Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation Through E2F1-Mediated Increase of Cyclins S

Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/12/21/mol.116.106757.DC1

1521-0111/91/3/189–196$25.00 http://dx.doi.org/10.1124/mol.116.106757 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:189–196, March 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation through E2F1-Mediated Increase of Cyclins s

Jonathan Choiniere, Jianguo Wu, and Li Wang Department of Physiology and Neurobiology, Institute for Systems Genomics, University of Connecticut, Storrs, Connecticut (J.C., J.W., L.W.); Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut (L.W.); Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut (L.W.); and School of Pharmaceutical

Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China (L.W.) Downloaded from Received August 31, 2016; accepted December 9, 2016

ABSTRACT Hepatocellular carcinoma (HCC) is a common form of cancer PDK4-knockdown HCC cells also progressed faster through with prevalence worldwide. There are many factors that lead to the cell cycle, which concurrently expressed high levels of molpharm.aspetjournals.org the development and progression of HCC. This study aimed to cyclins and E2F1 as seen in the Pdk42/2 mice. Interestingly, identify potential new tumor suppressors, examine their function the induced cyclin E1 and cyclin A2 caused by Pdk4 deficiency as cell cycle modulators, and investigate their impact on the was repressed by arsenic treatment in mouse liver and in HCC cyclin family of proteins and cyclin-dependent kinases (CDKs). cells. E2f1 deficiency in E2f12/2 mouse liver or knockdown E2F1 In this study, the pyruvate dehydrogenase kinase (PDK)4 gene using small hairpin RNAs in HCC cells significantly decreased was shown to have potential tumor suppressor characteristics. cyclin E1, cyclin A2, and E2F1 proteins. In contrast, inhibition of PDK4 expression was significantly downregulated in human PDK4 activity in HCC cells increased cyclin E1, cyclin A2, and HCC. Pdk42/2 mouse liver exhibited a consistent increase in E2F1 proteins. These findings demonstrate that PDK4 is a critical cell cycle regulator proteins, including cyclin D1, cyclin E1, cyclin regulator of hepatocyte proliferation via E2F1-mediated regula-

A2, some associated CDKs, and transcription factor E2F1. tion of cyclins. at ASPET Journals on September 23, 2021

Introduction 2003). Identification of cyclin modulators will aid in the understanding of HCC development and progression. Liver cancer is the second leading cause of cancer-related Pyruvate dehydrogenase kinase PDK4 is a mitochondrial death worldwide, with an estimated 745,500 deaths yearly. protein with a histidine kinase domain that inhibits the The majority of liver cancer deaths are identified as hepato- pyruvate dehydrogenase complex (PDC). Inhibition of the cellular carcinoma (HCC). The mechanisms behind HCC PDC results in reduced conversion of pyruvate to acetyl- progression are continually studied. Many factors that in- CoA. Acetyl-CoA is used in the citric acid cycle to carry out crease the risk of HCC have been identified, including cellular respiration (Sugden and Holness, 2003). PDK4 is a environmental factors, viral infection, alcohol consumption, major factor in cellular respiration, since it works to inhibit and smoking (Kanda et al., 2015). Cyclins are vital cell cycle the progression from glycolysis to the citric acid cycle. Cellular regulators that normally function to ensure the control of respiration is a major physiologic factor that was shown to be cellular proliferation. The cyclin family of proteins and the altered in cancer cells (Scatena, 2012); therefore, PDK4 is a associated cyclin-dependent kinases (CDKs) have been shown prime molecular suspect in crosstalk between cellular respi- to be significantly elevated in HCC tissues (Masaki et al., ration and cell cycle progression. Because liver cells are highly aerobic and metabolically active, liver tissue and associated cells serve as excellent test subjects. This research was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01-ES025909], the Arsenic is identified as a group 1 carcinogen and is the 20th National Institutes of Health National Institute of Diabetes and Digestive most common element in the earth’s crust (International and Kidney Diseases [R01-DK080440, R01-DK104656, and P30-DK34989 (to Agency for Research on Cancer, 2012). Over 100 million people Yale Liver Center)], the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants R21-AA022482 and R21-AA024935], worldwide are exposed to arsenic-contaminated drinking and the U.S. Department of Veterans Affairs [Merit Award 1I01BX002634]. water, making it a global health concern (Polya and Charlet, dx.doi.org/10.1124/mol.116.106757. s This article has supplemental material available at molpharm. 2009). Arsenic has been reported to induce epigenetic alter- aspetjournals.org. ations related to HCC progression (Liu et al., 2014) and to

ABBREVIATIONS: Aza, 59-aza-29-deoxycytidine; CDK, cyclin-dependent kinase; DNMT, DNA methyltransferase; HCC, hepatocellular carcinoma; MeDIP, methylated DNA immunoprecipitation on chromatin immunoprecipitation; PCNA, proliferating cell nuclear antigen; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; SHP, small heterodimer partner; shRNA, small hairpin RNA; WT, wild type.

189 190 Choiniere et al. silence hepatic PDK4 expression through activation of histone Liver Specimens. The coded human liver specimens were H3K9 methyltransferase G9a (Zhang et al., 2016). obtained through the Liver Tissue Procurement and Distribution Nuclear receptors are a class of proteins that mediate the System (Minneapolis, MN) and were described previously (He et al., activity of hormones and have been implicated in various 2008). Because we did not ascertain individual identities associated diseases, including HCC (Rudraiah et al., 2016). The small with the samples, the Institutional Review Board for Human Research at the University of Connecticut determined that the project was not heterodimer partner (SHP) is a unique transcriptional re- research involving human subjects. pressor (Zhou et al., 2010) and has been implicated as a critical Cell Culture Experiments, Fluorescence-Activated Cell inhibitor of HCC progression (Zhang et al., 2011b) by regulat- Sorting Analysis, and Luciferase Promoter Assay. Huh7 ing multiple pathways involved in tumor growth, including (Zhang and Wang, 2011) and 293T (Yang and Wang, 2012) cells cell proliferation (Zhang et al., 2008), apoptosis (Farhana were described previously. Cultured cells were maintained in et al., 2007; Zhang et al., 2010), and migration and invasion Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with (Yang et al., 2016). Intriguingly, SHP represses DNA meth- 10% fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin ylation via suppressing DNA methyltransferase (DNMT) (Mediatech, Manassas, VA). High- and low-efficiency (shPDK4 H expression (Zhang and Wang, 2011; Zhang et al., 2012), and shPDK4 L, respectively) PDK4 knockdown cell lines were suggesting that modulating SHP function by its agonist may generated by a lentiviral vector containing the small hairpin RNA (shRNA) along with packing vector and envelope vector. These be useful to develop epigenetic-based therapeutic treatment of

components were cotransfected into 293T cells using X-tremeGENE Downloaded from HCC. HP DNA transfection reagent (Sigma-Aldrich) according to the Using an unbiased approach that included a combination manufacturer’s protocol. The supernatant containing virus particles of methylated DNA immunoprecipitation on chromatin was collected and concentrated. Suspended virus was applied to immunoprecipitation (MeDIP) and RNA sequencing (RNA- target cells with 6 mg/ml polybrene. The shPDK4H cell line was seq) methods in Shp2/2 mice, this study identified PDK4 as generated using shRNA (TRCN0000006264, clone ID NM_002612.2- a potential tumor suppressor gene. The expression of PDK4 2954s1c) from Sigma-Aldrich, and the shPDK4 L was generated was markedly reduced in human HCC specimens and in using shRNA (TRCN0000194917, clone ID NM_002612.2-1297s1c1) molpharm.aspetjournals.org 2 2 m mouse liver tumors. The Pdk4 / liver had a significant also from Sigma-Aldrich. shPDK4L cells were treated with 15 M induction in multiple cyclin proteins, with the most striking arsenic for 24 hours. E2F1 shRNAs were from Sigma-Aldrich (shE2F1 1: TRCN0000039659, clone ID NM_005225.1-502s1c1; and shE2F1 2: elevation of cyclin E1, cyclin A2, and E2F1, whereas Pdk4- TRCN0000010328, clone ID NM_005225.x-1171s1c1). The PDK4 knockdown HCC cells exhibited a similar activation of cyclin inhibitor diisopropylamine dichloroacetate was from Fisher Scien- proteins and progressed faster through the G2/M phase of the tific (cat. no. AAH6134103, Hampton, NH). cell cycle compared with control cells. Interestingly, arsenic Western Blotting, Quantitative Polymerase Chain Reaction, decreased the levels of cyclin proteins in both mouse livers and Transient Transfection and Luciferase Assay. Western and cultured HCC cells that were induced by Pdk4 de- blotting and quantitative polymerase chain reaction (qPCR) methods ficiency. Overall, our study revealed a novel function of were well established in our laboratory, as described previously (Yang at ASPET Journals on September 23, 2021 PDK4 in cell cycle control, suggesting a critical role of PDK4 et al., 2013b; Tsuchiya et al., 2015). All antibodies used for Western in HCC progression. blotting were as follows: cyclin D1 (Cell Signaling Technology), cyclin E1 (Cell Signaling Technology), cyclin A2 (Cell Signaling Technology), cyclin D3 (Cell Signaling Technology), cyclin H (Cell Signaling Materials and Methods Technology), cyclin E2 (Cell Signaling Technology), CDK2 (Santa Cruz Biotechnology, Dallas, TX), CDK4 (Santa Cruz Biotechnology), Mouse Study and Treatment. Wild-type (WT) and Shp2/2 mice b-actin (Cell Signaling Technology), glyceraldehyde 3-phosphate were described previously (Wang et al., 2003; Lee et al., 2015). dehydrogenase (Cell Signaling Technology), p21 (Santa Cruz Biotech- Pdk42/2 mice were kindly provided by Dr. Robert Harris (emeritus nology), Rb (Santa Cruz Biotechnology), pRB (Cell Signaling Technol- professor from Indiana University) (Hwang et al., 2009). All mice were ogy), MyB (Millipore, Billerica, MA), glycogen synthase kinase-3b of C57BL/6J background, were fed a standard rodent chow with free (Abcam, Cambridge, UK), SMAD4 (Thermo Fisher Scientific, Waltham, access to water, and were maintained on a 12-hour light/dark cycle MA), and E2F1 (Thermo Fisher Scientific). All qPCR experiments (lights on from 6 AM to 6 PM) in a temperature-controlled (23°C) and were repeated three times with each sample run in triplicate. All virus-free facility. Experiments were performed on male mice at the Western blot experiments were repeated three times. For the mouse age of 8–12 weeks unless stated otherwise (n 5 5 per group). study, five mouse samples were pooled per group and the samples were Experimental groups included WT control (WT Con), WT with arsenic loaded as singlets or duplicates. For the transient transfection and exposure (WT As), Pdk42/2 control (Pdk42/2 Con), and Pdk42/2 with luciferase reporter assay, Huh7 cells were transfected with the plasmids arsenic treatment (Pdk42/2 As). For arsenic treatment, mice were fed as indicated in the figure legends. Transfection was carried out using with 50 ppm NaAsO2 (Sigma-Aldrich, St. Louis, MO) via distilled Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Luciferase activity was drinking water, which was replaced every other day for 4 weeks measured and normalized against Renilla activity (Promega, Madison, (Zhang et al., 2016). RNA-seq and MeDIP were conducted at the WI). Experiments were done in three independent triplicate transfec- Microarray and Genomic Analysis Core Facility at the University of tion assays (Yang et al., 2013a). Utah (Salt Lake City, UT) as described previously (Smalling et al., Statistical Analysis. Data are shown as means 6 S.E.M. 2013). Immunohistochemistry staining was performed on paraffin- Statistical analysis was carried out using the t test for unpaired embedded liver-section slides with proliferating cell nuclear antigen data to compare the values between the two groups and using one- (PCNA) antibody (13110; Cell Signaling Technology, Danvers, MA). wayanalysisofvariancetocomparethevaluesamongmultiple ImmPACT DAB peroxidase (SK-4105; Vector Laboratories, Burlin- groups. P , 0.05 was considered statistically significant. game, CA) was used for color detection with a hematoxylin counter- stain. PCNA staining was conducted on five mice per group with three liver tissue sections per mouse. ImageJ software (National Institutes Results of Health, Bethesda, MD) was used to quantify positive PCNA staining. Protocols for animal use were approved by the Institutional An Unbiased Approach to Identify New Tumor 2/2 Animal Care Committee at the University of Connecticut. Suppressors Using Shp Mice. In the past decade, our PDK4 and Arsenic Crosstalk in HCC Cell Proliferation 191 published results using both in vivo mouse and in vitro cell were also consistently low in Shp2/2 mouse tumor tissue models, as well as human HCC specimens, revealed a tumor- (Shp2/2-T). suppressive role of SHP in HCC (He et al., 2008; Zhang et al., Importantly, PDK4 mRNA was significantly diminished in 2008, 2010; Yang et al., 2013b). More recently, our studies human HCC specimens relative to surrounding liver tissue established a crucial inhibitory feedback loop between SHP (Fig. 2E), suggesting that a low level of PDK4 is associated and Dnmt1 (Zhang and Wang, 2011; Zhang et al., 2012). with the development of HCC. Analysis of the Oncomine Tumor suppressors are generally downregulated in cancers database revealed an overall diminished expression of PDK4 by DNA hypermethylation via the activity of DNMTs (Jin in a variety of human cancers (Supplemental Fig. 1). It and Robertson, 2013). We propose that the upregulated should be noted that other PDK isoenzymes (PDK1–PDK3) Dnmts in Shp2/2 mice due to the loss of SHP inhibition will, did not show such universal downregulation (not shown), in turn, methylate other tumor suppressor genes (Fig. 1A). suggesting a specific regulatory role of PDK4 in HCC and From this regulatory model, we developed an unbiased other cancers. approach to identify putative new tumor suppressors, as Treatment of multiple HCC cell lines with the demethylating described below. agent 59-aza-29-deoxycytidine (Aza) or histone deacetylase in- Two genome-wide high-throughput analyses were con- hibitor trichostatin revealed an induction of PDK4 mRNA in ducted in Shp2/2 mice. MeDIP was used to characterize Hep3B and HepG2 cells by Aza and in Hep3B, MH97H, and alterations in DNA methylation of specific genes (Fig. 1B) MH97L cells by TSA (Fig. 2F). In addition, Aza decreased PDK4 Downloaded from and RNA-seq was used to determine changes in gene gene promoter methylation in HepG2 cells (Fig. 2G, left), which expression (Fig. 1C). By integrating both datasets using was in agreement with the increased PDK4 mRNA (Fig. 2G, bioinformatics tools and appropriate validation, we aimed at right). Overall, the results suggest that PDK4 downregulation in identifying genes that met the criteria by showing promoter HCC is associated with epigenetic silencing. hypermethylation and mRNA downregulation, the charac- Arsenic Repressed Hepatocyte Proliferation Induced teristics of tumor suppressors, in Shp2/2 mice. by Pdk4 Deficiency. To examine the in vivo effect of molpharm.aspetjournals.org Integrating RNA-Seq and MeDIP Revealed PDK4 as Pdk4 in hepatocyte proliferation and the impact of arsenic, a Potential Tumor Suppressor. Shp2/2 mice were orig- WT and Pdk42/2 mice were fed with arsenic for 2 weeks. inally generated by replacing their exon 1 with the b-gal/pG- Immunohistochemistry staining for the PCNA protein in Kneo cassette (Wang et al., 2002). The accuracy of RNA-seq arsenic-treated and untreated mice showed that Pdk42/2 mice was validated by the missing exon 1 of the Shp gene (Fig. 2A, exhibited greater hepatocyte proliferation compared with WT left, top) and the upregulation of a known SHP target gene, control mice. The liver sections of arsenic-treated mice showed early growth response 1, in Shp2/2 mice (Zhang et al., 2011a; decreased PCNA staining compared with clean water–treated Smalling et al., 2013) (Fig. 2A, left, bottom). MeDIP analysis controlmiceinbothWTandPdk42/2 animals (Fig. 3A). revealed enhanced DNA methylation in the Shp gene pro- To further investigate the molecular components responsi- at ASPET Journals on September 23, 2021 moter in Shp2/2 mice (Fig. 2A, right), further supporting our ble for the cell proliferation changes among test groups, cell proposed feedback regulatory loop between SHP and Dnmts cycle proteins were analyzed via Western blotting. We first (Fig. 1A). We integrated MeDIP and RNA-seq data and examined three major cyclins and observed that Pdk42/2 identified the top 10 genes that showed hypermethylation control livers had a drastic increase in cyclin E1 and A2 and downregulation in Shp2/2 mice (Fig. 2B). We chose to proteins compared with that of WT control livers (Fig. 3B). In focus on the PDK4 gene due to its striking hypermethylated contrast, arsenic treatment prevented the induction of cyclin promoter and downregulated expression in Shp2/2 mice E1 and A2 proteins in Pdk42/2 As mice compared with (Fig. 2C). In agreement with the RNA-seq results, qPCR Pdk42/2 Con mice (Fig. 3B). confirmed a marked reduction of Pdk4 mRNA in Shp2/2 We continued to analyze other cell cycle regulatory pro- livers that were collected from both young and old mice (aged teins by Western blotting to gain a better understanding of 2 and 13 months, respectively) (Fig. 2D). Pdk4 mRNA levels the overall changes. Interestingly, cyclin D2 and D3 levels were

Fig. 1. Regulatory model and unbiased approach to identify new tumor suppressor candidates in HCC. (A) A feedback inhibitory loop between Shp and Dnmts. SHP downregulation by loss of heterozygosity (LOH) in HCC releases SHP repression of Dnmt and increases Dnmt activity. Upregulated Dnmts further methylate SHP and other tumor suppressor genes. (B) MeDIP arrays. The Mouse Promoter 1.0R Array (Affymetrix, Santa Clara, CA) is a single array composed of over 4.6 million probes tiled to interrogate over 28,000 mouse promoter regions. (C) RNA-seq. Pol II RNAs were isolated by a 59-capped purification method and analyzed by RNA-seq. Two-month-old male mice were used for this experiment (n = 5 per group). 192 Choiniere et al. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 2. Integrating MeDIP and RNA-seq results identified PDK4 as a putative tumor suppressor in HCC. (A) (Left) Integrated Genome Browser visualization tracks from RNA-seq reads depict complete loss of Shp exon 1 in Shp2/2 mice. Early growth response 1 (Egr-1), a known target of Shp, is increased 8-fold in Shp2/2 versus WT livers. (Right) Enhanced DNA methylation signal (blue lines) in the Shp gene promoter in Shp2/2 versus WT mice. (B) Genes showed good correlation between promoter methylation and gene expression in Shp2/2 mice and Pdk4 was among the top 10 genes. (C) MeDIP and RNA-seq revealed Pdk4 promoter hypermethylation and gene downregulation in Shp2/2 livers. (D) qPCR of Pdk4 mRNA in 2- and 13-month-old WT, Shp2/2, and Shp2/2-T (tumor) livers (n = 5 per group). (E) qPCR of PDK4 mRNA in 21 pairs of human HCC specimens and the corresponding controls. (F) qPCR of PDK4 mRNA in HCC cells treated with Aza or trichostatin. (G) (Left) The methylated DNA levels of the PDK4 promoter CpG island were quantified using the EpiTect methyl-profiler qPCR primer assay (QIAGEN). (Right) qPCR of PDK4 mRNA. In the bar graphs, data are shown as means 6 S.E.M. (triplicate assays). *P , 0.05 versus corresponding controls. DMSO, dimethylsulfoxide; TSA, trichostatin. moderately increased in WT As versus WT Con mice (Fig. 3C). On hydroxyurea to synchronize all cells at the G0/G1 phase (Fig. the other hand, CDK2 and CDK4 were noticeably elevated in 4A). When PDK4-knockdown (shPDK4) cells were released into Pdk42/2 mice relative to WT mice. Among other cell cycle the cell cycle, they progressed faster through all phases of the regulators we analyzed, transcription factor E2F1 protein was cell cycle. At 6 hours, shPDK4 cells had a higher percentage in found to be highly induced in Pdk42/2 Con mice compared with the S phase compared with shControl cells. By the 9-hour and WT Con mice, and its induction was prevented by arsenic 12-hour time points, shPDK4 cells showed a greater transition treatment (Fig. 3D). Because E2F1 is a well established tran- to the G2/M phase compared with shCon cells. scriptional activator of cyclins (Wu et al., 2001), the similar Nocodazole was used to arrest Huh7 cells in the G2/M phase, expression pattern between E2F1 and cyclins E1 and A2 in where a higher percentage of shPDK4 cells accumulated in the Pdk42/2 mice suggests that E2F1 may be responsible for the G2/M phase compared with shCon cells (Fig. 4A). As time upregulation of both cyclins. progressed, shPDK4 cells appeared to maintain a constant high Knockdown of PDK4 Expedited HCC Cell Progression. percentage of G2/M cells compared with shCon cells (Fig. 4B). We next examined the effect of PDK4 in HCC cell proliferation Because cyclins E1 and A2 were the two most highly using in vitro cell models. Huh7 cells were treated with induced cyclins in Pdk42/2 liver (Fig. 3B), we examined their PDK4 and Arsenic Crosstalk in HCC Cell Proliferation 193 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 3. Pdk42/2 livers showed increased hepatocyte proliferation, which was diminished by arsenic treatment. (A) (Left) Immunohistochemistry staining for PCNA protein. Dark brown indicates positively stained nuclei. (Right) Histogram showing quantification of positive PCNA staining results. *P , 0.05 versus WT Con; #P , 0.05 versus Pdk42/2 Con. (B–D) Western blot of cyclin and CDK proteins and cell cycle modulators in WT and Pdk42/2 mice treated with arsenic (50 ppm for 1 month). Pooled samples (n = 5 per group) were run in duplicate lanes or single lane. Numbers indicate density relative to WT control (Con). protein expression in PDK4-knockdown Huh7 cells. Two that established a feedback regulatory loop between DNMTs knockdown cell lines were established with moderate (Fig. and SHP (Zhang and Wang, 2011; Zhang et al., 2012). Tumor 5A, top) to strong knockdown efficiency (Fig. 5A, bottom). In suppressors are often downregulated in cancers due to cells with moderate Pdk4 knockdown, small increases in their promoter hypermethylation. PDK4 was identified as cyclin A2, CDK2, and E2F1 were observed (Fig. 5B). In high a putative tumor suppressor because its expression was Pdk4-knockdown cells, a more striking elevation of cyclin E1, largely diminished in human HCC specimens and other CDK2, and E2F1 was observed (Fig. 5C). Therefore, the more cancers. efficient knockdown of PDK4 resulted in greater induction of cell One prime characteristic of tumor suppressor genes is cycle regulator proteins. As expected, E2F1 dose-dependently their ability to inhibit tumor cell growth. Thus, we exam- transactivated cyclin E1 promoter activity (Fig. 5D). ined the role of PDK4 in hepatic cell proliferation. Consis- The levels of cyclin E1 and A2 proteins were markedly tent with our hypothesis, Pdk4-deficient mouse liver showed diminished in E2f12/2 mouse liver compared with WT mice increased hepatocyte proliferation, as evidenced by the activa- (Fig. 5E). In addition, knockdown of E2F1 using shRNAs in tion and induction of cyclins. In addition, HCC cells with PDK4 Huh7 cells significantly decreased cyclin E1, cyclin A2, and knockdown exhibited a similar activation of cell cycle regula- E2F1 proteins (Fig. 5F). Furthermore, treatment of Huh7 cells tors, which was accompanied by enhanced cell cycle progres- with PDK4 inhibitor diisopropylamine dichloroacetate increased sion. It was noted that results in HCC cells were less striking cyclin E1, cyclin A2, and E2F1 proteins dose dependently (Fig. than in mouse livers, which was likely caused by the different 5G). Taken together, the results suggest an E2F1-dependent cellular environment in HCC cells versus mouse livers. Overall, activation of cyclins E1 and A2 by PDK4. these results strongly suggest PDK4 as an important new regulator of the cell cycle. Discussion To date, very little research has been done analyzing PDK4 outside of its known metabolic function. The Warburg effect This study aimed to identify potential new tumor suppres- suggests that cancer cells rely highly on glycolysis rather sor genes using Shp2/2 mice, based on our previous findings than oxidative phosphorylation (Liberti and Locasale, 2016). 194 Choiniere et al. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 4. FACS analysis showed rapid G2/M cell cycle progression in shPDK4 HCC cells. (A) shCTRL and shPDK4 cells synchronized in G1/S phase by hydroxyurea treatment (3 mM for 24 hours) followed by FACS. (B) shCTRL and shPDK4 cells synchronized in the G2/M phase by nocodazole (50 ng/ml for 16 hours) treatment followed by FACS. FACS, fluorescence-activated cell sorting.

Since PDK4 functions to inhibit PDC, which converts Previous studies have shown in different model organisms that pyruvate into acetyl-CoA and therefore feeds the citric acid inorganic arsenic treatment inhibits cell cycle progression and cycle, PDK4 is expected to interact with other proteins induces apoptosis (Sidhu et al., 2006; Wang et al., 2007). Our leading to the subsequent inhibition in cellular proliferation. results were in agreement with the prior observations. The Because the levels of multiple cyclins were highly induced by inhibitory effect of arsenic in HCC cell proliferation is in contrast PDK4 deficiency, it is postulated that PDK4 may repress the with the general knowledge that arsenic is often considered to expression of cyclins via interaction with other transcription have oncogenic properties by predisposed cells for cancer devel- factors. E2F1 is a major transcriptional activator of cyclins opment. We also noted that despite reduced hepatocyte pro- that we observed to be strongly induced in Pdk42/2 liver and liferation by arsenic treatment, the overall cyclin levels were not PDK4-knockdown HCC cells. Therefore, we examined a decreased. Future studies are warranted to fully understand the direct protein-protein interaction between PDK4 and E2F1 action of arsenic. The results suggest that the final outcome of by coimmunoprecipitation and Western blotting but failed to arsenic exposure would be largely depending on the cellular validate their interaction (not shown). As expected, E2F1 context. dose-dependently activated cyclin E1 promoter activity (Fig. In summary, this study shows that Pdk4 deficiency results 5E). Unfortunately, coexpression of PDK4 with E2F1 did not in increased hepatocyte proliferation as a consequence of alter E2F1 transactivation (not shown). These results sug- induction of cyclins and E2F1. These results suggest that gest that PDK4, a mitochondria protein, does not seem to act PDK4 may function as a potential tumor suppressor. Un- as a transcriptional factor to enhance E2F1 activity. It is fortunately, we are unable to establish an in vivo HCC likely that there are intermediate players between PDK4 model, because Pdk42/2 mice injected with a single dose of and E2F1. Future studies may focus on identifying the diethylnitrosamine died within a week. We are currently molecular intermediates leading to increased levels of cyclin investigating the molecular mechanisms that may explain E1 caused by Pdk4 deficiency. the causes of death. Nonetheless, identification of tumor In this study, arsenic was found to decrease cyclin protein levels suppressor genes and their molecular mechanisms will and in both mouse and cultured HCC cells. This decrease in cyclin has led to therapies and anticancer treatments. Future protein levels indicates a reduction in cellular proliferation. studies may focus on further elucidating the molecular PDK4 and Arsenic Crosstalk in HCC Cell Proliferation 195 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 5. Knockdown of PDK4 in HCC cells induced cell cycle proteins, which was diminished by arsenic. (A) Western blot of PDK4 protein and qPCR of PDK4 mRNA in Huh7 cells showing lower (top) and higher (bottom) knockdown efficiency (shPDK4L). (B) Western blot of cell cycle proteins in control (shCTRL) or shPDK4L cells in the presence or absence of arsenic (As) (15 mM for 24 hours). (C) Western blot of cell cycle proteins in shCTRL and shPDK4H cells. (D) Transient transfection assay to determine cyclin E1 promoter reporter activity by E2F1. (E) Western blot of protein expression in WT and E2f12/2 mouse liver. (F) Western blot of protein expression in Huh7 cells with E2F1 knockdown using two different shRNAs against E2F1. (G) Western blot of protein expression in Huh7 cells treated with PDK4 inhibitor diisopropylamine dichloroacetate (DADA). pathway in which Pdk4 deficiency increases the level of cyclin International Agency for Research on Cancer (2012). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans—Arsenic, Metals, Fibres, and Dusts: proteins, as well as the effects of arsenic and Pdk4 on apoptosis A Review of Human Carcinogens, vol 100C. International Agency for Research on in the liver. Cancer, Lyon, France. Jin B and Robertson KD (2013) DNA methyltransferases, DNA damage repair, and cancer. Adv Exp Med Biol 754:3–29. Authorship Contributions Kanda M, Sugimoto H, and Kodera Y (2015) Genetic and epigenetic aspects of ini- tiation and progression of hepatocellular carcinoma. World J Gastroenterol 21: Participated in research design: Choiniere, Wu, Wang. 10584–10597. Conducted experiments: Choiniere, Wu. Lee SM, Zhang Y, Tsuchiya H, Smalling R, Jetten AM, and Wang L (2015) Small Performed data analysis: Choiniere, Wang. heterodimer partner/neuronal PAS domain protein 2 axis regulates the oscillation of liver lipid metabolism. Hepatology 61:497–505. Wrote or contributed to the writing of the manuscript: Choiniere, Liberti MV and Locasale JW (2016) The Warburg effect: how does it benefit cancer Wang. cells? Trends Biochem Sci 41:211–218. Liu M, Jiang L, and Guan XY (2014) The genetic and epigenetic alterations in human – References hepatocellular carcinoma: a recent update. Protein Cell 5:673 691. Masaki T, Shiratori Y, Rengifo W, Igarashi K, Yamagata M, Kurokohchi K, Uchida N, Farhana L, Dawson MI, Leid M, Wang L, Moore DD, Liu G, Xia Z, and Fontana JA Miyauchi Y, Yoshiji H, Watanabe S, et al. (2003) Cyclins and cyclin-dependent (2007) Adamantyl-substituted retinoid-related molecules bind small heterodimer kinases: comparative study of hepatocellular carcinoma versus cirrhosis. Hepatology partner and modulate the Sin3A repressor. Cancer Res 67:318–325. 37:534–543. He N, Park K, Zhang Y, Huang J, Lu S, and Wang L (2008) Epigenetic inhibition of Polya D and Charlet L (2009) Environmental science: rising arsenic risk? Nat Geosci nuclear receptor small heterodimer partner is associated with and regulates he- 2:383–384. patocellular carcinoma growth. Gastroenterology 134:793–802. Rudraiah S, Zhang X, and Wang L (2016) Nuclear receptors as therapeutic targets in Hwang B, Jeoung NH, and Harris RA (2009) Pyruvate dehydrogenase kinase iso- liver disease: are we there yet? Annu Rev Pharmacol Toxicol 56:605–626. enzyme 4 (PDHK4) deficiency attenuates the long-term negative effects of a high- Scatena R (2012) Mitochondria and cancer: a growing role in apoptosis, cancer cell saturated fat diet. Biochem J 423:243–252. metabolism and dedifferentiation. Adv Exp Med Biol 942:287–308. 196 Choiniere et al.

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that fine tunes Mdm2 and SHP stability. FEBS Lett 586:1135–1140. molpharm.aspetjournals.org at ASPET Journals on September 23, 2021 Supplementary Figure 1

PDK4-deficiency results in expedited cellular proliferation through increased cyclins

Jonathan Choiniere, Jianguo Wu, and Li Wang

Molecular Pharmacology

Cancer Analysis Type by Cancer /Normal Bladder Cancer 1 Brain and CNS Cancer Breast Cancer 5 Cervical Cancer Colorectal Cancer 18 Esophageal Cancer 21 Gastric Cancer 2 Head and Neck Cancer 1 Kidney Cancer Leukemia Liver Cancer 13 Lung Cancer 7 Lymphoma Melanoma Myeloma Other Cancer 1 Ovarian Cancer 1 Pancreatic Cancer 2 Prostate Cancer 2 Sarcoma 4 348 Significant Unique Analyses 322 Total Unique Analyses

Supp. Fig. 1 PDK4 gene is down-regulated in various human cancers. The data from 48 different studies in Oncomine are analyzed. Cell color is determined by the best gene rank percentile for the analyses within the cell. Blue: downregulation; red: upregulation. Threshold (P value): 1x10-4; Threshold (Gene rank): Top 10%; Threshold (Fold change): 2.