermo,Italy 2Fondazione Ri.MED, Palermo, Italy 3Section of Pharmacology, Department of Internal Medicine (DiMI) and Center of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy 4Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy 5IRCCS-AOU San Martino-IST, Genova, Italy

Correspondence should be addressed to Serena Vella; [email protected]

Received 2 August 2016; Accepted 14 September 2016

Academic Editor: Daniele Fanale

Copyright © 2016 Serena Vella et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neuroblastoma (NB) is the most common and aggressive pediatric cancer, characterized by a remarkable phenotypic diversity and high malignancy. The heterogeneous clinical behavior, ranging from spontaneous remission to fatal metastatic disease, is attributable to NB biology and genetics. Despite major advances in therapies, NB is still associated with a high morbidity and mortality. Thus, novel diagnostic, prognostic, and therapeutic approaches are required, mainly to improve treatment outcomes of high-risk NB patients. Among neuroepithelial cancers, NB is the most studied tumor as far as PPAR ligands are concerned. PPAR ligands are endowed with antitumoral effects, mainly acting on cancer stem cells, and constitute a possible add-on therapy to antiblastic drugs, in particular for NB with unfavourable prognosis. While discussing clinical background, this review will provide a synopsis of the major studies about PPAR expression in NB, focusing on the potential beneficial effects of hypoglycemic drugs, thiazolidinediones and metformin, to reduce the occurrence of relapses as well as tumor regrowth in NB patients.

1. Introduction region, often in the adrenal medulla. Other common sites of disease include the neck and head (5%), chest (20%), and (1) Neuroblastoma. Neuroblastoma (NB) is a tumor of the pelvis (5%) [1]. developing sympathetic nervous system observed in early NB is a disease of the sympaticoadrenal lineage of the childhood, which is characterized by a broad spectrum of neural crest and originates from neuroblasts in the develop- clinical behaviors, ranging from complete regression to death. ing peripheral nervous system [2]. NB represents the second most common extracranial In recent years, it has been suggested that NB tumori- malignancy of childhood, accounting for 8 to 10% of all genesis is dependent on the presence of cancer stem cells childhood cancers (NB prevalence is about one case in 7,000– (CSCs), which have been also isolated from NB cell lines 10,000 live births) and for approximately 15% of the pediatric [3, 4]. CSCs are thought to be also responsible for metastasis deaths for malignant conditions [1]. and recurrence in NB patients [5, 6]. The clinical presentation of NB ranges from asymp- Cellular heterogeneity is a hallmark of NB nodules and tomatic masses to primary tumors that cause critical illness the prognosis of these tumors depends on their differentia- due to local invasion and/or widely disseminated disease. tion levels [7]. Interestingly, cell lines established from several Most primary NB (65%) usually present in the abdominal human NB retain similar cellular heterogeneity. 2 PPAR Research

Biedler et al. described three cell subtypes, often dis- PPARs are activated by fatty acids, eicosanoids, other cernible also in NB cell line cultures, based on cell morphol- dietary lipids, and their metabolites, or synthetic ligands [40], ogy, biochemical features, and growth patterns [8]: (i) N-type which have been pharmacologically used in several diseases, (neuroblastic: aggregated, poorly attached, and rounded cells making PPARs attractive therapeutic targets. with short neurites); (ii) S-type (substrate-adherent and non- There are three PPAR isoforms (𝛼, 𝛽/𝛿,and𝛾)which neuronal cells); and (iii) I-type (intermediate: mildly adher- are encoded by separate genes [41] and are expressed during ent cells, showing marked stem-like traits, representing can- different stages of prenatal development [42]. cer stem-like cells population, and being thought to originate Through the regulation of the expression of multiple both S- and N-type cells) [7]. genes [43], PPARs control several physiological processes, Several studies have shown that these cell types derive including cell proliferation, morphogenesis, differentiation, from a common precursor and are able to bidirectionally and cellular homeostasis [44, 45], and have been implicated differentiate. This bidirectional conversion between well- in different human diseases such as hyperlipedimia, diabetes, defined differentiation lineages of the neural crest has been obesity, inflammation, neurodegenerative disorder, cardio- termed “transdifferentiation” [9]. vascular diseases, and cancer [46–49]. Because the transdifferentiation process is able to also Although all PPAR isoforms display a partially overlap- allow the differentiating of malignant CSCs into benign ping spectrum of activity, essentially as far as the control of phenotype, a novel concept in cancer biology was intro- lipid and energy metabolism is concerned, they differ in tissue expression pattern and functional roles [50–52]. duced: “induction of differentiation” as possible treatment 𝛼 (e.g., using retinoids to treat NB and acute promyelocytic PPAR- is predominantly expressed in metabolically leukaemia [10]). active tissues, such as liver, skeletal muscle, heart, intestinal mucosa, brown adipose tissue, adrenal gland, pancreas, and The cause of NB development is still unclear occurring kidney. This receptor regulates catabolism of fatty acids and mostly as sporadic disease but also rare (about 1% of all cases) promotes lipolysis and fatty acid oxidation [53–56]. familial cases were reported [1]. Genomic alterations are PPAR-𝛼 endogenous ligands (fatty acids and several fatty- associated with NB development and/or progression, many of acid-derived compounds) or synthetic pharmacological ago- whichhaveproventobecorrelatedwithclinicaloutcome.The nists (fibrate drugs, WY14643 and GW7647) have been iden- most widely studied cytogenetic alterations, associated with tified[57],andsomeofthemarecurrentlyusedforthetreat- poor outcome in NB, include N-myc oncogene amplification, ment of hypertriglyceridemia and cardiovascular diseases loss or rearrangement of the distal portion of the short arm [58]. of chromosomes 1 (1p31-term), 3 (3p22), and 11 (11q23), gains PPAR-𝛽/𝛿 is ubiquitously expressed particularly in liver, of chromosome arm 1q or 17q, and the expression of the intestine, kidney, abdominal adipose tissue, skeletal muscle, TrkB neurotrophin receptor and its ligand [34–38]. Other and macrophages. It regulates energy expenditure, participat- cytogenetic and molecular abnormalities are likely involved ing in fatty acid oxidation and regulating blood cholesterol in NB pathogenesis and their identification could be useful concentrations and glucose levels [41, 54, 59]. for diagnosis, prognosis, and therapy of NB patients. PPAR-𝛽/𝛿 agonists are prostacyclin PGI2, oleic acid, and Traditional NB treatments include surgery, chemother- synthetic agents, such as GW501516, GW7842, and GW0742, apy, radiotherapy, and biotherapy [34]. However, the majority which attenuate hepatic steatosis [60]. of NB patients (50%) have poor outcomes and relapses, PPAR-𝛾 is expressed within adipose tissue, the large remaining a clinical challenge. intestine, spleen, skeletal muscle, liver, pancreas, endothelial Unfortunately, in many cases, by the time of diagnosis, cells, immune cells, various cancer cells, and brain [61–63]. the disease has usually spread already. In these cases, the It regulates energy storage and has a key role in fatty mainstay treatment is frequently intensive regimens includ- acid metabolism and glucose homeostasis [55, 64–66], ing combinations of high doses of chemotherapeutics [39] mitochondrial biogenesis, and ROS metabolism [67, 68]. that often are accompanied by unacceptable high toxicities Many lipids, including eicosanoids and the cyclopentenone and no long-term improvements. Innovative approaches are prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15-deoxy- therefore needed for this disease. The new treatment proto- PGJ2), are endogenous PPAR-𝛾 ligands, while synthetic ago- cols for NB currently under investigation consist of targeted nists include thiazolidinediones (TZDs), GI262570, GW1929, radiotherapy and retinoid compounds (to induce terminal and GW7845 [69]. PPAR-𝛾 has been extensively studied as a differentiation of NB cells), immunological treatment, such pharmacological target in several diseases. as using antidisialoganglioside 2 with or without associa- TZDs are the best-characterized pharmacological PPAR- tion with cytokines (GM-CSF, IL-2), antiangiogenics, neuro- 𝛾 agonists, and, among them, pioglitazone and rosiglitazone trophin-signaling inhibitors, proapoptotic agents, allogeneic have been approved by FDA for treatment of type II diabetes haemopoietic stem cell transplantation, and new chemother- [70–72]. apeutics [34, 39]. In addition, another oral hypoglycemic drug, metformin, (2) PPARs. Peroxisome proliferator-activated receptors which directly improves insulin action, modulating AMPK (PPARs) are ligand-activated transcription factors belonging activity (a key energy regulator), increases PPAR-𝛾 mRNA to the nuclear receptor superfamily. levels [73], acting similarly to rosiglitazone [74]. PPAR Research 3

2. PPARs in Neuroblastoma GW1929 prodifferentiating effect was shown to be dependent on PPAR-𝛾 activation, as demonstrated by the use of Interestingly, it has been suggested to use PPARs as target for specific antagonists [96]. cancer treatment, and several PPAR agonists, in particular 𝛾 In2005,Valentineretal.testedtheeffectsoffourTZDs acting on PPAR- , represent promising therapeutic tools as (ciglitazone, pioglitazone, troglitazone, and rosiglitazone) in 𝛾 antitumoral agents [75]; PPAR- agonists were reported to seven NB cell lines (i.e., Kelly, LAN-1, LAN-5, LS, IMR-32, inhibit cell growth and to induce apoptosis in several cancer SK-N-SH, and SH-SY5Y) [17]. All the ligands, in particular cell lines in vitro and in vivo [76–86], including NB cells ciglitazone and rosiglitazone, inhibited cell proliferation and [87, 88]. viability in a dose-dependent manner, with different drug AllthreePPARsisoformshavebeenidentifiedinNB, effectiveness among cell lines. Moreover, drug potency was although human NB cell lines express PPAR-𝛼 (mRNA or not related to PPAR-𝛾 protein amount in NB cell lines, proteins) at very low level [11, 89–91], and PPAR-𝛽/𝛿 expres- but rather to various cellular conditions associated with the sion data are still incomplete [11, 92]. Conversely, PPAR-𝛾 is receptor function. highly expressed in NB cell lines [11, 12] and in primary NB The antiproliferative effect of rosiglitazone was confirmed cell cultures [75], being mainly localized in the nuclei rather bythesamegroupin vivo, in a metastatic xenograft mouse than in the cytoplasm and being particularly expressed in model [18], although its antitumor effect was very limited. cells showing ganglionic differentiation [11, 89]. Ciglitazone was also used in association with 15-deoxy- In addition, it has been documented that embryonic rat PGJ2 to overexpress Rb protein and inhibit PPAR-𝛾 activity, brain and neural stem cells have higher concentration of reducing NB cell growth [13]. PPAR-𝛾 than adult rat brain [42, 93]. In neural stem cells, Servidei et al. tested 15-deoxy-PGJ2 and rosiglitazone on PPAR-𝛾 is involved in the regulation of proliferation and 8 NB cell lines, with different phenotypes, including N- and differentiation [94]. S-types [12]. The two PPAR-𝛾 ligands inhibit cell growth in Interestingly, PPAR-𝛾 expression is correlated to the mat- all cell lines, and the sensitivity seems to be more associated 𝛾 urational stage of NB and therefore to NB patients’ outcome with the cell phenotype than with PPAR- expression: indeed, [85], and PPAR-𝛾 agonists induce NB cell differentiation, N-type cells are more susceptible to treatment than S-type cells, partly because of their higher capability of undergoing inhibiting proliferation, neurite outgrowth, and reducing N- apoptosis. myclevels[11]. Many studies have documented that the inhibitory effects 2.1. PPARs Agonists in Neuroblastoma. Several studies have of TZDs on neuroblastoma cell growth are partially due to 𝛾 an increase of apoptosis. Indeed, troglitazone induced PPAR- assessed the activity of PPAR- in NB, evaluating the effects of 𝛾 several natural or synthetic ligands on cellular proliferation, -dependent apoptosis in NB-1 cells [19] and in SHEP NB [20].Incontrast,onlytwostudies,toourknowledge,reported apoptosis, and differentiation (Table 1). antiapoptotic effects of rosiglitazone which protected NB cells 15-deoxy-Δ12,14-prostaglandin J2 (15-deoxy-PGJ2), a + 𝛾 subjected to MPP -induced mitochondrial injury reducing high-affinity natural ligand of PPAR- ,inhibitsin vitro ROS production [21, 22]. growth and induces apoptosis in NB cells [11–16], through Proapoptotic effects of rosiglitazone were also reported PPAR-𝛾-dependent ERK2 activation, although PPAR-𝛾-inde- [23]. This PPAR-𝛾 ligand significantly inhibits cell adhesion pendent effects of 15-deoxy-PGJ2 have been also described and invasiveness and induces apoptosis, more effectively in [14]. SK-N-AS than in SH-SY5Y cell lines. The distinct response In addition, Rodway et al. have found that the inhibition ofthetwoNBcelllinesislikelyduetoareducedphospho- of NB growth induced by 15-deoxy-PGJ2 can be reduced rylation of PPAR-𝛾 and consequently its increased activity by the presence of serum lysolipids in the culture medium in SK-N-AS cells. Cellai and colleagues also evaluated the [14], while Emmans et al. reported that the degree of PPAR in vivo effect of TZDs in NB xenograft models, confirming activation, due to 15-deoxy-PGJ2, in a NB cell line, is atten- their previous in vitro observations [24]. Indeed rosiglitazone uated in the presence of the retinoblastoma protein (Rb) and (150 mg/kg/day) for 4 weeks significantly reduced tumor restored by treatment with the histone deacetylase inhibitor growth (−70%) as compared to control mice [24]. trichostatin A (TSA). The combination treatment with 15- In addition, rosiglitazone induces differentiation, increas- deoxy-PGJ2 and TSA enhances the inhibition of NB growth, ing density of dendritic spines in rat primary cortical neurons suggesting a synergistic activity of the two compounds [13]. [25]. Furthermore, 15-deoxy-PGJ2 promotes NB cell differenti- Moreover, in neural stem cells (NSC) from adult mam- ation, which may be mediated by the p38 MAP kinase malian brain, pioglitazone and rosiglitazone directly regulate activation and the AP-1 signaling pathway [95]. proliferation, differentiation, and migration [26]. Synthetic PPAR-𝛾 ligands have been also tested to con- Accordingly, Miglio et al. described the effects of piogli- trast NB cell growth. tazone on SH-SY5Y NB cells, in which this agonist promotes Han and coworkers firstly evaluated the effect of the differentiation and outgrowth of cell processes, in a dose- synthetic ligand, GW1929, in the NB cell line LA-N-5, and dependent manner [27]. In 2014, Chiang et al. evaluated found that this compound induces cell differentiation and the effects of rosiglitazone in the mouse NB Neuro 2a inhibits proliferation [11]. (N2A)cellline.Thisagoniststimulatesneuriteoutgrowthand 4 PPAR Research

Table 1: Preclinical and experimental studies on PPAR agonists in neuroblastoma.

Study Drug/s Reference/s Year Target Cell lines/animal model Effects types Inhibition of growth and 2001, NB cell lines and primary apoptosis induction, through 15-deoxy-PGJ2 [11–16] 2003, PPAR-𝛾 In vitro cultures of cortical neurons PPAR-𝛾-dependent and 2004 PPAR-𝛾-independent effects. Prodifferentiating effect and GW1929 [11] 2001 PPAR-𝛾 In vitro LA-N-5 inhibition of proliferation. Rosiglitazone SH-SY5Y, SH-EP1, SK-N-AS, Inhibition of cell growth with and [12] 2004 PPAR-𝛾 In vitro SK-N-FI, LA-N-5, SMS-KCNR, different sensitivity related to the 15-deoxy-PGJ2 SK-N-DZ, and LA-N-1 cell phenotype. Overexpression of Rb protein Ciglitazone and SK-N-AS, IMR-32, SK-N-SH, [13] 2004 PPAR-𝛾 In vitro andinhibitionofPPAR-𝛾 15-deoxy-PGJ2 and ND-7 activity, reducing NB cell growth. Ciglitazone, pioglitazone, Inhibition of cell proliferation Kelly, LA-N-1, LA-N-5, LS, troglitazone, [17] 2005 PPAR-𝛾 In vitro and viability in a dose-dependent IMR-32, SK-N-SH, and SH-SY5Y and manner. rosiglitazone SK-N-SH xenograft NB mouse Rosiglitazone [18] 2010 PPAR-𝛾 In vivo Inhibition of tumor growth. model Increase of PPAR-𝛾-dependent Troglitazone [19] 2002 PPAR-𝛾 In vitro NB-1 cell line apoptosis. Increase of PPAR-𝛾-dependent Troglitazone [20] 2006 PPAR-𝛾 In vitro SHEP NB cell line apoptosis. Antiapoptotic effects of rosiglitazone which protected NB 2006, + Rosiglitazone [21, 22] PPAR-𝛾 In vitro SH-SY5Y cell line cells subjected to MPP -induced 2007 mitochondrial injury reducing ROS production. Inhibition of cell adhesion, Rosiglitazone [23] 2006 PPAR-𝛾 In vitro SK-N-AS and SH-SY5Y cell lines invasiveness, and proapoptotic effects. Significant decrease of tumor SK-N-ASxenograftNBmouse Rosiglitazone [24] 2010 PPAR-𝛾 In vivo growth (−70%) as compared to model control mice. Induction of cell differentiation, Rosiglitazone [25] 2008 PPAR-𝛾 In vitro Rat primary cortical neurons increasing dendritic spine density. Induction of proliferation, Both in Pioglitazone and differentiation, and migration of [26] 2011 PPAR-𝛾 vitro and Adult male Wistar rats rosiglitazone neural stem cells in vitro and in in vivo vivo. Induction of differentiation and neurite outgrowth, promoting Pioglitazone [27] 2009 PPAR-𝛾 In vitro SH-SY5Y cell line differentiation and outgrowth of cell processes. Stimulation of neurite outgrowth Mouse NB Neuro 2a (N2A) cell and significant increase of the Rosiglitazone [28] 2014 PPAR-𝛾 In vitro line population of neurite-bearing cells, via PPAR-𝛾 pathway. Induction of G1 cell cycle arrest, Oleic acid or reduction of cell migration and [29] 2007 PPAR-𝛽/𝛿 In vitro SH-NH-5YSY cell line GW0742 invasiveness, and an increase of neuronal differentiation. PPAR Research 5 significantly increases the population of neurite-bearing cells, We reported that the effects of metformin treatment in via PPAR-𝛾 pathway [28]. human SKNBE2 and SH-SY5Y NB cell lines are a significant All these observations are in agreement with previous reduction in the proliferation rate and cell viability, due to findings indicating that PPAR-𝛾 activation contributes to inhibition of AKT phosphorylation and an increased cell neuronal differentiation [11, 94–99]. death, via apoptosis-independent pathways. These effects While PPAR-𝛾 activation mainly results in apoptosis were more pronounced in SKNBE2, which are less differen- promotion in proliferating cells, PPAR-𝛽/𝛿 natural (i.e., oleic tiated, highly proliferative cells than SH-SY5Y cells. Notably, acid) and synthetic (i.e., GW0742) agonists induce G1 cell metformin effects were different depending on the differen- cycle arrest, reduce cell migration and invasiveness, and tiating stimuli, being abolished by retinoic acid, but were increase neuronal differentiation in SH-NH-5YSY [29]. potentiated by overexpression of NDM29, a noncoding RNA In summary, all these results suggest the possible use of affecting NB malignancy, although both conditions were PPAR agonists as novel therapy for NB, but to date clinical tri- characterized by a neuron-like differentiated phenotype [30, als are not yet underway (http://www.who.int/topics/clinical 123–128]. trials/en/). These in vitro results are in agreement with those of Kumar and coworkers who evaluated the antitumor activ- 2.2. Metformin and Neuroblastoma. Beyond TZDs, met- ity of metformin against neuroblastoma in vivo [31]. Oral formin is another hypoglycemic drug able to modulate PPAR administration of metformin, in both SH-SY5Y and SK- expression or activity, although these effects are rather cell N-BE xenograft NB mouse models, significantly inhibited specific and mainly indirectly mediated by the activation of the growth of the tumors. NB cell viability is reduced by AMPK. metformin, which also interferes with spheroid formation in Metformin is biguanide with a well-known safety profile, 3D cultures, confirming that its antitumor effect could also mainly used as oral antidiabetic drug [100, 101], whose involve the inhibition of CSC self-renewal. Moreover, in this promising anticancer activity was recently discovered [102]. study, it was confirmed that SKNBE2 cells are more suscep- It is well-documented that metformin inhibits tumor growth tible to metformin than SH-SY5Y cells. In addition, studying in in vitro and in vivo preclinical cancer models [103–110], and the underlying signaling mechanisms, they highlighted that various human clinical trials are in progress (WHO Interna- a modulation of Rho-GTPases and MAP kinase activation tional Clinical Trials Registry Platform, http://www.who.int/ mediate metformin effects on NB cell survival. topics/clinical trials/en/). Inthesameyear,Vujicetal.successfullyusedmetformin In particular, metformin seems to selectively affect cancer to inhibit cell proliferation and induce apoptosis in NRAS stem cell survival, inhibiting cancer metastases and thus mutant NB cell lines (SK-N-AS and CHP-212), in which represents a good potential adjuvant agent for chemotherapy NRAS signaling is constitutively active through the PI3K/ (as reviewed by [111]). AKT/mTOR pathway [32]. However, the molecular mechanisms of action of met- In 2015, Mouhieddine and colleagues found that metformin are still not completely defined, although it seems that formin reduces proliferation rate, viability, and invasive the antiproliferative mechanisms induced by this drug are at potential of NB cell lines SH-SY5Y [33]. least partially diverging from those regulating glucose home- Interestingly, focusing on metformin effect on stem cell ostasis. While the latter is mainly dependent on the AMPK population within a 3D culture model, these authors reported activation, the antitumor activity of metformin is mediated by that this drug is able to decrease, but not abolish, cell sphere- inhibition of AKT/mTOR (also involving AMPK), the inhi- forming ability, significantly targeting and reducing cancer bition of TK activity, or the regulation of chloride channels stem/progenitor cell population and thus possibly preventing [112–116]. recurrence. As far as the effects of metformin on PPAR activity are Notably, metformin also reduces MMP-2, a protein concerned, several studies were performed but the results involved in extracellular matrix degradation, favoring metas- are extremely dependent on the receptor subtype and the tasis and cancer progression [33]. cells analyzed. For example, metformin increased PPAR-𝛽/𝛿 Thanks to the highly selective and cytotoxic effects of expressioninmusclecellsandactivityinendothelialcells metforminonNBcellsandonstemcellpopulationin [117, 118], reducing the effects of ER stress and increasing particular (Table 2), these studies suggest that high-risk NB the bioavailability of nitric oxide [119]. On the other hand, patients could have great benefit by using metformin in metformin counteracts antiosteogenic PPAR-𝛾 activation by terms of increasing disease prognosis and propose that this rosiglitazone in bone marrow progenitor cells [120] and both drug can also be used as a novel therapeutic agent against PPAR-𝛾 and PPAR-𝛼 activity in hepatoma cells [121]. Finally, neuroblastoma. However, at the moment, there are no clinical aPPAR-𝛼 activation role of metformin was identified to trials evaluating metformin efficacy for the treatment of NB increase GLP-1 receptor levels [122]. (WHO International Clinical Trials Registry Platform, http:// However, although demonstrated in several models, the www.who.int/topics/clinical trials/en/). role of the modulation of PPAR expression and/or activity in the antiproliferative effects of metformin in neuroblastoma 3. Conclusion has not been addressed yet. The effect of metformin on NB was firstly demonstrated Despite advances in standard therapeutic protocols, the prog- by our groups [30]. nosis of NB has not gained significant progress, especially 6 PPAR Research

Table 2: Studies on metformin treatment to date in neuroblastoma. Reference/s Year Study types Cell lines/animal model Effects Significant reduction in the proliferation rate and cell [30] 2014 In vitro SKNBE2 and SH-SY5Y cell viability, due to inhibition of AKT phosphorylation, lines and an increased cell death, via apoptosis-independent pathways. Significant inhibition of tumor growth and NB cell SH-SY5Y and SK-N-BE viability, interfering with spheroid formation in 3D [31] 2014 In vivo xenograft NB mouse cultures. Modulation of Rho-GTPases and AMPK models activation mediate metformin effects on NB cell survival. [32] 2015 In vitro SK-N-AS and CHP-212 cell Inhibition of cell proliferation and induction of lines apoptosis. Reduction of proliferation rate, viability, and invasive [33] 2015 In vitro SH-SY5Y cell line potential.

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Review Article Commonalities in the Association between PPARG and Vitamin D Related with Obesity and Carcinogenesis

Borja Bandera Merchan,1 Francisco José Tinahones,1,2 and Manuel Macías-González1,2

1 Unidad de Gestion´ Cl´ınica Endocrinolog´ıa y Nutricion,´ Instituto de Investigacion´ Biomedica´ de Malaga´ (IBIMA), Complejo Hospitalario de Malaga´ (Virgen de la Victoria), Universidad de Malaga,´ 29010 Malaga, Spain 2CIBER Pathophysiology of Obesity and Nutrition (CB06/03), 28029 Madrid, Spain

Correspondence should be addressed to Francisco Jose´ Tinahones; [email protected] and Manuel Mac´ıas-Gonzalez;´ [email protected]

Received 25 March 2016; Accepted 15 May 2016

Academic Editor: Daniele Fanale

Copyright © 2016 Borja Bandera Merchan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The PPAR nuclear receptor family has acquired great relevance in the last decade, which is formed by three different isoforms (PPAR𝛼,PPAR𝛽/𝛿,andPPARΥ). Those nuclear receptors are members of the steroid receptor superfamily which take part in essential metabolic and life-sustaining actions. Specifically, PPARG has been implicated in the regulation of processes concerning metabolism, inflammation, atherosclerosis, cell differentiation, and proliferation. Thus, a considerable amount of literature has emerged in the last ten years linking PPARG signalling with metabolic conditions such as obesity and diabetes, cardiovascular disease, and, more recently, cancer. This review paper, at crossroads of basic sciences, preclinical, and clinical data, intends to analyse the last research concerning PPARG signalling in obesity and cancer. Afterwards, possible links between four interrelated actors will be established: PPARG, the vitamin D/VDR system, obesity, and cancer, opening up the door to further investigation and new hypothesis in this fascinating area of research.

1. Introduction Once in the nucleus, several molecules known as corepressors and coactivators, which show histone modifying There are three subtypes of PPARG, known as PPARG1, activities by themselves [7], bind the PPARG-RXR complex, PPARG2, and PPARG3. It has been established that PPARG2 showing some control over the genetic expression-repression leads in potency as a transcription factor [1]. PPARG per- interplay. Some known corepressors are SMRT or NCOR. forms its functions mainly through PPARG1 and PPARG2 When it comes to coactivators, we can mention p300/CRRB- [2]. Moreover, it shares lots of additional features with its binding protein (CBP) or SRC/p160 [8]. Importantly, dif- other counterparts. Concerning that, the parallelism found ferential recruitment of coactivators implies different gene betweenthePPARGsystemandthevitaminD/vitaminD receptor (VD/VDR) system will be further explored later on. expression patterns [9], wherefrom it can be deduced that In order to modulate gene expression, the PPAR NRs fam- the corepressors and coactivators comprise another gene ily, and specifically the PPARG, after binding with either nat- expression regulatory point which is worth studying. PPREs ural or synthetic ligands, heterodimerizes with the Retinoid are normally found in the promoter of those genes, which X Receptor (RXR) as vitamin D receptor (VDR) does. is regulated by PPARG activity [3]. The direct nucleotide Later on, the complex PPARG-RXR translocates to the sequences which PPARG-RXR will be bound to are known nucleus in order to get attached to PPREs (PPAR Response as DR-1 motifs (direct hexanucleotide repeats) of PPRE Elements), genome nucleotides sequences wherefrom the [8]. Some PPARG target genes are those codifying CD36, PPARs will coordinate the expression or repression of some FABP4 (Fatty Acid Binding Protein 4), adiponectin, or the genes involved in metabolism, immunity, differentiation, or CCAAT/enhancer binding protein 𝛼 [10], all being genes cellular proliferation, to cite some [3–6]. involved in adipose tissue homeostasis. However, afar of its 2 PPAR Research adipose functions PPARG is also vital for development of we have already addressed, adipogenesis and lipid storage are some important organs such as heart and the placenta [11]. some of them. Illustrating this, a high-fat feeding augments PPARG expression while fasting diminishes it [38]. 2. The PPARG Physiology Remarkably, PPARG performs different functions in metabolically sick rodents and metabolically healthy ones. PPARG behaves as a transcription factor, as many other In disease, PPARG activation seems to improve metabolic nuclear receptors (NRs) do. Then, it modulates the expression parameters, but in the healthy population its downregulation andrepressionofamyriadofgenesinvolvedinmetabolic shows antiobesity effects [39]. homeostasis, regulating energy expenditure and storage [12, Inthesameway,moredifferenteffectshavebeen 13]. Some PPARG target genes are those codifying CD36, described in metabolic health and disease regarding PPARG FABP4 (Fatty Acid Binding Protein 4), adiponectin, or the expression. For instance, in healthy subjects a high-fat meal CCAAT/enhancer binding protein 𝛼 [10], all being genes greatly induced the expression of PPARG while the same involvedinadiposetissuehomeostasis.However,afarof high-fat feeding diminished PPARG expression in a group of its adipose functions PPARG is also vital for development morbidly obese patients [40]. of some important organs such as heart and the placenta In like manner, an indirect correlation between IR and [11]. Although most research on PPARG has been focused PPARG expression, measured by glucose status, HOMA- on its metabolic action, some of them are neurogenesis, IR index, and insulin levels, can be set in morbidly obese osteogenesis, cancer, or cardiovascular disease [14]. Such persons,whosevisceraladiposeandmuscletissuesshowless pleiotropism of actions gives us a clue of the relevance of this PPARG expression as IR increases [40]. transcription factor regarding health and disease. We know During placentation and intrauterine development, the forinstancethatuniversalPPARGdeletionandlifearenot PPARG gene methylation patterns could be altered by compatible [11]. maternal nutrition, which actually exerts long-term effects The considerable host of actions performed by PPARG upon the receptor status in the offspring, as indicated very can be compared to those of vitamin D and VDR [15], recently by Lendvai et al. [41]. This is preliminary evidence which has been implicated in neurologic disorders [16–18], about the early programming of our lifelong metabolism set autoimmune pathologies [19–21], cardiovascular disease [22], points through nutritional inputs, which could easily leave us diabetes mellitus [23, 24], psoriasis [15] or infectious disease susceptible to obesity and metabolic disease in later stages of [25, 26], and, above all of what is mentioned, cancer [27, 28]. life.

3. PPARG and Obesity 4. PPARG and Cancer Much has been already written about PPARG signalling and its role in conditions such as obesity or diabetes. In PPARG is highly expressed in lung, prostate, colorectal, obesity, PPARG orchestrates adipocyte maturation and dif- bladder, and breast tumours [42]. Furthermore, we can find ferentiation,harmonisingtheroleofmanyotherplayersin in the literature compelling evidence for PPARG having thatprocess[29].Remarkably,itistheonlyknownfactor, antineoplastic actions in colon, prostate, breast, and lung which is completely necessary and sufficient for the adipocyte cancers[43,44],whichhappentobethemostprevalentforms differentiation process to occur [11, 30]. This nuclear receptor of cancer in occident (Figure 1). acts, then, as a master regulator of adipogenesis. Solid evidence backs up that epigenetic events frequently In addition, it is widely known that PPARG has an found in cancer can hamper nuclear receptors responsiveness important whole-body insulin-sensitizer role. For example, toward their ligands. In that respect, increased levels of core- muscle-PPARG knocked-out mice are insulin resistant [31]. pressor NCOR in prostate cancer can silence the expression In adipose tissue, PPARG deletion leads to increases in of target genes and constitute a potential epigenetic lesion, bone mass, lipoatrophy, and insulin resistance (IR) [32]. which selectively distorts the actions of PPARG/PPAR𝛼 [45]. In the same fashion, PPARG induces the proliferation of Inthesameline,PPARGpromotermethylationincol- adipocytes progenitors into mature adipocytes and dimin- orectal carcinoma (CRC) is associated with poor prognosis ishes the osteoblasts population likewise [33]. [46]. This transcriptional silencing of PPARG is operated The specific deletion of PPARG in liver conduces toIR through HDAC1 (Histone Deacetylase 1), EZH2 (Enhancer and decrease of hepatic fat depots [34]. Even in macrophages, of Zeste 2 Polycomb Repressive Complex 2 Subunit), and thepresenceofPPARGisimportanttokeepadequateinsulin MeCP2 (Methyl CpG Binding Protein 2) recruitment, leading sensitivity levels throughout the body [35, 36]. It is then easy to repressive chromatin states that eventually increase cell to deduce that one of the main objectives of PPARG activity is proliferation and invasive potential [46]. Correspondingly, min/+ the insulin sensitivity maintenance through different tissues. APC mice which have undergone PPARG genetic abla- Thiazolidinediones (TZD), a family of synthetic PPARG tion demonstrate increased colon tumour growth [47]. agonistwidelyusedindiabetestreatment,showclear In the literature, some mutations and variations in improvements in insulin sensitivity, enhanced adipocyte PPARG expression have been associated with cancer in our differentiation, reduction of leptin levels, and upregulation of specie [48, 49]. Beyond that, its expression comprises an adiponectin [37]. independent prognostic factor in CRC [50, 51]. Contrary to the catabolic actions elicited by the PPAR𝛼 Apartfromepigenetics,weshouldnotlosesightofthe and PPAR𝛿,thePPARGisinchargeofanabolicfunctions.As fact that metabolic syndrome, insulin resistance, obesity, PPAR Research 3

Angiogenesis Cell differentiation

Cell cycle arrest

Cancer Cell proliferation

Dyslipidemia Apoptosis

Metabolic syndrome Cardiometabolic disease Inflammation PPARG Obesity Oxidative stress

Type 2 diabetes mellitus Cell migration and invasiveness

Hypertension

Vitamin D system crosstalk Coronary heart disease

Figure 1: PPARG actions: PPARG plays an important role in cardiometabolic disease and cancer. The noteworthy crosstalk between vitamin D system and PPARG is also considered. Arrow’s width exemplifies the level of consistency found in the literature regarding each association in the mind picture.

and inflammation, importantly interrelated conditions in In human pancreatic cancer cells the same phenomenon which PPARG has modifying and regulatory actions, increase is observed: PPARG is able to trigger cell cycle arrest of the cancer risk [52–59], which adds weight to PPARG and cancer malignant cells through activation by thiazolidinediones [64]. research (Figure 1). Through PPARG activation, its ligands increase the There is some evidence linking PPARG agonist’s actions expression of the cyclin-dependent kinase inhibitors p21 [64, to better cancer treatment responsiveness as well. PPARG 65] and p27 [65–69], enhance the turnover of 𝛽-catenin, and agonist Rosiglitazone, in this phase II clinical trial, raised the downregulate the expression of cyclin D1 [70–74]. radioiodine uptake in differentiated thyroid cancer [60]. IFN-𝛽 treated pancreatic cancer cells were more affected 4.2. Differentiation. In vitro activation of PPARG by its when Troglitazone was added to the therapy, showing syner- ligands correlates with increased expression of carcinoem- gistic effects between IFN-𝛽 and TGZ [61]. But it is necessary bryonic antigen (CEA), E-cadherin, developmentally regu- to be careful in some studies, in which PPARG agonist lated GTP-binding protein 1 (DRG), alkaline phosphatase, likeRosiglitazoneactsasagreatpromoterofhydroxybutyl or keratins, all of them being molecules expressed in well nitrosamine-induced urinary bladder cancers [62]. differentiated cells, opposing to the undifferentiated cell state In the following paragraphs, we will review what we know commonly found in most cancers [48, 64, 75–77]. about the specific molecular actions of PPARG in cancer Tontonoz et al. gave us the first evidence about the effec- biology. Cell cycle arrest, cell differentiation, angiogenesis, tiveness of PPARG ligands inducing differentiation in human proliferation, invasiveness, migration capacity, apoptosis, cancer cells, concretely in liposarcoma cancer cells [75]. inflammation, and oxidative stress should be evaluated. Again, in human liposarcoma, treatment with Troglitazone raised the level of differentiation of its cells [78]. 4.1. Cell Cycle Arrests. Some evidence suggests that PPARG More evidence that PPARG enhances terminal differen- and its agonists have the ability to interfere with the cellular tiation in cells is reviewed in papers of Grommes et al. and cycle and then, likely, with malignancies development. Koeffler, respectively [43, 44]. In renal cell carcinoma, Troglitazone (TGZ) was able to induce G2/M cell cycle arrest via activation of p38 MAPK 4.3. Angiogenesis. It is common knowledge that angiogenesis (Mitogen-Activated Protein Kinase) [63]. is a vital step in malignant development. The complex 4 PPAR Research process by which new vessels are formed, angiogenesis, has cancer. The milieu found in chronic inflammation acts as beenfeverishlystudiedasanewpossibletargetincancer a facilitator for carcinogenesis and cancer development [52, treatment. 113]. This has been shown in colorectal, liver, bladder, lung, In vitro and in vivo angiogenesis-modulating functions and gastric neoplasms [114, 115] and investigated in several have been described for PPARG [79]. In spite of that, more.Therangeofprocessesinwhichinflammationpartakes differential effects regarding angiogenesis have been observed in carcinogenesis goes from cell growth and survival, metas- for PPARG in vitro and in vivo, showing either pro- or tasis and cell invasion, treatment response, angiogenesis, and antiangiogenic actions dependent on cell context [80–85]. tumour immunity [115, 116]. PPARG agonist can also enhance VEGF expression in cancer There is evidence of PPARG having anti-inflammatory cells, as some studies reveal [86, 87]. activity in several cell lines [117, 118]. In models of experi- The mechanisms deciding whether PPARG will act as mentally induced colitis PPARG expressed in macrophages a proangiogenic factor or as an antiangiogenic one are is capable of inhibiting inflammation [119]. still elusive to us, but we believe that cellular context and ItiswidelyknownthatsomePPARligandssuchas environment are likely the controllers of such process. omega-3 fatty acids EPA and DHA have anti-inflammatory properties. Those and other natural and synthetic ligands 4.4. Proliferation. Antiproliferative actions are also attributed couldbeusedinthefutureaschemopreventiveagentsin to PPARG and its ligands. TZD, for example, has shown a vast range of conditions linked to inflammation, that is, antiproliferative effects [88, 89]. cancer [105, 120, 121]. Modulation of PPARG can have differential effects on Activation of PPARG by its ligands reduces cytokines carcinogenesis depending on the cellular microenvironment such as TNF𝛼 and NF-𝜅𝛽 in monocytes, turning down the [90]. Therefore, depending on the cellular environment inflammatory milieu [120, 122]. PPARG can behave as a proliferative or antiproliferative The epigenetic process of sumoylation has been linked to factor, as happened with angiogenesis. PPARG transrepression of inflammation. After ligand acti- Tumour cells are frequently in shortage of polyunsatu- vation, PPARG binds to a SUMO protein (Small Ubiquitin- rated fatty acids. Docosahexaenoic acid (DHA), a well-known like Modifier) and both join a nuclear corepressor complex, ligand of the PPAR family, has been shown to reduce tumour reducing the proinflammatory gene expression [123]. proliferation in lung tumour cell cultures [91]. Along with The NF-𝜅𝛽 transcription factor has repeatedly been that, DHA in breast cancer cells diminishes proliferation and associated with tumour development and thriving [52]. increases apoptosis [92, 93]. Interacting with this factor, PPARG inhibits the genesis of In prostate cancer, PPARG ligand activation effect was proinflammatory molecules such as IL-6, TNF, and MCP1 assessed in a phase II clinical trial. The results showed a through transrepression [3, 117]. hampered cancer cell growth [94]. Again, a word of caution must be said due to the Eukaryotic initiation factor 2 is a target of inhibition seemingly tumour-promoting effects of PPARG found spo- for PPARG agonists (i.e., thiazolidinediones). Such factor radically [124–127]. Therefore, it seems as if the effects carried inhibition, which is mediated in a PPARG-independent way, on by the cell depend of cell context and environment. truncates the translation process [95]. Environment is, usually, at the helm of cellular functions. In liposarcoma patients, treatment with Rosiglitazone increased the necessary time to double tumour volume in 4.7. Oxidative Stress. PPARG has demonstrated an antioxi- this clinical trial [96]. In other studies, however, Troglitazone dant effect [128, 129]. SOD (Superoxide Dismutase) expres- (another member of the thiazolidinedione family) had low or sion might well be regulated by PPAR because a PPRE is no effects in prostate cancer [97] or breast or colorectal cancer found in the Cu/Zn-SOD promoter [40]. [98, 99]. IR found in diabetes mellitus and metabolic disease is 4.5. Apoptosis. The combined effect of an RXR agonist and certainly correlated with increased oxidative stress, which Troglitazone curtailed gastric cancer cells proliferation in eventually could lead to an increased risk of cancer through vitro by enhancing apoptotic mechanisms [100]. nongenomic carcinogenesis [130–133]. PPARG agonists increased the expression of PTEN [101– In macrophages, PPARG mediates some notable abilities: 105], BAX, BAD [106, 107], and the turnover of the FLICE uptake and reverse transport of cholesterol, macrophage inhibitory protein (FLIP) [108, 109], known for its antiapop- subtype specification (enhancing the M2 macrophage pheno- totic role. type, which is associated with higher insulin sensitivity and Conversely, PPARG agonists can inhibit BCL-XL and lower inflammation levels), and anti-inflammation proper- BCL-2 expression [107, 110], PI3K activity, and AKT phos- ties [36, 134, 135]. phorylation [101, 111, 112] and restrain the activation of JUN Postprandial hypertriglyceridemia is associated with N-terminal protein kinase [107]. It is worth mentioning that lower PPARG expression in metabolic syndrome patients many of those actions were elicited in a PPARG-independent while in healthy subjects the same “insult” leads to overex- manner. The exact mechanisms by which these effects are pression of PPARG [136]. We could hypothesize that since performed are still unknown. the PPARG system is injured in the metabolically ill patients, after an oxidative stress insult (a high-fat feeding), it cannot 4.6. Inflammation. Nowadays, it is common knowledge in the respond, leaving us more susceptible to oxidative actions scientific community that chronic inflammation promotes and its consequences (hypothesis coined as “nuclear receptor PPAR Research 5 exhaustion theory”). In the healthy group, the PPARG would Moreover, there is enough evidence to assert that epi- perfectly be capable of managing the lipid storage and would genetic events can influence both PPARG and VDR/VD act as an oxidative stress buffer. systems behaviour. In this study, Fujiki et al. showed that in a diabetic mouse 4.8. Cell Migration and Invasiveness. Less evidence is avail- model PPARG promoter methylation levels are higher than able with respect to invasiveness and PPARG. However, we those of the control mice [191], along with the possibility should pay attention to some preliminary data. of methylation reversal when the animals were exposed 󸀠 The PPARG gene modulates the invasion of cytotro- to 5AZA (5 -aza-cytidine). At least three messages can be phoblast into uterine tissue, which could be a novel indicator drawn from this study: (1) the PPARG system is susceptible of some invasion-related function of PPARG [137]. to epigenetic regulation, (2) diabetes and other metabolic Going further, this study by Yoshizumi et al. showed conditions could alter the PPARG epigenetic landscape and how PPARG ligand thiazolidinedione (TZD) is able to inhibit then disrupt its proper functioning, and (3) this disruption growth and metastasis of HT-29 human colon cancer cells, via canbereversedbydrug-inducedchangesor,likely,bylifestyle the induction of cell differentiation. The use of the TZD drives changes. to G1 arrest, in association with a great increase in p21Waf-1, The vitamin D system is likewise susceptible to epige- Drg-1, and E-cadherin expression [77]. netic regulation [192–195] and, interestingly, in cancer this epigenetic repression of the vitamin D system is almost Paradoxically, molecules with PPARG antagonist actions always present [196–204], which compellingly leaves the door are able to inhibit invasiveness and proliferation of some opened to the possibility of the same phenomena happening cancer cell lines [26, 138–140]. Again, one nuclear receptor in the PPARG system. can exert one or just the opposite function depending on the In fact, PPARG promoter hypermethylation is a prognos- cellular environment and ligand exposure. ticfactorofadverseoutcomeincolorectalcancer[46,205]. Higher levels of PPARG promoter methylation were found 5. Connecting the Dots: PPARG, Vitamin D in advanced tumour stages while earlier stages showed lower System, Obesity, and Cancer methylationlevels.Thissuggeststhatashappenswithvitamin D, advanced cancer stages can epigenetically repress PPARG Often in biology and medicine research, we tend to focus expression and then nullify its antineoplastic actions. on the individualities of separated molecules or molecule systems in order to explain their functions, forgetting the intermolecular communication, which is ever-present in 5.2. The PPARG/VDR Crosstalk: What an Interesting Con- every biological system. More frequent than not, that sepa- versation! Some studies have clearly shown the existence rateness gives us a rather limited perspective of the matter at of some communication between PPARG and VD/VDR. hand. For instance, the interconnectedness of biology systems Interestingly, potent VDRE (Vitamin D Response Elements) 𝛿 and the emerging properties of such interconnectedness have been discovered in human PPAR promoter, which shouldbefurtherexaminedandtakenintoaccount. opens the door to VDR/VD influence over the PPAR system[206].Intheoppositedirection,somestudieshave The crosstalk between different NRs, the “dance” and demonstrated the ability of PPARG to bind VDR and inhibit messages they give one another, is recently becoming an vitamin D-mediated transactivation [207]. This data might excitingnewareawhichwillbeexplored.Thisisthecaseof be an indicator of bidirectional or reciprocal actions of both the VDR/VD and the PPARG system, in which both have systems influencing each other, which have deep implications beenshowntobeinvolvedinsomerelationshipwedonot and introduce new and interesting questions to ponder upon. utterly understand yet. Even between PPAR subtypes some modulation of expressionhavebeenfound:PPAR𝛿 could repress PPAR𝛼 5.1. PPARG and VDR/VD System: Commonalities in Cancer. and PPARG gene expression [208], illustrating the complexity Noteworthy, great parallelism exists between PPARG and of PPAR system regulation. the VDR/VD system regarding its protective role in carcino- In the adipocyte cell, the VD/VDR system has shown genesis. There are a vast number of studies describing the anti-PPARG activity, inhibiting its expression and then anticancer properties of vitamin D. The majority of them are adipogenesis [209, 210], which is contradictory with the brilliantlyanalysedinthisreviewbyFeldmanetal.[28]. commonly found proadipogenesis effects of vitamin D [211], Vitamin D has been extensively associated with anti- at least in human. The factors leading to either pro- or inflammatory actions [141–143], apoptotic mechanisms [144– antiadipogenesis effects are completely uncharted. 150], antiproliferative functions [151–159], prodifferentia- In melanoma cell lines, administration of calcitriol and tion effects [160–166], antiangiogenic properties [167–171], a several PPAR ligands modified the expression of both PPARG potential role-managing invasion and metastasis [172–184], and VDR, demonstrating again this intriguing connection microRNAmodulation[185–189],andevensomeroleinthe [212]. Sertznig et al. conclude in this article that calcitriol and Hedgehog signalling pathway modulation [190]. Remarkably, some PPAR ligands can inhibit proliferation of the human most of those actions have been attributed to PPARG sig- melanoma cell line MeWo [213]. nalling in a somewhat lesser extent, as reviewed in this work. Such similarity and overlap in anticancer actions are worth 5.3. PPARG and VD/VDR System: Metabolic Commonalities. studying. We are about to discuss the metabolic effects of vitamin D 6 PPAR Research and their analogy with those of PPARG, establishing again system. The underlying mechanism behind this deterioration the parallelism. should be further studied. As contradictory as it seems, VDR or CYP27B1 knocked- AdisruptedVDR/VDsystemleadsmicetolossof out mice show great fat mass loss [211] while obesity in fat deposits and great increase of energy expenditure. In −/− humans is commonly associated with poor vitamin D plas- relation to that, VDR mice increase the expression of matic levels [214]. Actually, an indirect relationship between UCP1 (uncoupling protein 1 or Thermogenin) twenty-five- Body Mass Index (BMI) and 25OHD3 has been amply fold [211], with the consequent energy consumption. Is described in the literature [215]. vitamin D, along with PPARG, an energy-conserving and In addition, low plasmatic vitamin D levels are associated metabolic homeostasis-maintaining hormone? with increased risk of type 2 diabetes mellitus (T2DM) inde- However,adiposetissueisnottheonlyoneaffectedby pendently of BMI [24] and with hypertension, dyslipidemia disruption of the VD system. A shortage of calcitriol in rats (DLP), and metabolic syndrome (MS) [216, 217]. Besides, was related with increased skeletal muscle ubiquitination and vitamin D deficiency predisposes to diabetes in animal loss of total muscle mass [224]. On the PPARG side, its models, while its supplementation prevents the disease [214]. activation through TZD in growing pigs increased muscle Concerning PPARG, we have extensively discussed before in fiber oxidative capacity independently of fiber type [225]. the review its orchestrating actions regarding adipogenesis Overexpression of PPAR𝛿 in mice almost doubles the animal andadipocytemetabolism.Bothcalcitriol(theactiveformof endurance and exercise capacity [226]. We should not lose vitamin D) and PPARG seem to oppose metabolic homeosta- sight of the important role the muscle has in obesity and sis disruption. metabolic disease pathogenesis, being a potential target for Another paradoxical event is found in the fact that in calcitriol and PPAR modulating actions. humans calcitriol enhances adipogenesis while in mice the Taken all data together, the vitamin D system seems to same hormone diminishes it via downregulation of C/EBP𝛽 team up with PPARG in order to maintain proper metabolic mRNA and upregulation of CBFA2T1 (a corepressor) [218, homeostasis. Notwithstanding, in some occasions this love 219]. With reference to PPARG, it enhances adipogenesis [10]. relationship breaks apart and both partners seem to bother In human subcutaneous preadipocytes, calcitriol elicits one another in ways that we utterly ignore but, likely, have actions impressively similar to those of PPARG in adipocyte somethingtodowithepigeneticregulation. maturation and differentiation. For instance, calcitriol is able to increase the expression of the enzyme Fatty Acid Synthase 6. Conclusions (FASN) increasing lipogenesis in like manner as PPARG The PPARG transcription factor has been classically associ- [210]. ated with metabolic homeostasis and lipid storage functions. The storage capacity theory introduces the idea that lipid Recently, newfound anticancer actions are assigned to this storagecapacityandtheabilityofPPARGtomanagethe nuclear receptor. processes leading to lipid storage are limited. As to that, when However, its anticancer actions are not always consistent; the organism reaches a lipid level threshold lipotoxicity shows in some studies some oncogenic effects have been described. up, PPARG is no more capable of lipid handling, and the We believe that cellular environment is the guiding factor harmful hormonal environment of obesity starts to spread behind PPARG actions and cells are controlled “from outside through the organism [220]. in.” In alignment with this, the PPARG and other nuclear Transferring the same concept of “nuclear receptor receptors would only be “cellular effectors,” carriers of outside exhaustion” to VD/VDR anticancer actions we could estab- messages of health or disease. lish a parallelism. It has shown that the VD/VDR is epigenet- When a “disease threshold” is reached, in either obe- ically downregulated in late cancer stages but overexpressed sity or cancer, PPARG and VDR expression, respectively, or normally expressed in early stages [221, 222]. As the diminishes. However, in early stages of those diseases, the aforementioned studies show, in those later stages epigenetic expression of those nuclear receptors is higher than normal. downregulation of the VD system molecules occurs, leaving Derived from these observations, we have coined the so- it unable to exert its antineoplastic functions properly. Is called “nuclear receptor exhaustion theory,”bywhich,inan obesity, as cancer does with vitamin D, acting as a negative early disease stage, nuclear receptors PPARG and VDR coun- epigenetic driver when it comes to PPARG signalling? That terbalance the harmful effects that obesity and cancer exert could answer why in most morbidly obese patients expression upon the organism, their expression being high. However, of PPARG is greatly lower in comparison to healthy subjects. sadly, if disease progresses, it generates epigenetic silencing Accordingly, PPARG1 and PPARG2 expression in visceral mechanisms upon both transcription factors, whose expres- adipose tissue (VAT) from morbidly obese (MO) subjects is sion decreases radically. This silencing leaves us increasingly significantly downregulated when compared to metabolically susceptible to disease. The positive side is that through drugs healthy subjects [223]. Not only that, in insulin resistant MO or, better yet, lifestyle changes reversal of epigenetic changes subjects PPARG expression is even lower [220] compared is possible. with noninsulin resistant MO patients, whichever interest- There is an exciting function overlap between PPARG and ingly correlates with the lower vitamin D levels found in VDR/VD system, both of which wield oncoprotective and MO with IR compared to their insulin sensitive counterparts metabolic actions. Actually, parallel metabolic and anticancer [24]. Somehow, the metabolic impairment caused by insulin actions are described in the literature, suggesting that they resistance is able to deteriorate both PPARG and VD/VDR team up to keep at bay those diseases. Maybe the detailed PPAR Research 7

Disease Health

(i) Cell proliferation (ii) Cell differentiation (iii) Apoptosis Obesity Vitamin D system (iv) Cell migration and invasiveness (v) Cell cycle regulation (vi) Angiogenesis (vii) Inﬂammation (viii) Energy and metabolism homeostasis (ix) Lipid storage (x) Insulin sensitivity

Cancer PPARG system

Figure 2: The four players: this figure shows the interrelation between the four players: obesity, cancer, vitamin D system, and PPARG. Red arrow: harmful effects, which contribute to disease. Green arrow:positive effects, which contribute to health. Disease perpetuates itself damaging both nuclear receptors. Arrow’s width is in proportion with the strength and consistency of each association found in the literature. Dashed line: yet-to-be determined, preliminary, or hypothetical effects. Continuous line: in vitro/in vivo demonstrated effects. Right box: in green, actions mainly attributed to vitamin D, and in purple, actions classically attributed to PPARG. However, it is known that both agents exert every action illustrated in this box, in a higher or lower extent. studyofthisoverlapcouldgiveuscluesinrespecttothe studies. The author focused mainly on systematic and narra- molecular pathogenesis of important conditions as metabolic tive reviews. Data sources are medline (Pubmed), Jabega´ 2.0 disease and cancer. Further study in this new area is necessary (Malaga´ University Search Engine Software), Gerion´ search to elucidate those questions. engine, and screening of citations and references. Regarding Obesity, a first-order problem in our society, is linked eligibility criteria, we focused on papers published in mag- with increased risk of cancer incidence and progression. azines considered to be in the first impact factor quartile The debatable factors behind this risk are an increment in without restrictions regarding publishing date. Keywords are oxidative stress, chronic inflammation, poorer vitamin D PPARG; Obesity; Transcription factor; Vitamin D; Calcitriol; status, hormone misbalance, and, arguably, PPARG silencing Vitamin D Receptor; Epigenetics; Nuclear Receptor; Cancer; through unknown mechanisms. As we know, PPARG and the Methylation. vitamin D system play conjunctly a yet-to-elucidate role in cancer, so it is not surprising at all that their hypothetical Competing Interests epigenetic repression in obesity could be another mechanism The authors declare that there are no competing interests. linking this metabolic disorder to malignancies. It has been shown, both in vitro and in vitro, that the tumours are capable of epigenetically silencing both the Acknowledgments vitamin D and the PPARG system. This silencing could lead This study was supported byCentros “ de Investigacion´ En to the deterioration of their anticancer and metabolic actions. Red” (CIBER, CB06/03/0018), the “Instituto de Salud Carlos Finally, a worse known crosstalk between the two NRs III” (ISCIII), and grants from ISCIII (PI11/01661) and from exists. Its usefulness, purpose, and message are (almost) Consejer´ıa de Innovacion, Ciencia y Empresa de la Junta de utterly unexplored to us and should be studied more dili- Andaluc´ıa (PI11-CTS-8181) and cofinanced by the European gently. The interrelation, reciprocity, and interdependence of Regional Development Fund (FEDER). M. Mac´ıas-Gonzalez´ all four actors examined here might be the starting point wasrecipientoftheNicolasMonardeprogramfromtheSer- of new fascinating research linking epigenetic signalling and vicioAndaluzdeSalud,JuntadeAndaluc´ıa, Spain (C0029- twoofthemosthurtfuldiseasesofourtime(Figure2). 2014). Additional Points References Design is literature review across preclinical studies, descrip- [1] J. N. Feige, L. Gelman, L. Michalik, B. Desvergne, and W.Wahli, tive studies, analytic studies, and reference lists of selected “From molecular action to physiological outputs: peroxisome 8 PPAR Research

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