The Role of Peroxisome Proliferator-Activated Receptor

The Pennsylvania State University

The Graduate School

The Huck Institutes for Life Sciences

THE ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR

β/δ IN VIRAL PROTEIN-INDUCED LIVER PATHOGENESIS

A Dissertation in

Molecular Medicine

Gayathri Balandaram

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2015

The dissertation of Gayathri Balandaram was reviewed and approved* by the following:

Jeffrey M. Peters Distinguished Professor of Molecular Toxicology and Carcinogenesis Dissertation Advisor Chair of Committee

Gary H. Perdew John T. and Paige S. Smith Professor in Agricultural Sciences

Connie J. Rogers Assistant Professor and Occupant of the Broadhurst Career Development Professorship for the Study of Health Promotion and Disease Prevention

Andrew Patterson Associate Professor of Molecular Toxicology

Adam B. Glick Associate Professor of Veterinary and Biomedical Sciences Chair, Molecular Medicine Program Associate Chair, Molecular Cellular and Integrative Biosciences

*Signatures are on file in the Graduate School

ABSTRACT

PPARβ/δ has an established role in attenuating preneoplastic conditions in the liver such as inflammation, steatosis, proliferation and oxidative stress pointing to a chemopreventive role for this nuclear receptor in the liver. However, not much is known about its part in orchestrating the progression of hepatocellular carcinoma. The present study examined the potential of this nuclear receptor in delaying the progression of chronic hepatocellular injury-induced liver cancer. In hepatitis B virus (HBV) transgenic mice, which develop progressive inflammation and cellular injury at the early stages (2-3 months of age), followed by hyperplasia, neoplasia and eventually hepatocellular carcinoma at later stages (12-18 months of age), long-term ligand activation (8 months) with PPARβ/δ ligand (GW0742), displayed a significant attenuation in tumor multiplicity and the average number of liver foci, which was not observed in HBV mice not treated with GW0742. Further, this attenuation in tumorigenesis was accompanied by a reduction in hepatic steatosis and markers of cellular proliferation CYCLIN D1 and c-

MYC as well as an increase in a marker of apoptosis, cleaved PARP. Ligand activation of PPARβ/δ attenuated mRNA expression of hepatic inflammatory cytokine Tnfa and serum ALT at the early stages of disease pathogenesis in these mice. Ligand activation of PPARβ/δ attenuated LPS-induced mRNA expression of Tnfa in primary Kupffer cell cultures from wild-type but not in Kupffer cells from Pparβ/δ-null mice. LPS-induced mRNA expression of Tnfa was also found to be attenuated in Pparβ/δ-DBM (DNA binding mutant form of PPARβ/δ) Kupffer cells treated with GW0742, indicating that the anti-inflammatory effects of ligand activated PPARβ/δ occurs independent of the nuclear receptor binding to its PPREs. These effects were not observed in hepatocytes. Further,

iii co-immunoprecipitation studies confirmed interaction of PPARβ/δ with p65 subunit of pro-inflammatory transcription factor NF-κB in LPS and GW0742 activated KC13-2

Kupffer cells. In the hepatitis C virus (HCV) transgenic mice expressing the core protein of the virus, preliminary histopathological analyses did not yield a conclusive role for

PPARβ/δ in modulating hepatic inflammation and steatosis. However, PPARβ/δ was found to significantly increase the life span of HCV wild-type mice compared to that of

HCV Pparβ/δ-null mice, independent of ligand activation of PPARβ/δ. Protein microarray displayed a consistent increase in Cofilin 1 and a decrease in SODD,

SOCS2 and p27Kip1 expression levels, as well as a decrease in phosphorylation of

PKCγ in livers of 18-month-old HCV Pparβ/δ-null mice compared to that of HCV wild- type mice. Combined, the present study confirms a hepatoprotective role for PPARβ/δ in mouse models of chronic hepatocellular injury-induced liver cancer and its potential as a possible therapeutic approach to treat hepatocarcinogenesis is promising and warrants further investigation.

TABLE OF CONTENTS

LIST OF FIGURES ...... vii LIST OF TABLES ...... viii ACKNOWLEDGEMENTS ...... ix Chapter 1 Literature review ...... 1 1.1 Introduction to nuclear hormone receptors and peroxisome proliferator-activated receptors ...... 1 1.1.1 Nuclear hormone receptors ...... 1 1.1.2 PPARα, PPARβ/δ and PPARγ ...... 5 1.2 The role of peroxisome proliferator-activated receptors in the liver ...... 8 1.2.1 Characteristics of PPARα and its function in the liver ...... 8 1.2.2 Characteristics of PPARγ and its function in adipose tissue ...... 10 1.2.3 Characteristics of PPARβ/δ and its function in the liver ...... 13 1.3 PPARβ/δ and carcinogenesis ...... 20 1.4 Hepatocarcinogenesis ...... 26 1.4.1 The liver and its physiological role ...... 26 1.4.1.1 General function structure of the liver ...... 26 1.4.1.2 Functions of parenchymal cells in the liver ...... 26 1.4.1.3 Functions of non-parenchymal cells in the liver ...... 29 1.4.2 Factors that predispose the liver to hepatocarcinogenesis ...... 31 1.4.2.1 Hepatic inflammatory cytokines and oxidative stress ...... 32 1.4.2.2 Hepatic steatosis ...... 34 1.4.2.3 Viral hepatitis ...... 35 1.4.3 Current mouse models of hepatitis viral protein-induced hepatocarcinogenesis ...... 37 1.5 PPARβ/δ and hepatocarcinogenesis ...... 40 1.6 Objective of the study and hypothesis ...... 42 Chapter 2 PPARβ/δ attenuates hepatic tumorigenesis in hepatitis B transgenic (HBV) mice……... ……………………………………………………………………………………...46 2.1 Abstract ...... 46 2.2 Introduction ...... 48 2.3 Materials and Methods ...... 51 2.4 Results ...... 58

2.4.1 PPARβ/δ-dependent signaling attenuates tumorigenesis in HBV mice ...... 58 2.4.2 PPARβ/δ-dependent signaling attenuates markers of proliferation and enhances apoptosis in HBV mice ...... 60 2.4.3 PPARβ/δ-dependent signaling attenuates hepatic steatosis in HBV mice ... 62 2.4.4 PPARβ/δ-dependent signaling attenuates hepatotoxicity and hepatic Tnfa in HBV mice ...... 64 2.4.5 PPARβ/δ-dependent attenuation of Tnfa is modulated in the Kupffer cells and not the hepatocytes ...... 66 2.4.6 PPARβ/δ-dependent attenuation of Tnfa in the KC13-2 Kupffer cell line is most likely mediated by binding of PPARβ/δ to NF-κB ...... 69 2.5 Discussion ...... 72 Chapter 3 PPARβ/δ improves survival of hepatitis C transgenic (HCV) mice ...... 81 3.1 Abstract ...... 81 3.2 Introduction ...... 83 3.3 Materials and Methods ...... 86 3.4 Results ...... 92 3.4.1 PPARβ/δ-dependent signaling improves survival in HCV mice in vivo ...... 92 3.4.2 PPARβ/δ-dependent signaling does not modulate hepatic inflammatory cell infiltration and steatosis in HCV mice in vivo ...... 95 3.4.3 Hepatic protein expression and phosphorylation in response to targeted disruption of PPARβ/δ function in HCV mice in vivo ...... 98 3.5 Discussion ...... 104 Chapter 4 Conclusions and Future Directions ...... 110 Bibliography ...... 131

LIST OF FIGURES

Figure 1-1: DNA Structure of nuclear hormone receptors ...... 3 Figure 1-2: Pathways proposed for PPARβ/δ-dependent attenuation of NF-κB activity 18 Figure 1-3: Hepatoprotective functions of PPARβ/δ ...... 42 Figure 1-4: Schematic of Hypothesis ...... 45 Figure 2-1: Ligand activation of PPARβ/δ attenuates liver tumorigenesis in HBV transgenic mice ...... 59 Figure 2-2: Ligand activation of PPARβ/δ attenuates markers of proliferation and increases apoptosis in livers of HBV transgenic mice ...... 61 Figure 2-3: Ligand activation of PPARβ/δ attenuates liver steatosis in HBV transgenic mice ...... 63 Figure 2-4: Ligand activation of PPARβ/δ attenuates hepatic Tnfa and a marker of liver damage in HBV transgenic mice ...... 65 Figure 2-5: Ligand activation of PPARβ/δ inhibits pro-inflammatory cytokine Tnfa mRNA expression in wild-type and a PPARβ/δ DNA binding mutant Kupffer cells but not in Pparβ/δ-null Kupffer cells. This effect is not observed in hepatocytes ...... 68 Figure 2-6: Ligand activation of PPARβ/δ inhibits pro-inflammatory cytokine Tnfa in the KC13-2 Kupffer cell line via protein-protein interaction with p65 subunit of NF-κB ...... 70 Figure 3-1: PPARβ/δ promotes survival in HCV transgenic mice ...... 94 Figure 3-2: PPARβ/δ-dependent modulation of hepatic inflammatory cytokines in HCV transgenic mice ...... 97 Figure 3-3: Western blot quantification of changes in microarray analysis of SOCS2 and p27Kip1 ...... 103 Figure 4-1: Schematic of potential pathway by which PPARβ/δ could modulate PKCα activity ...... 121 Figure 4-2: Schematic of potential pathway by which PPARβ/δ could modulate oxidative stress ...... 128

vii

LIST OF TABLES

Table 1-1: Peroxisome proliferator-activated receptor isoforms and their functions (↑- Increase, ↓- Decrease) ...... 8 Table 3-1: Histopathological analyses do not confirm a role for PPARβ/δ in modulating hepatic inflammation, steatosis and centrilobular hypertrophy in HCV mice in vivo ...... 96 Table 3-2: Partial lists from 2 anti-body microarrays of target proteins significantly modulated by PPARβ/δ as identified by KinexTM anti-body microarray analysis ...... 101 Table 3-3: List of combined fold changes in expression and phosphorylation of proteins with consistent expression patterns between antibody microarray I and II ...... 102

viii

ACKNOWLEDGEMENTS

I would like to take this opportunity to extend my sincere gratitude to my thesis advisor

Dr. Jeffrey M. Peters for his guidance, patience and invaluable advice during my PhD career. I am thankful to him for taking the time to review and revise my thesis. Without his help I would not have been able to complete my PhD. Apart from research guidance, he also goes the extra mile to make sure his students understand the importance of dedication and a strong work ethic. His constant encouragement for me to keep challenging myself and to always strive to be the best, has taught me not to settle for anything in life, and for that I am very thankful, Dr. Peters.

I would also like to thank my committee members Dr. Gary H. Perdew, Dr. Connie J.

Rogers and Dr. Andrew Patterson for being a part of my thesis committee and providing me with their time, valuable suggestions and guidance throughout all of my committee meetings.

I have been very privileged to be a part of the Peters Laboratory and have been fortunate to work with many great past and present members of this lab. I am grateful to past graduate students, Dr. Michael G. Borland and Dr. Bokai Zhu who took me under their wings when I first joined the laboratory and showed me the ropes. I would like to thank Dr. Jennifer Foreman, Dr. Prajakta Albrecht, Dr. Combiz Khozoie, Dr. Luis

Morales, Dr. Pei-Li Yao, Dr. Takayuki Koga, Dr. Prasad Krishnan and Dr. Kartick

Pramanik for all their valuable scientific advice, motivation, support, laughter and friendship throughout these five years that lightened the load of graduate school.

I would like to extend my gratitude to members of the Perdew laboratory for all their support, friendship and warmth, especially Dr. Iain A. Murray, Dr. Ann Kusnadi and Dr.

Nicholas Blazanin for always making time to discuss any research questions that I had.

I am also grateful to Dr. Ann Kusnadi for trying to make me a part of her family whenever I got terribly homesick. I am also thankful to Cherish McAulay for her invaluable administrative assistance and friendship.

I would also like to pay my deepest gratitude to my mother, Murugaiyan Tamilchelvi and my father Balandaram Nathan. Everything I have achieved this far would not have been possible if not for the constant love, motivation and high expectations that they have for me. My father has always been and will always be my pillar of strength. I am also greatly indebted to my husband, Vijay Navaneethakrishnan who has stood by my side not only through the good times, but also through times of frustration and self-doubt. I am truly blessed to have such a loving family and a wonderful support system. I owe my

PhD to my family.

Chapter 1 Literature review

1.1 Introduction to nuclear hormone receptors and peroxisome

proliferator-activated receptors

1.1.1 Nuclear hormone receptors

The discovery of hormones such as cortisone, thyroxine and estrogen in the early

1900s and the physiological reactions they induced in the body, opened doors to the investigation of the mechanisms by which small molecule hormones had the ability to modulate specific gene expression in target tissues. Then, for the first time in 1958,

Elwood Jensen utilizing the hormone estradiol showed that hormones act as ligands and can bind certain proteins and be localized in the nuclei of the cells where genes reside [1]. This led Jensen to the isolation and characterization of the first nuclear hormone receptor (NR), the estrogen receptor (ER) in 1967 [2-5]. Following this, subsequent discoveries of other receptors soon ensued, such as the glucocorticoid [6,

7], retinoic acid [8, 9] and thyroid hormone [10] receptors. In addition, there was also an effort to investigate orphan nuclear receptors for which regulatory ligands had not yet been discovered. The discovery of the retinoid X receptors (RXR) [11, 12] that was initially deemed an orphan nuclear receptor shed light on novel regulatory mechanisms not known before. The term “orphan” is used for nuclear receptors identified based on sequence homology to classic receptors, but for which activating ligands and their signaling pathways are still unknown. RXR heterodimerizes with a variety of nuclear receptors to modulate gene expression [13]. Hence, search for ligands that activate

1 orphan nuclear receptors and the mechanism by which these receptors regulate cellular processes is actively underway as they have been found to be involved in regulating various biological processes including but not limited to inflammation [14], metabolism

[15, 16] and cell proliferation [17].

Phylogenetic analyses of the nuclear receptor superfamily groups them into six different subfamilies (classes), even though, evolutionarily they share a common ancestor [18].

Class I receptors include thyroid hormone receptors (TRs), retinoic acid receptors

(RARs), vitamin D receptors (VDRs) and peroxisome proliferator-activated receptors

(PPARs). Class II receptors include retinoid X receptor (RXR), chicken ovalbumin upstream promoter transcription factor (COUP-TF) and hepatocyte nuclear factor 4

(HNF4). Steroid receptors such as the testis receptor (TR2), glucocorticoid receptor

(GR), androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER) and the estrogen-related receptor (ERR) come under class III. Classes IV, V and VI contain orphan nuclear receptors [19, 20]. Although orphan nuclear receptors can be found in the first three classes as well, these groups also contain receptors with known ligands that activate them.

The structure of a typical nuclear receptor consists of 4 main regions (Figure 1-1), namely, the A/B domain, the DNA-binding domain (DBD), the hinge region and the ligand-binding domain (LBD). The A/B region which is located close the variable NH2- terminal of the molecule often contains an AF-1 ligand independent transcriptional activation domain which can induce constitutive activation of the receptor. The AF-1

2 domain is also a target of phosphorylation by several molecules. For example, the mitogen-activated protein kinase (MAPK) has been shown to phosphorylate the ER at serine residue 118 as well as the PPARα at serine residues 12 and 21 to modulate gene expression [21, 22]. The DBD region is highly conserved. It is has the ability to recognize and bind to specific target sequences on genes (response elements or RE) and induce their transcription. It has two zinc fingers, one of which contains the “P” box, which is responsible for recognition and differentiation of core DNA motifs [23] and the other, which has the “D” box that is involved in dimerization [24]. A flexible hinge region connects the A/B domain and the DBD to the LBD. The LBD is located closer to the

COOH-terminal of the nuclear receptor and contains the AF-2 domain that is responsible for ligand dependent activation of the nuclear receptor. This region forms a hydrophobic ligand binding pocket which varies in size across different nuclear receptors to accommodate different types of ligands [25].

Figure 1-1: DNA Structure of nuclear hormone receptors. The general structure of a nuclear receptor consists of 4 main regions: an N-terminal transactivation domain (A/B), a DNA-binding domain (C), a hinge region (D) followed by a ligand binding domain (E/F) at the carboxyl terminal. The A/B domain includes the AF-1 region that is involved in ligand-independent transactivation and the E/F domain includes the AF-2 region involved in ligand-dependent activation.

Nuclear receptor ligands are often small lipophilic molecules that can fit into the hydrophobic pockets of the LBD. Endogenous ligands are usually hormones or pre-

3 cursors to hormones as well as physiological metabolites. Exogenous ligands include xenobiotics and synthetic drugs. Their hydrophobic nature allows them to cross the plasma membrane to bind nuclear receptors [26]. The general mechanism of ligand dependent activation of a nuclear receptor consists of a ligand binding to the LBD domain of a nuclear receptor, which is bound to co-repressors that render it inactive.

This binding causes the nuclear receptor to undergo a conformational change and subsequent release of the co-repressors and recruitment of co-activators, thus allowing binding to the associated RE as recognized by its DBD. This subsequently results in ligand dependent activation of gene expression. Nuclear receptors can also negatively modulate gene expression by 1) binding to RE along with co-repressors in absence of ligand, 2) forming transcriptionally inactive heterodimers, 3) ligand-induced binding to

REs that silence gene expression [19]. Recent studies, however, have demonstrated that nuclear receptors bind chromatin in a dynamic and transient manner as opposed to the previously described static manner, such that the duration of receptor-chromatin interaction lasts for mere seconds, with the nucleosome localization and/or chromatin structure playing a big role in the binding of nuclear receptors to chromatin. This binding can be greatly enhanced or diminished by the presence of endogenous and exogenous ligands [27-29]. Interestingly, nuclear receptors have also been found to attenuate the proliferative and inflammatory effects of transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) respectively in presence of ligands via transrepression [30, 31]. This mechanism of transrepression is thought to be occurring independent of binding of ligand activated nuclear receptor to its consensus RE, but more likely by forming a complex via physical interaction [32-34] with the transcription

4 factors and thus inhibiting their transcriptional activity. This transrepressive activity has also been found to be reciprocal whereby the transcription factors themselves can impose inhibitory effects on the nuclear receptors.

Deregulation of cellular signaling pathways can lead to a myriad of disease pathogenesis including but not limited to cancer. Hence, the opportunity to modulate cell signaling via the use of small molecules that can be generated synthetically, to target nuclear receptors to manipulate gene expression, as a potential avenue for drug therapy is very enticing. Some of the nuclear receptors with currently marketed drugs include that of TRα/β for treatment of hypothyroidism and obesity, ERα/β for breast cancer and osteoporosis, PPAR α, β/δ and γ for diabetes and obesity as well as RAR α,

β and γ for inflammatory skin disorders and leukemia [35].

1.1.2 PPARα, PPARβ/δ and PPARγ

Peroxisomes are single membrane organelles that contain oxidase and catalase activity involved in the generation and degradation of hydrogen peroxide [36]. In addition, they are well known for their roles in β-oxidation of fatty acids [37]. In the late 1960s, it was observed that administration of hypolipidermic drugs led to massive hepatomegaly in rodents [38-40]. Interestingly this phenomenon correlated with increased hepatic peroxisome levels and enhanced fatty-acid oxidation in rodents. Because the administration of five structurally dissimilar hypolipidermic drugs caused an increase in peroxisome proliferation and as well as hepatocarcinogenesis, they formed a novel class of chemical carcinogens [41]. Initially, there was interest in studying these

5 peroxisome proliferators because they seemed to be a good model for investigating non-genotoxic hepatocarcinogens. However, because they seemed to promote hepatocarcinogenesis independent of DNA damage [42], and because it was found that peroxisome proliferators bound to protein, and had the ability to modulate specific gene expression [43], it became likely to investigators that they could potentially be ligands activating a nuclear receptor. Then after the cloning of a mouse nuclear receptor

(mPPARα) that was activated by peroxisome proliferators in 1990 [44] came the discovery of the peroxisome proliferator-activated receptors, consisting of PPARα

(NR1C1), PPARβ/δ (NR1C2) and PPARγ (NR1C3), cloned from Xenopus laevis in

1992, which formed a new family in the nuclear receptor superfamily [45].

Peroxisome proliferator-activated receptors (PPAR) are soluble ligand activated transcription factors that regulate a variety of cellular processes including glucose and lipid metabolism, cellular differentiation, inflammation [46] and carcinogenesis [47].

Similar to other nuclear receptors they contain an amino terminal DBD and a carboxyl terminal LBD connected by a flexible hinge region. The DBD and the LBD of PPARs are highly conserved amongst the three PPAR isoforms. Upon ligand activation, PPARs undergo a conformational change, which releases their co-repressors like the silencing mediator of retinoid and thyroid hormone receptor (SMRT) and the nuclear receptor co- repressor (N-CoR) [48] allowing the receptor to heterodimerize with RXR. Together, the

PPAR-RXR complex recruits co-activators and binds to DNA in regions of target genes known as peroxisome proliferator-activated response elements (PPREs) [49]. PPREs are usually found in the 5’ flanking site of target genes and consist of a direct repeat of

6 the TGACCT separated by a single base, and also called a DR-1 site [50]. After binding to consensus PPRE sites, RNA polymerase is recruited and transcription of target genes is initiated. However, as mentioned above, it is likely that interaction of PPARs with PPREs occur in a dynamic and transient manner, whereby the nuclear receptor binds and dissociates from their respective response elements in a matter of seconds as opposed to static binding of minutes or hours [27, 28]. In addition, the non-random conformation of coiled chromatin in the nucleus can enhance proximity of distally located enhancers to promoters of nuclear receptor response elements, thus allowing nuclear receptors to bind sites of up to 200 kb distal to transcriptional start sites and initiate transcription of target genes [29, 51]. A recent study investigating the modulation of gene expression by PPARβ/δ on keratinocytes demonstrated that PPARβ/δ could modulate gene expression in several ways giving rise to eight potential response types.

What is more interesting is that not all the genes modulated by PPARβ/δ contained

PPREs in their promoter regions [52]. In addition, PPARs also have the ability to crosstalk with other transcription factors such as AP-1, NF-κB [34, 53] and specificity protein-1 (Sp-1) [54] via a transrepressive mechanism mentioned earlier. Also, it has been found that ligand activated transcriptional activity of PPARα and PPARγ is strongly inhibited by the expression of PPARβ/δ. The mechanism underlying this inhibitory effect of PPARβ/δ was shown to be the competitive binding of PPARβ/δ on PPREs along with co-repressors. This repressive effect of PPARβ/δ was abolished when a functional

PPARβ/δ mutant lacking DNA binding ability was used. This mechanism seems to be unique to PPARβ/δ, as PPARα and PPARγ were not found to be associated with co- repressors when bound to DNA [55]. Each of the PPARα, PPARβ/δ and PPARγ

7 receptors have their own tissue expression distribution and can be activated by both endogenous and synthetic ligands that are unique to them. This difference in expression and activation profiles can lead to different clinical outcomes. A summary of some of the well-known functions of the three PPAR isoforms and the ligands that activate them can be found on Table 1-1.

Table 1-1: Peroxisome proliferator-activated receptor isoforms and their functions (↑- Increase, ↓- Decrease)

1.2 The role of peroxisome proliferator-activated receptors in the liver

1.2.1 Characteristics of PPARα and its function in the liver

With the identification of mPPARα by Isseman et al. in 1990 there was a lot of controversy as to whether hypolipidermic drugs which activated PPARα and promoted hepatic tumorigenesis in rodents would induce the same effect in humans. However, liver biopsies from fibrate treated patients showed no signs of toxicity or carcinogenicity.

In an attempt to determine if differences in species accounted for the observed differences, the human PPARα was cloned from a human liver cDNA library in 1993

[56]. PPARα is highly expressed in metabolically active tissues such as liver, kidney,

8 intestinal mucosa [57] and skeletal muscles [58]. Endogenous ligands of PPARα include endogenous saturated and unsaturated fatty acids, Leukotriene B4 (LTB4), 8-hydroxy- eicosatetraenoic acid (8-HETE), Prostaglandin D1 and D2 [59, 60]. Apart from these a variety synthetic ligands have been generated to target the activation of PPARα such as

Clofibrate, Bezafibrate, Ciprofibrate, Wy-14643, Perfluorooctanoic acid (PFOA),

Perfluorooctanesulfonic acid (PFOS) and GW7647 [61]. The degradation of a pro- inflammatory molecule LTB4 that activates PPARα via a negative feedback loop [62], the delay in would healing in skin keratinocytes in Pparα-null mice due to impaired recruitment of inflammatory cells, the inhibition of pro-inflammatory inducible-nitric oxide synthase (iNOS) by Wy-14643 activated PPARα in macrophages [63], and the attenuation of inflammatory gene expression in vascular walls by fibrate treatment [53], all collectively point to an anti-inflammatory role for PPARα. PPARα has also been found to play a role in glucose homeostasis and insulin resistance.

However, one of the most important and best studied functions of PPARα is that of the regulation of fatty acid uptake and catabolism, which may explain the high level of expression of PPARα in metabolically active tissues. Interestingly the activation of

PPARα is mainly manifested in the liver. The activation of PPARα in the liver induces expression of target genes involved in fatty acid uptake such as the fatty acid transporter CD36 (FAT/CD36), fatty acid binding protein 1 (FABP-1) and carnitine palmitoyl transferase 1 (CPT-1) as well as target genes involved in fatty acid oxidation like acyl-CoA oxidase 1(ACOX-1), acyl-CoA synthase, (ACS), acyl-CoA dehydrogenase

(ACD) and acetyl-CoA acyltransferase 2 (ACAA2) [59, 64, 65]. In addition, PPARα is

9 also believed to regulate hepatic glucose homeostasis by modulating carbohydrate metabolism [66].

Although it has been well established that the activation of PPARα promotes hepatic peroxisome and hepatocyte proliferation, followed by induction of hepatocellular carcinoma in mice [67-69] this phenomenon was not observed in humans. It has been proposed that the PPARα-dependent regulation of microRNA let7c induces the expression of C-MYC, which contributes to hepatocyte proliferation and hepatocellular carcinoma [70]. The lower expression of hepatic PPARα and the differential induction of target genes [59] in humans compared to mice could potentially explain the species difference in pathogenesis [71, 72].

1.2.2 Characteristics of PPARγ and its function in adipose tissue

After the identification of PPARγ from Xenopus laevis, came the identification of mPPARγ in 1993 [73] followed by hPPARγ in 1995 [74]. PPARγ is highly expressed in adipose tissue, large intestines [75] and immune cells such as macrophages and dendritic cells [76]. Due to alternative splicing PPARγ exists in two isoforms, namely,

PPARγ1 and PPARγ2 [77, 78]. Some well-characterized endogenous ligands for

PPARγ include poly-unsaturated fatty acids (PUFAs), 15-hydroxy-eicosatetraenoic acid

(15-HETE), 13-hydroxy-octadecadienoic acid (13-HODE), prostaglandin (PGJ2) and its derivative 15-deoxy-Delta 12-14-PGJ2 [79, 80]. Whereas, compounds such as

L766499, S26948 and the thiazolidinediones like troglitazone and rosiglitazone fall under the group of highly specific synthetic ligands for PPARγ [61, 81].

One of the very well-studied functions of PPARγ is in regulating adipogenesis. PPARγ has been shown to promote adipocyte differentiation in different fibroblast cells lines

[82]. The inability of Pparγ-null mice to generate adipocytes in vivo [83], and the progressive development of lipodystrophy in mice [84] and humans [85] lacking functional PPARγ, point to the importance of this receptor in development and survival of mature adipocytes. Activation of PPARγ is also involved in uptake of fatty acids via up-regulation of lipoprotein lipase (LPL) both in vitro and in vivo [86] and retaining them in adipocytes via induction of phosphoenolpyruvate carboxykinase (PEPCK) [87].

Recent work have shown PPARγ is expressed prenatally in a white adipose tissue progenitor pool in mice [88] and that ligand activation of PPARγ promotes the transition of adipose progenitors to adipocytes by reducing the expression of adipogenic stem cell markers Sca1, CD29 and CD24 [89] thus, demonstrating a role for PPARγ in regulating adipocyte renewal. In fact, there are numerous studies, which show that PPARγ activity promotes adipocyte differentiation [83, 90-92]. PPARγ has also been demonstrated to convert non-adipose cells to cells with an adipocytic phenotype [93, 94]. Additionally, activation of PPARγ has been shown to increase insulin sensitivity by directly up- regulating glucose transporter type 4 (GLUT4) [95] and c-Cbl-associated protein (CAP), which has allowed this receptor to be targeted for treating insulin resistance [96].

Recent studies have also showed that adipocyte fatty-acid binding protein-dependent degradation of PPARγ, correlates with an increase in insulin resistance [97, 98].

Being highly expressed in macrophages, the activation of PPARγ in these cells can attenuate inflammatory signaling by negatively regulating the expression of

11 inflammatory cytokines [99]. It is most likely that the underlying mechanism for this is via repression of pro-inflammatory signaling of transcription factors such as AP-1, signal transducer and activator of transcription (STAT) and NF-κB [100]. Because of their anti- inflammatory properties, ligands of PPARγ are also targeted for treatment of inflammatory diseases like ulcerative colitis [101, 102]. Apart from its role in regulating adipogenesis, insulin sensitivity and inflammation, PPARγ has also been strongly linked to anti-fibrotic activities. Reduced expression of PPARγ or targeted deletion of PPARγ, frequently coincide with enhanced fibrosis [103-106]. Similarly, ligand activation of

PPARγ increased its expression and attenuated fibrotic markers α-SMA, type-1 collagen and connective tissue growth factor (CTGF) in dermal fibroblasts scleroderma lesions in culture in vitro [107] and was also found to inhibit fibrogenesis in animal models in-vivo

[108, 109].

It is not surprising that given the adipogenic potential of PPARγ, hepatic expression of this receptor in the liver contributes to hepatic steatosis [110] with exacerbation of the condition upon ligand activation. The observation that PPARγ ligand activation in Pparγ- null mice, do not exhibit hepatic steatosis is strong evidence that this condition is the result of modulation of lipogenic genes by PPARγ [111]. Consequently elevated expression levels of PPARγ have been observed in steatotic livers of mice fed a high-fat diet [112].

1.2.3 Characteristics of PPARβ/δ and its function in the liver

The identification of PPARβ/δ in Xenopus laevis led to the cloning of human [113] and mouse [114-116] PPARβ/δ in the early 1900s. Although, not as extensively studied as

PPARα and PPARγ, the finding that it improved obese conditions led to a lot of interest in studying this nuclear receptor. The last decade has seen prolific research in exploring this nuclear receptor. PPARβ/δ is found to be highly expressed in the colon, small intestines, liver and keratinocytes, and localized more in the nucleus than in the cytosol

[117]. Endogenous ligands of PPARβ/δ include arachidonic acid, linoleic acid and oleic acid whereas synthetic ligands include L-165041, GW501516 and GW0742 [118].

PPARβ/δ has been well studied for its role in skeletal muscle metabolism. Several in vitro experiments have shown that activation of PPARβ/δ up-regulates genes involved in fatty acid uptake, processing and breakdown in cultured myotubes and skeletal muscle cells [119, 120]. Gain of function experiments have shown that expression of

PPARβ/δ contributes to reduced lipid accumulation in serum and adipose tissue resulting in a leaner phenotype in mice in vivo. Ligand activation of PPARβ/δ in obese mice also attenuated obesity by up regulation genes long chain acyl-CoA synthetase

(LCAS), acyl-Co-A oxidase (AOX), muscle carnitine palmitoyltransferase-1 (mCPT-1) and long chain acyl-CoA dehydrogenase (LCAD) that are involved in fatty acid β- oxidation as well as uncoupling proteins 1 and 3 (UCP1 and UCP3). Reduced energy coupling and increased obesity in Pparβ/δ-null mice fed a high fat diet indicate that regulation of fatty acid catabolism in obesity is dependent on PPARβ/δ [121]. Although the earliest and well-known physiological role of PPARβ/δ is to act as lipid sensors in

13 regulating metabolism of fatty acids and cholesterol, there are now other significant roles that this nuclear receptor has been found to have.

Ligand activation of PPARβ/δ has been shown to attenuate proliferation in a variety cancer models such as breast carcinoma (MCF-7), melanoma (UACC903) [122], colon carcinoma (HCT116), hepatoma (HepG2) [123] and lung carcinoma (A549 NSC) [124] cell lines in vitro. The skin is a model with the most convincing evidence of the anti- proliferative effects of PPARβ/δ. PPARβ/δ expression in dermal fibroblasts attenuates proliferative potential of keratinocytes via autocrine signaling. The underlying mechanism for this was shown to be PPARβ/δ-dependent inhibition of Il-β and subsequent attenuation of TGF-activated kinase 1 (TAK1) and AP-1 activity [125].

Activation of PPARβ/δ was shown to inhibit cell proliferation in primary mouse keratinocytes and the human keratinocyte cell line N/TERT-1 by blocking the G1/S phase transition of cell cycle [126]. Enhanced keratinocyte proliferation of Pparβ/δ-null keratinocytes and Pparβ/δ-null keratinocytes treated with 12-O-tetradecanoylphorbol-

13-acetate (TPA) compared to their wild-type controls, further strengthens the anti- proliferative role of PPARβ/δ. This effect is mediated by PPARβ/δ target gene ubiquitin

C (UbC), which increases the turnover of protein kinase C alpha (PKC α) that subsequently reduces activities of downstream MAPK/ extracellular-signal-regulated kinase (ERK) pathway known to induce cell proliferation [127]. The anti-proliferative effects of PPARβ/δ have also been shown in vivo using Pparβ/δ-null mice. In a study utilizing HRAS expressing wild-type and Pparβ/δ-null keratinocytes as a model of

Harvey rat sarcoma virus (Hras) dependent tumorigenesis, PPARβ/δ-dependent

14 signaling was found to attenuate proliferation by inducing a G2/M arrest. The mechanism underlying this mitotic block was identified as repression of E2F activity brought about by direct binding of ligand activated PPARβ/δ to p130/107 complex, which subsequently enhances nuclear translocation and promoter recruitment to repressor E2F4 binding sites. This results in inhibition of E2F1, which consequently attenuates mitosis [128]. Interestingly ligand activation of PPARβ/δ was also seen promoting keratinocyte terminal differentiation as well in the same model, via induction of differentiation markers transglutaminase-I (TG-I), involucrin, small proline-rich protein

1A (SPR1A), small proline-rich protein 2H (SPR2H) and adipocyte differentiation-related protein (ADRP) [129]. This supports similar findings from another group that used a different ligand in primary keratinocytes [130]. Activation of PPARβ/δ also promotes wound-healing in vivo likely via modulation of transforming growth factor (TGF)-β1 signaling [131]. Additionally activation of PPARβ/δ has also been found to inhibit proliferation in rat neonatal cardiac fibroblasts via induction of cell cycle inhibitor gene

G0/G1 switch gene 2 (G0S2), which most likely is a direct target gene of PPARβ/δ

[132].

There is abundant literature showing that PPARβ/δ is a significant player in modulating inflammation [133-137]. Ligand activation of PPARβ/δ has been shown to attenuate inflammatory signaling in a number of different models [138-143]. PPARβ/δ is highly expressed in both human and murine macrophages and its activation by ligand has been shown to turn on the switch of macrophages from a pro-inflammatory “M1” to an anti-inflammatory “M2” state [144]. In a zymosan induced non-septic shock model,

15 activation of PPARβ/δ attenuated inflammatory cytokines tumor necrosis factor alpha

(TNFa), interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) by reducing

NF-κB activity brought about by inhibition of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) degradation [145]. Also, in a cecal ligation and puncture (CLP) mouse model PPARβ/δ attenuated plasma TNFa, interleukin-6 (IL-6),

IL-1β and monocyte chemoattractant protein-1 (MCP-1) by negatively modulating NF-κB signaling. This study further went to show that PPARβ/δ agonist activity reduced the binding of the p65 subunit of NF-κB to TNFa promoter [146]. Using Ldlr−/− mice fed an atherogenic diet, to induce atherosclerosis, it was found that activation of PPARβ/δ with ligand attenuated the expression of pro-inflammatory cytokines MCP-1, TNFa and intercellular adhesion molecule-1 (ICAM-1) in the aorta as well as TNFa and cluster of differentiation 36 (CD36) in the peritoneal macrophages [147]. A recent study, using

PPARβ/δ antagonist has shown that PPARβ/δ is required for the anti-inflammatory inhibition of TNFa, IL-1β and iNOS in a murine spinal cord injury model [148]. Thus, it is clear that PPARβ/δ has a potent anti-inflammatory role and there is also a strong consensus in the field that PPARβ/δ mediates its anti-inflammatory activities by modulating the activities of the NF-κB transcription factor. However, the underlying mechanism by which this occurs is still not clear. A few different mechanisms have been proposed. One study showed that PPARβ/δ attenuates LPS induced NF-κB activity by preventing upstream ERK1/2 phosphorylation in white adipose tissue of Zucker diabetic fatty rats [149]. Another study demonstrated that PPARβ/δ attenuated LPS induced

TNFa production by inhibiting the degradation of NF-κB inhibitors, IκBα and IκBβ, which prevents nuclear translocation, DNA-binding and subsequent transcription of NF-κB

16 target genes [150]. This study also went on to show the importance of the ligand-binding domain of PPARβ/δ in mediating anti-inflammatory activities, as a mutation in this domain, did not significantly attenuate LPS-induced TNFa. Interestingly, a recent study has demonstrated that PPARβ/δ-dependent signaling in an acute lung injury model, not only attenuated ERK 1/2 phosphorylation and NF-κB expression and activity, in addition, it also increased expression levels of NF-κB inhibitor IκB [151]. On the other hand, another group found contradictory results, whereby PPARβ/δ was not found to modulate NF-κB activity by inhibiting IκBα and IκBβ proteolysis. Rather PPARβ/δ was found to physically interact with the NF-κB transcription factor, and this was proposed as a potential mechanism by which PPARβ/δ attenuated LPS induced NF-κB activity

[152]. Interestingly, there have been other studies by other independent laboratories that have demonstrated similar findings, which support this hypothesis [153-155].

Interaction with the B-cell lymphoma 6 (BCL6) transcriptional repressor is another mechanism by which PPARβ/δ has been demonstrated to modulate inflammatory activities of NF-κB and has been shown to be required for PPARβ/δ-dependent modulation of NF-κB activity [156]. Studies show that in the absence of ligand, PPARβ/δ is bound to and sequesters BCL6 co-repressors, and upon ligand activation, PPARβ/δ releases BCL6 which binds NF-κB-dependent promoters to inhibit transcriptional activity of inflammatory molecules [157, 158]. Interestingly, another study demonstrated that upon ligand activation, PPARβ/δ and BCL6 are both recruited to NF-κB-dependent target genes to repress transcription [146]. More recently, another mechanism was demonstrated, whereby, upon ligand activation, PPARβ/δ indirectly attenuates acetylation of p65 subunit of NF-κB by increasing AMPK activity and subsequent

17 phosphorylation of p300 transcriptional co-activator that is involved in acetylation of the p65 subunit [159]. Thus, it seems likely that PPARβ/δ might modulate NF-κB-dependent inflammatory signaling by more than one pathway and further studies are needed to shed light on these potential mechanisms (Figure 1-2). It is also possible that the discrepancies observed between different laboratories are due to the types of models that were used in these studies.

Figure 1-2: Pathways proposed for PPARβ/δ-dependent attenuation of NF- κB activity. A summary of the current mechanisms proposed to explain the PPARβ/δ- dependent attenuation of inflammatory signaling via modulation of NF-κB activity. Briefly, PPARβ/δ is thought to attenuate NF-κB activity by 1) reducing ERK1/2 activity, 2) increasing recruitment of BCL6 co-repressor to NF-κB response elements, 3) reducing the degradation of IκB inhibitory proteins, 4) reducing acetylation of NF-κB and 5) reducing promoter occupancy of NF-κB at its response elements by introducing steric hindrance brought about by PPARβ/δ-NF-κB protein-protein interaction. (↑- Increase, ↓- Decrease)

There is strong evidence in the literature that PPARβ/δ plays a protective role in the liver. For example, previous studies from our laboratory showed that PPARβ/δ was hepatoprotective in a chemically-induced liver toxicity model of azoxymethane and carbon tetrachloride (CCl4) treated mice, by reducing bile duct and regenerative

18 hyperplasia, serum alanine aminotransferase (ALT) and NF-κB activity [160]. In a follow up study it was shown that activation of PPARβ/δ attenuates hepatotoxicity and inflammation by inhibiting TNFa and MCP-1. Interestingly, it was found that PPARβ/δ did not mediate these anti-inflammatory effects via hepatocytes or stellate cells, indicating other cell types such as the pro-inflammatory Kupffer cells might be the cell- type responsible [140]. In a recent study it was shown that overexpression of PPARβ/δ and its activation by ligand attenuated TNFa and oxidative stress in a copper-induced liver injury mouse model and this effect was not seen in mice treated with PPARβ/δ antagonist [161]. In a non-alcoholic steatohepatitis model of mice fed a methionine and choline deficient diet, PPARβ/δ ligand attenuated inflammation by down-regulating

MCP-1, TNFa, IL-1β, IL-6 and NF-κB expression as well as pro-inflammatory lipoperoxide levels [162]. The same study also observed reduced hepatic steatosis in mice treated with PPARβ/δ ligand. A recent transcriptional profiling of livers of wild-type and Pparβ/δ-null mice showed that the absence of PPARβ/δ in the liver enhanced pathways of innate immunity, inflammation, as well as Kupffer cell marker genes [163].

In addition, PPARβ/δ has also been found to protect against fibrosis in a cholestasis induced liver injury mouse model, which also coincided with a significant reduction in hepatic TNFa and IL-1β expression [164]. All of which point towards an anti- inflammatory role for PPARβ/δ in the liver likely mediated by modulation of NF-κB signaling in the Kupffer cells.

There are also ample studies indicating that the anti-inflammatory activities of PPARβ/δ in the liver can contribute to reduced hepatic steatosis [165]. In fact, in the

19 transcriptional profiling study mentioned above, Pparβ/δ-null mice displayed a decreased expression of genes required for lipoprotein metabolism in the liver, which coincided with significantly increased plasma triglyceride levels. It was also shown that in mice with a Kupffer cell specific deletion of PPARβ/δ, there was increased hepatic steatosis and hepatic triglyceride levels [166]. Another mechanism for PPARβ/δ- dependent attenuation of hepatic steatosis has also been described. For example, one study showed that the in obese mice, activation of PPARβ/δ, up-regulated its target gene insulin-induced gene-1 (Insig-1) in the liver, which prevented the processing and nuclear localization of sterol regulatory element-binding protein-1 (SREBP-1), resulting in reduced expression of lipogenic genes [167].

1.3 PPARβ/δ and carcinogenesis

The six hallmarks of cancer being; 1) self-sufficiency in growth signals, 2) limitless replicative potential, 3) evasion of apoptosis, 4) insensitivity to anti-growth signals, 5) sustained angiogenesis and 6) tissue invasion and metastasis were coined by Weinberg and Hanahan in 2000 [168]. Almost a decade later, they introduced four other characteristics to this list as emerging hallmarks, namely, 1) evasion of immune destruction, 2) tumor-promoting inflammation, 3) genomic instability and mutations and

4) deregulation of cellular energetics [169]. As expected, there has been a lot of effort to identify potential molecular targets to modulate one or more of these hallmarks as therapeutic approaches to treat various types of carcinogenesis. Given the beneficial role of PPARβ/δ in attenuating inflammation and proliferation as well as positively modulating fatty acid metabolism, it seemed likely that targeting PPARβ/δ dependent

20 signaling could prove promising in chemoprevention. However, the role of PPARβ/δ in carcinogenesis is highly controversial and the proposed mechanisms for tumor promotion and tumor prevention by PPARβ/δ in modulating some of these hallmarks will be discussed below. Most of the extensive studies utilizing in vivo mouse models to investigate the role of PPARβ/δ in carcinogenesis have been in colon and skin tumorigenesis models. Hence, I will discuss the role of PPARβ/δ in these two models to better explain the controversial findings.

An initial finding that PPARβ/δ expression was increased in the skin of psoriatic patients and psoriatic lesions [170-172], and decreased in expression with the correction of psoriasis led to the hypothesis that PPARβ/δ plays a role in promoting the hyper- proliferative condition of psoriasis and potentially skin tumorigenesis. However, because psoriasis occurs in a setting of chronic inflammation, and PPARβ/δ has been shown to be up-regulated by inflammatory cytokines via the AP-1 pathway [173], its presence in psoriatic lesion could most likely be to attenuate the inflammation. As has been discussed before, PPARβ/δ has been demonstrated to have anti-inflammatory activities, and in support of this idea, it was observed that anti-inflammatory treatment strategies in psoriatic patients resulted in reduced PPARβ/δ expression [174].

A recent study demonstrated that in a UV-induced skin cancer model, PPARβ/δ promotes skin tumorigenesis via expression and activation of proto-oncogene tyrosine- protein kinase sarcoma (Src) and subsequent downstream epidermal growth factor receptor (EGFR)/ERK 1/2 signaling enhancing epithelial to mesenchymal transition.

This observation was not detected in the presence of PPARβ/δ antagonist [175].

However, the PPARβ/δ antagonist GSK0660 that was used in this study absorbs UV light and was applied topically on the dorsal skin of mice prior to UV irradiation. Thus, it is highly possible that the antagonist itself prevented Src expression and its subsequent activation in vivo by absorbing the UV irradiation. In this study, the antagonist was also used in high millimolar doses instead of in micromolar quantities, which could lead to other non-specific interactions. In fact, there is overwhelming evidence in the literature that PPARβ/δ dependent signaling contributes to attenuation of skin tumorigenesis. A number of different mechanisms have been proposed for PPARβ/δ dependent attenuation of skin tumorigenesis. As mentioned before, PPARβ/δ attenuates skin hyperplasia by inducing ubiquitin dependent turnover of PKC α, which attenuates the downstream MAPK/ERK kinase pathway, resulting in attenuation of proliferation [127].

Also, in addition to a PPARβ/δ-dependent inhibition of proliferation in Hras expressing wild-type keratinocytes as mentioned earlier, the findings of the study also correlated with skin tumors from wild-type mice exhibiting reduced HRAS expression and a lower mitotic index compared to that of Pparβ/δ-null samples [128]. In a more recent study, it was shown that PPARβ/δ promotes Hras-induced senescence by inhibiting phosphoinositide-dependent protein kinase-1 (PDPK1) and integrin-linked kinase (ILK) expression levels, which subsequently attenuates the PI3K/ protein kinase B (AKT) pathway that has been shown to inhibit senescence in keratinocytes in vitro. These findings were also confirmed in skin tumors in vivo [176]. In another study by the same group, utilizing a model of Hras induced (endoplasmic reticulum) ER stress, investigators showed that in vitro 1) HRAS promotes ER stress and its associated

22 unfolding protein response (UPR), contributing to decreased cellular senescence by increasing the p-AKT/ mammalian (or mechanistic) target of rapamycin (mTOR) signaling pathway and that 2) PPARβ/δ attenuates the increase in Hras induced ER stress by inhibiting p-AKT / mTOR activity, thereby contributing to increased senescence. Importantly in a dimethylbenz (a) anthracene (DMBA) induced skin tumor model, the study further went to show increased skin tumorigenesis and reduced senescence in skin tumors Pparβ/δ-null mice compared to wild-type controls [177].

The role of PPARβ/δ in colon carcinogenesis has been actively investigated in the last decade, but it is still hard to reach a consensus as to whether PPARβ/δ plays a pro- tumorigenic or an anti-tumorigenic role in colon carcinogenesis. The finding of increased expression of PPARβ/δ in colon tumors compared to adjacent non-tumor tissues and the subsequent link of PPARβ/δ to be a direct target of the β catenin/ transcription factor

4 (TCF4) pathway that promotes colon tumorigenesis put forth the initial hypothesis that

PPARβ/δ could promote tumorigenesis in the colon [178]. This study proposed that adenomatous polyposis coli (APC) deficiency up-regulates PPARβ/δ expression via the

β catenin-TCF4 pathway similar to the induction of known proto-oncogenes of colon tumorigenesis such as Cyclin d1 and c-Myc by this pathway [179, 180]. However, other more recent studies utilizing Apcmin mice have shown that PPARβ/δ is not a target of the

APC/β catenin pathway and whilst CYCLIN D1 was overexpressed in intestinal adenomas, no difference was observed in PPARβ/δ expression between normal and tumor tissues [181, 182]. In support of this finding, intestinal specific deletion of β

23 catenin in mice attenuated CYCLIN D1 and C-MYC expression but did not modulate

PPARβ/δ expression [183].

In another recent study, human colorectal carcinoma samples, displayed a high expression of PPARβ/δ with Cyclooxygenase-2 (COX-2) and went further to show that tissues with increased PPARβ/δ and COX-2 expression together also displayed a high vascular endothelial growth factor-A (VEGF-A) expression that positively correlated with increased microvessel density and venous vessel invasion [184]. In a follow-up study, the same group showed that the combined expression of PPARβ/δ with COX-2 resulted in high incidence of liver metastasis and poor outcomes for colorectal cancer patients

[185]. In contrast, in a 15 year long-term follow up study of 141 primary rectal cancer patients, it was found that 1) PPARβ/δ was significantly related to reduced levels of a marker of cellular proliferation Ki67, 2) there was no correlation between PPARβ/δ and

COX-2 expression and that 3) High levels of PPARβ/δ expression in primary cancers promoted patient survival [186]. Other studies have observed an increased expression of VEGF, through a PPARβ/δ dependent mechanism that has been directly linked inhibition of apoptosis and promotion of cell survival via induction of phosphorylated

AKT (p-AKT) [187, 188]. However, studies from our laboratory did not find any of the above-mentioned changes in VEGF, COX-2 or p-AKT levels [123].

PPARγ mediates apoptosis in colorectal cancer cell lines via attenuation of survivin and induction of caspase-3 activity. Studies have shown that PPARβ/δ crosstalk with PPARγ

[189] suppress its apoptotic effect, hence promoting survival and conferring resistance

24 to colorectal cancer cell lines [190]. However, studies have also shown that ligand activation of PPARβ/δ did not modulate apoptosis in colon carcinoma cell line HT-29 and that the similar expression in cells that did and did not respond to PPARγ mediated apoptosis likely indicates PPARβ/δ does not modulate PPARγ activity in mediating apoptosis [181, 191]. Also, in a chemically induced colon carcinogenesis model in vivo, activation of PPARβ/δ attenuated colon polyp multiplicity by inducing apoptosis and up- regulating the expression differentiation markers fatty acid-binding protein (FABP), and cathepsin E. These effects were not observed in Pparβ/δ-null mice [192].

Hence, it can be seen that even in the two most well studied models of carcinogenesis there is conflicting data that does not allow for a strong consensus as to whether or not

PPARβ/δ attenuates tumorigenesis. However, the role of PPARβ/δ does seem to lean towards a more protective role against skin tumorigenesis. Reasons for the observed discrepancies could lie in the differences in types of animal models and cell lines, treatment durations, and types and doses of agonists used. Also, some of these studies used immunohistochemistry to detect PPARβ/δ expression levels, which is not a reliable method for protein quantification.

1.4 Hepatocarcinogenesis

1.4.1 The liver and its physiological role

1.4.1.1 General function structure of the liver

The liver is one of the larger organs and a major site of detoxification and systemic metabolic regulation in our bodies. The “classic microscopic unit” of the liver is the hepatic lobule, with its central hepatic vein and peripheral portal triads with intervening sinusoids. The portal triad is made up of the portal vein, hepatic artery, and bile duct

[193]. The liver is made up of both parenchymal (hepatocytes) and non-parenchymal cells (Kupffer cells, hepatic stellate cells and sinusoidal endothelial cells). The blood supply from the hepatic portal vein leads blood directly from the gastrointestinal tract to the liver and as a result the liver encounters a variety of inflammatory stimuli in the form of nutritive toxins, pathogenic bacteria and viruses that induce hepatic inflammatory responses [194].

1.4.1.2 Functions of parenchymal cells in the liver

The hepatocytes are the parenchymal cells (PCs) of the liver. They make up 80% of the liver mass. They contain a high number of mitochondria and rough endoplasmic reticulum, as well as free ribosomes [194]. The hepatocytes carry out various functions such as protein synthesis and storage, nutrient (amino acid, carbohydrate and lipid) metabolism and redistribution, particulate filtration, bioactivation and bile formation

[193]. They are organized into continuous plates that are 1 cell thick, and separated by fenestrated vascular channels called sinusoids [195].

The hepatocytes carry a group of important enzymes that are also involved in biotransformation of endogenous and exogenous substances, as well as excretion of toxic and xenobiotic substances. Because most xenobiotics encountered by the body are non-polar and lipophilic, there is a need to convert them into more polar hydrophilic compounds to facilitate their excretion [196]. The enzymes responsible for biotransformation have a broad range of substrate specificity and are divided into two groups known as the phase I and phase II enzymes [197]. Phase I enzymes are usually involved in hydrolysis, oxidation and reduction reactions which modify the xenobiotics to introduce or expose a functional group (-OH, -COOH, -NH2, etc.) in non-polar molecules. The cytochrome P450 (CYP) is a superfamily of haeme proteins that are phase I oxidative membrane-bound enzymes. Among the CYP isozymes that have been identified CYP1A2, CYP2B6, CYP2C9, 2C19, 2D6, 2E1 and 3A4/5/7 account for the majority of all oxidation reactions [198]. Other phase I enzymes include epoxide hydrolase and flavin monooxygeneases. Phase II enzymes mainly consist of transferases such as UDP-glucuronosyltransferases, glutathione S-transferases, sulfotransferases, N-acetyltransferases and methyl transferases that participate in the conjugation reactions. Examples of such conjugation reactions include glucuronidation, sulfation, methylation, acetylation, amino-acid and glutathione conjugation, all of which can further increase the hydrophilicity of the modified xenobiotic to facilitate excretion

[199].

Hepatocytes have a long average life span of approximately five months. However, these cells have a high regenerative potential in response to cellular injury which can be mediated by factors such as dietary toxins and viral infections [200]. Partial hepatectomy, a widely accepted model to study liver regeneration has allowed for the elucidation of the mechanisms and factors modulating liver regeneration [201]. Growth factors and cytokines play an important role in regulation of liver regeneration.

Inflammatory cytokines like TNFa and IL-6 released by Kupffer cells in response to liver damage can stimulate proliferation in hepatocytes. TNFa stimulates NF-κB activity to induce IL-6 expression, which in turn activates other transcription pathways such as

STAT3 and MAPK to promote hepatocyte proliferation [202]. Anti- TNFa treatment to rats prior to partial hepatectomy significantly inhibited DNA synthesis and liver regeneration in rats [203]. In another model of hepatocyte proliferation induced by ethylene dibromide, inhibition of TNFa production by dexamethasone prevented hepatocyte proliferation [204]. Similarly, Il-6-null mice prevented hepatocyte proliferation after partial hepatectomy, whereas post-operative treatment with IL-6 rescued the DNA synthesis and proliferative potential of hepatocytes [205]. Apart from cytokines, growth factors like hepatocyte growth factor (HGF) [206], fibroblast growth factor (FGF) [207], insulin-like growth factor (IGF) [208], vascular endothelial growth factor (VEGF) [209] and epidermal growth factor (EGF) [210] have also been demonstrated to play an important role in regulation of hepatocyte proliferation as loss of function experiments have exhibited impaired hepatocyte proliferation. Regulation of hepatocyte proliferation by cytokines and growth factors is well reviewed in [211].

1.4.1.3 Functions of non-parenchymal cells in the liver

Compared to the hepatocytes, the non-parenchymal cells (NPCs) make up only 6.5 % of the liver mass, but account for 40% of total liver cell population. The NPCs consist of the Kupffer cells, the sinusoidal endothelial cells and the hepatic stellate cells. Although, not the main cell-type involved in carrying out many of the physiological functions of the liver, these cells secrete various substances such as cytokines and growth factors, which regulate hepatocyte functions.

The Kupffer cells are specialized resident macrophages of the liver that line the sinusoids. They represent 35% of NPCs and they have been reported to have a life span of approximately 3.8 days [212]. However, other contradicting studies have demonstrated that the life span of Kupffer cells in the liver can be anywhere from a few weeks to 14 months and these discrepancies could be due to species and model differences [213]. The number of Kupffer cells localized in the periportal area is much higher than that found to be localized in centrilobuluar regions of the liver. Further, it was found that Kupffer cells of the periportal area were larger in size with a greater capacity for lysosomal activities and phagocytic activities, but a lesser capacity for inflammatory activities [213-215]. This suggests that within the Kupffer cell population in the liver, there are distinct subsets that may have specific functions. In support of this idea, recent studies have demonstrated that Kupffer cells do in fact have phenotypic and functional heterogeneity, but whether or not they can be distinctly classified as separate subsets based on specific cell surface markers is still a matter of debate [216-

219]. The main physiological role Kupffer cells in the liver is in participating in host defense via the following ways: 1) functioning as antigen-presenting cells, 2) phagocytic elimination of microorganism, apoptotic cells, immune complexes and toxins and 3) secretion of soluble inflammatory mediators (TNFa, IL-6, IL-1, ROS etc.), and in the process participating in liver regeneration [220-222]. These cells also contain hemeoxygenase-1 (HO-1), a key enzyme in bilirubin metabolism [212]. However, long- term production of these inflammatory mediators in cases of unresolved chronic inflammation, have been shown to have oncogenic potential, predisposing the liver to hepatocellular carcinoma [223].

The hepatic stellate cells also known as Ito cells account for 5-8% of the hepatic cell population and are located in the region between the endothelial layer of the sinusoids and the hepatocytes, called the space of Disse. These cells are usually in a quiescent state, but can become activated by inflammatory signals from Kupffer cells. They secrete growth factors, extracellular matrix proteins, matrix metalloproteinases and collagen upon activation to regulate turnover of extracellular matrix [224]. Activated hepatic stellate cells have been found to enhance inflammation via the secretion of inflammatory cytokines and chemokines. Uncontrolled activation of these cells has been linked to hepatic fibrosis. They also contain large stores of vitamin A in lipid droplets and thus also participate in retinoid metabolism as well [225]. Apart from contributing to extracellular matrix deposition and inflammatory signaling, hepatic stellate cell can also participate in liver regeneration by promoting angiogenesis via secretion of Angiopoietin

1 [226], as well stimulating hepatocyte proliferation [227, 228]. Supporting these

30 findings, recent studies have demonstrated that inhibition of activated stellate cells shows attenuation of angiogenesis [229] and impaired liver regeneration [230, 231].

Thus, it is possible that chronic activation of stellate cells can induce an inflammatory, fibrotic and proliferative state in the liver, which can contribute to hepatocellular carcinoma. Recent studies supporting this notion have also shown that hepatic stellate cell-dependent signaling promotes hepatocarcinogenesis [232-234].

The liver sinusoidal endothelial cells (LSECs) are small cells of about 6.5 microns in size that form the lining of the liver sinusoids. Their main role in the liver is in scavenger function and blood clearance via receptor mediated endocytosis [235]. They have also been found to play a role in immunologic tolerance [236]. Similar to the hepatic stellate cells and Kupffer cells, the LSECs also have the potential to contribute to angiogenesis and hepatocyte proliferation via secretion of HGF and wingless-type MMTV integration site family, member (Wnt2) [237].

1.4.2 Factors that predispose the liver to hepatocarcinogenesis

Hepatocellular carcinoma is the third largest cause of mortality worldwide, accounting for approximately 745, 000 deaths annually [238]. A number of factors can contribute to the development of hepatocellular carcinoma including unresolved chronic inflammation, prolonged oxidative stress, hepatic steatosis and viral infections, all of which can systemically promote neoplastic transformations in the liver. In this section I will discuss how some of these factors, predispose the liver to hepatocellular carcinoma.

1.4.2.1 Hepatic inflammatory cytokines and oxidative stress

During acute inflammation (hepatitis), the activated Kupffer cells release a milieu of inflammatory cytokines (TNFa, Il-6, IL-1β, iNOS etc.) and reactive oxygen and nitrogen species, as well as recruit other inflammatory cells (lymphocytes, natural killer cells, neutrophils, monocytes etc.), which can cause further oxidative damage to neighboring hepatocytes. The anti-oxidant glutathione and superoxide dismutase pathways can counter-effect this oxidation in healthy liver tissue. Appropriate regeneration of liver tissue, resolution of inflammation and recovery of normal hepatic structure and function follow the death of damaged hepatocytes. However, in chronic inflammation, when the inflammatory insult is sustained over a prolonged period, this balance is offset and the liver is overwhelmed by the influx of immune cells and their inflammatory response leading to oxidative damage of hepatocytes and induction of apoptosis and subsequent necrosis. Combined these events lead to chromosome instability, oncogenic mutations and deregulated cell signaling, ultimately leading to the development of hepatocellular carcinoma [239]. TNFa and Il-6 are the two cytokines that are thought to play a crucial role in liver regeneration, however, there is now evidence indicating that the long-term production of these inflammatory mediators such as TNFa, IL-6 have oncogenic potential [223].

During inflammation the inflammatory cells of the liver promptly produce IL-6 and this drives compensatory proliferation of hepatocytes. High circulating levels of IL-6 have been associated with liver cirrhosis and hepatosteatosis [240]. IL-6 levels have also been found to be elevated in human hepatocellular carcinoma tissues and positively

32 correlate with tumor grade. The underlying mechanism was shown to be IL-6 induced activation of the STAT3 pathway to promote proliferation of hepatocellular carcinoma stem cells [241]. IL-6 also plays a crucial role in the development of hepatocellular carcinoma as IL-6 knockout mice are resistant to N-nitrosodiethylamine (DEN) induced liver carcinogenesis [242].

TNFa is another significant tumor promoting cytokine that often acts in concert with IL-6 in carcinogenesis. Inhibiting TNFa signaling in vivo via neutralizing antibodies showed a decrease in hepatocellular carcinoma progression due to increased apoptosis of transformed hepatocytes. Interestingly, further analysis of this study indicated that the source of TNFa in the inflamed livers was from non-hepatocyte cells indicating that the non-parenchymal cells play a major role in modulating tumorigenesis [243]. TNFa was also shown to induce proliferation-specific transcription factor Forkhead box M1

(FoxM1) to promote proliferation and apoptosis resistance in hepatoma cells [244].

TNFa production from tumor associated macrophages (TAMs) was found to positively correlate with angiogenesis, as well as invasive and metastatic properties [245]. They also have been found to play a role in immune escape of tumor cells. There is also evidence showing that TNFa induces TRAIL (tumor necrosis factor-related apoptosis- inducing ligand) expression on surface of hepatocellular carcinoma cells, and B7-H1

(B7 homolog 1) expression on Kupffer cells, and in the process promoting apoptosis of activated cytotoxic T lymphocytes (CTLs), thus preventing anti-tumor response and allowing immune evasion of the transformed cells [246-248]

The production of iNOS in response to inflammatory stimuli is responsible for the increased production of nitric oxide (NO) by Kupffer cells, which is a well-known mediator of chronic inflammation and can cause oxidative damage to surrounding cells via induction of reactive oxygen species (ROS) [249]. There is strong evidence in the literature that ROS can induce hepatocellular carcinoma [250, 251]. Common ROS

- - include superoxide anion (O2 ), hydroxyl radical (OH ), and hydrogen peroxide (H2O2).

ROS can induce oxidative DNA damage in replicating cells by inducing mutations via oxidation of nucleotides and generation of single strand breaks, ultimately promoting genetic instability [252, 253]. It has been observed that the GT transversions are common in oxidative DNA damage [254, 255]. Such mutations in tumor suppressor genes can promote tumorigenesis [256]. In addition, ROS can also regulate the activity of redox-sensitive kinases such as Src, PI3K/AKT and Janus kinase (JAK) to promote hepatocellular carcinoma [257]. From a tumorigenic point of view, iNOS induces angiogenesis, genetic instability and has also been shown to cross talk with transcription factors such as NF-κB that is known to have oncogenic potential. This up- regulates cell proliferation, promotes transformation and inhibits apoptosis. Elevated plasma levels of iNOS are consistently found in patients with liver cirrhosis and hepatocellular carcinoma [258].

1.4.2.2 Hepatic steatosis

Hepatic steatosis or fatty liver is characterized by the accumulation of lipids in the liver and can have a variety of etiologies, such as obesity, alcoholism or viral hepatitis infections. Hepatic steatosis is often a pre-cursor to steatohepatitis and non-alcoholic

34 fatty liver disease (NAFLD). There is clear evidence in the literature that NAFLD [259] and steatohepatitis [260] can both lead to the development of hepatocellular carcinoma.

A well-established mechanism for this has been the release of pro-inflammatory adipokines such as TNFa and IL-6, which promote a constant low-grade chronic inflammation, as well as related oxidative stress that can promote tumorigenesis in the liver [261]. Interestingly, it has been found that the anti-inflammatory adipokine adiponectin is reduced [262] and the pro-inflammatory adipokine leptin is increased

[263, 264] in NAFLD, further contributing to progression of inflammation. Hepatic steatosis and its subsequent increase in inflammation and lipotoxicity [265] have also been shown to promote insulin resistance and Type 2 diabetes and there is evidence that diabetes increases risk of hepatocellular carcinoma [266, 267]. Surprisingly, it has also been shown that NAFLD, can progress to hepatocellular carcinoma in the absence of steatohepatitis or liver fibrosis, in other words, purely by accumulation of lipids in the liver [268]. A potential mechanism put forth to explain this phenomenon is that the accumulation of fatty acids can interfere with cellular signaling, activating or repressing certain receptor or kinase activity that can promote an oncogenic signaling [269].

1.4.2.3 Viral hepatitis

Viral hepatitis infections such as hepatitis B and hepatitis C infections have long been shown to promote hepatocellular carcinoma by inducing all of the above-mentioned conditions such as chronic inflammation, oxidative stress, hepatic steatosis and liver fibrosis. They do this via a variety of different mechanisms. The two most common

35 causes of hepatitis induced hepatocellular carcinoma are Hepatitis B and Hepatitis C infections.

Hepatitis B virus infection

Hepatitis B virus (HBV) infection is associated with approximately 50% of hepatocellular carcinoma cases worldwide, and this infection is an endemic in Africa and Asia contributing to 60% of hepatocellular carcinoma cases there. HBV associated hepatocellular carcinoma has poor prognosis with the median survival being less than

16 months [270] HBV belongs to the family of hepadnaviruses known to cause liver infections. It is an enveloped, partially double stranded 3.2 kb DNA virus [271] Although the virus itself is not cytopathic, chronic HBV infection induces chronic necroinflammation of hepatocytes, thereby setting the stage for hepatocellular carcinoma.

Chronic HBV infection culminates in DNA damage, uncontrolled cellular proliferation, fibrosis, angiogenesis and eventually hepatocellular carcinoma [272]. It has also been shown that viral integration at cancer-associated genes promotes hepatocellular carcinoma [273]. Specifically the HBV X protein (Hbx) protein of the virus has been strongly linked to hepatocellular carcinoma [274] and promotes tumorigenesis via transactivation of oncogenes, transrepression of tumor suppressors and direct activation of cellular pathways promoting inflammation [275] and cell survival [276, 277].

Hepatitis C virus infection

Hepatitis C virus (HCV) is a small, enveloped positive stranded RNA virus, 9.6 kb in size and belonging to the Flaviviridae family. It is blood borne and was first identified in 1989

[278]. HCV has long been implicated in the development and progression of hepatocellular carcinoma [279]. The virus consists of three structural proteins, namely, the core protein and 2 envelope proteins as well as seven non-structural proteins. Like

HBV, HCV has also been shown to promote inflammation by activating pro- inflammatory transcription factors like NF-κB, which progresses to hepatocellular carcinoma [280, 281]. The HCV core and 3 of the non-structural proteins (NS3, NS5A and NS5B) have been heavily implicated in contributing to the tumorigenesis process.

The HCV core protein has been implicated in promoting oxidative stress and inflammation [282, 283]. NS5B has been show to promote cell proliferation by induction of ubiquitin-depend degradation of retinoblastoma protein (Rb) tumor suppressor gene

[284] as well as promote inflammation by inducing NF-κB- dependent secretion of type

1interferon and IL-6 [285]. NS5A has been shown to enhance stabilization and activation of β-catenin, a transcription factor with oncogenic potential [286]. NS3 has been found to inhibit apoptosis via suppression of tumor suppressor p53 activity without affecting its expression, by direct binding with p53 and inhibiting its transcriptional activity [287]. There is also evidence that NS3 impairs DNA damage repair by attenuating double strand break repair via co-localization with ataxia-telangiectasia mutated kinase (ATM) and interfering with its signaling [288].

1.4.3 Current mouse models of hepatitis viral protein-induced

hepatocarcinogenesis

Many mouse models have been generated to investigate the various etiologies that contribute to hepatocellular carcinoma. I will discuss the significance and uses of

37 hepatitis mouse models, more specifically, common hepatitis B and hepatitis C mouse models.

Francis V. Chisari was the first to develop a HBV mouse model (Tg(Alb-1,HBV)Bri44) in 1989, to mimic the disease progression and pathogenesis of a HBV infection. In this model, the large envelope protein of HBV was over-expressed which led to accumulation of non-secretable HBV surface antigen in the endoplasmic reticulum giving hepatocytes a “ground glass appearance”. This induced progressive liver injury and secondary inflammation, hyperplasia and ultimately culminating in hepatocellular carcinoma at 15 months of age [289]. This was a good model to show the cellular injury induced by HBV proteins and the subsequent inflammation they caused in a chronic state. In addition, it was also a good model to show that the continuous hepatocyte damage and regenerative proliferation in a setting of inflammation and oxidative stress can lead to hepatocellular carcinoma.

Interestingly, it was observed that in patients with acute HBV infection, there was a strong T cell response that cleared the virus, but in patients with chronic HBV infection a weak T cell response was displayed. Hence, later, this group developed another HBV mouse model (Tg(Alb-1,HBV)Bri66), in an attempt to determine the pathogenic mechanisms that contribute to HBV infections reflecting this immune condition. In this model, the expressed non-toxic concentrations of HBV envelope proteins, and the HBV surface antigens were designed to be immunologically tolerant to T cells, so that the mechanism by which chronic immune response against a viral antigen that drives a

38 severe MHC class I restricted necroinflammation resulting in hepatocellular carcinoma could be investigated [290]. This model showed that chronic immune stimulus alone was sufficient to drive hepatocellular carcinoma.

With the realization of the oncogenic potential of the HBx protein of the HBV virus, another laboratory developed a mouse model that had HBx protein integrated into the host genome and showed that this criterion alone was sufficient to drive hepatocellular carcinoma. In this mouse model, the liver expressed high levels of HBx and the mice developed pre-neoplastic altered foci at 4 months of age followed by adenoma and subsequent hepatocellular carcinoma at 11-15 months of age, all in the absence of liver damage, indicating that viral protein alone was sufficient to induce hepatocellular carcinoma [291]

Similar to the HBx, the oncogenic potential of HCV core protein led investigators to develop a HCV mouse model that expressed this protein. These mice develop hepatic steatosis at 3 months of age. This steatotic feature progressed with age, developing hepatocellular carcinoma at 16-19 months of age. The tumors in these mice had a characteristic “nodule in nodule” feature whereby the hepatocellular carcinoma developed from within pre-existing adenomas. Another surprising feature of this model is that disease pathogenesis occurs in absence of inflammation and tumor development can be directly linked to the presence of HCV core protein [292].

Interestingly, more recently a humanized mouse model of HCV was developed. This model involves the engraftment of human progenitor and hematopoietic stem cells from fetal liver into AFC8 immunodeficient humanized mice. In these mice HCV infection with subsequent human immune response and hepatic fibrosis was observed after approximately 2 months. Although this is a good model to study adaptive immunity and pathogenesis in human HCV infection, because these mice lack human B cells, the ability of these mice to generate antibodies to viral pathogen is impaired [293].

The mouse models discussed above (except for humanized HCV mice) are commonly used because they were designed to specifically address known mechanisms by which

HBV and HCV infections promote hepatocellular carcinoma pathogenesis characteristic of human infections. Apart from these, many other mouse models have been developed in recent times to investigate the disease pathogenesis of HBV and HCV infections and they are well reviewed in [294-296]. Because these viruses do not infect mice, there has been a lot of effort to create humanized mouse models for these virus infections [297].

However, as mentioned above, these models come with their own set of limitations as well.

1.5 PPARβ/δ and hepatocarcinogenesis

In spite of active investigations on the role of PPARβ/δ in skin and colon tumorigenesis, its modulation of hepatocarcinogenesis still lags behind. This is surprising, considering

PPARβ/δ has been shown to attenuate pre-neoplastic conditions such as hepatic inflammation, steatosis and fibrosis discussed above. Recently, in a model of NAFLD,

40 the pan PPARα /PPARβ/δ agonist GFT505 was shown to exhibit hepatoprotective effects in vivo. GFT505 greatly attenuated hepatic steatosis and fibrosis in a PPARβ/δ- dependent manner, as these effects were observed in the hApoE2-KI/ Pparα-null obese mice fed a western diet [298]. It has also been shown that PPARβ/δ is hepatoprotective in CCl4 and azoxymethane treated mice by attenuating inflammation and regenerative hyperplasia respectively [160].

In terms of the limited data investigating the role of PPARβ/δ in hepatocellular carcinoma, there have been some controversial studies that show PPARβ/δ promotes hepatic proliferation [299, 300] and tumorigenesis in multiple hepatoma cell lines. A potential mechanism proposed for this has been PPARβ/δ dependent up-regulation of

COX-2 expression [301]. Because COX-2 has been shown to promote hepatocellular proliferation via activation of EGFR and AKT signaling pathways [302], it has been postulated that PPARβ/δ promotes hepatic tumorigenesis via COX-2 [303]. However, studies on two different hepatoma cell lines treated with two highly specific ligands for

PPARβ/δ showed no induction of COX-2 or AKT activity as well as cell growth [123].

A more recent study investigating the nuclear receptor transcriptome shows that

PPARβ/δ is suppressed at the protein level in both liver regeneration and human hepatocellular carcinoma. They further go on to show that activation of PPARβ/δ by a highly specific agonist attenuates the proliferative potential of hepatoma cells [304].

However, more studies need to be done to investigate the role of PPARβ/δ in hepatocellular carcinoma models in vivo. The overwhelming literature that supports the

41 ant-inflammatory, anti-proliferative and anti-steatotic effects of PPARβ/δ in the liver shows great promise of this nuclear receptor in being targeted as a potential therapeutic avenue for preventing hepatocellular carcinoma disease progression (Figure 1-3).

Figure 1-3: Hepatoprotective functions of PPARβ/δ. Progressive inflammation, oxidative stress, fibrosis and steatosis promote the development of hepatocellular carcinoma. PPARβ/δ inhibits these pre-neoplastic stages in the liver, and hence, has the potential to attenuate the progression of hepatocellular carcinoma.

1.6 Objective of the study and hypothesis

The objective of this study is focused on investigating the hepatic anti-inflammatory potential of PPARβ/δ and the specific liver cell type involved, as non-parenchymal cells have been implicated in modulating inflammatory responses in the liver. Chronic

Inflammation promotes liver steatohepatitis, fibrosis and cirrhosis, thus setting the stage for development of hepatocellular carcinoma. Hence these studies aim to specifically target inflammatory signaling in the liver with the intention of reducing its adverse effects and potentially attenuating liver tumorigenesis

The central hypothesis of this research is that ligand activation of PPARβ/δ in Kupffer cells attenuates both acute and chronic inflammation in the liver via direct protein- protein interaction with NF-κB transcription factor and in the process contributes to attenuated tumor progression (Figure 1-4). Although the role of PPARβ/δ in carcinogenesis remains debatable [47], there have not been any studies looking at the role of PPARβ/δ in hepatocellular carcinoma in vivo. The rationale for my hypothesis lies in recent findings that, ligand activation of PPARβ/δ attenuates liver inflammation, steatosis, fibrosis and cirrhosis, all of which are preneoplastic stages of hepatocellular carcinoma. In addition the physical interaction of PPARβ/δ with a transcription factor that has oncogenic potential like NF-κB would most likely interfere with inflammatory signaling pathways that would promote tumorigenesis in inflammatory tumor models.

This transrepression mechanism could elicit anti-tumor effects. Specifically, I intend to target the Kupffer cells because there is evidence that among the nonparenchymal cells of the liver, the Kupffer cells have been primarily implicated in mediating proliferative and anti-apoptotic effects of NF-κB during liver regeneration [305] as well as in promoting and sustaining the inflammatory process that leads to hepatocarcinogenesis through NF-κB activation [280]. For these studies male mice from two different mouse models of chronic hepatocellular injury-induced liver cancer will be utilized. The first is

43 the Tg(Alb1-HBV)Bri44 hepatitis B (HBV) transgenic mouse line. These mice contain the large envelope protein of the HBV sequence inserted downstream of the albumin promoter, which allows for over-expression of this protein in the hepatocytes. This results in the mice developing hepatocellular injury and resultant inflammation by 2-3 months of age, followed by regenerative hyperplasia and subsequent development of liver tumors by 12-15 months. The second mouse model is the hepatitis C transgenic mouse strain (HCV) in which these mice express the core protein of HCV genotype 1b driven by the transcriptional regulatory region from hepatitis B virus [292]. These mice start to develop hepatic steatosis by 3 months of age with progressive hepatic lipid accumulation, as they get older. They eventually develop hepatocellular adenomas at

16 months and hepatocellular carcinomas between 19-24 months of age. In addition, in an effort to determine the mechanism of PPARβ/δ-dependent attenuation of inflammatory signaling and the specific liver cell type involved, male wild-type and

Pparβ/δ-null mice previously described in [306] as well Pparβ/δ DBM mice were used for the isolation of primary Kupffer cells and hepatocytes. Pparβ/δ DBM mice have a mutation in the DNA binding domain whereby two conserved cysteine residues, Cys-90 and Cys-93 were mutated to alanine residues thereby preventing PPARβ/δ from binding its respective PPREs [55].

Figure 1-4: Schematic of Hypothesis. Ligand activated PPARβ/δ binds NF-κB transcription factor and inhibits its binding to NF- κB response elements. This attenuates pro-inflammatory signaling in the Kupffer cells of the liver and possibly contributes to the attenuation of the tumorigenesis process.

Hepatocellular carcinoma is an aggressive disease and the third leading cause of cancer deaths worldwide [238]. Approximately 80% of hepatocarcinogenesis incidents are due to viral hepatitis infections [307]. Although surgical resection is the most promising treatment, it is not an option for most patients because of the extent of tumor progression. Alternative treatment strategies include liver transplantation, radiation therapy, embolization, etc. However, even with these, the prognosis remains poor. The significance of this study lies in the hope of developing a cell-type specific treatment, which might prove more effective in targeting the disease and improving prognosis.

Chapter 2 PPARβ/δ attenuates hepatic tumorigenesis in hepatitis B

transgenic (HBV) mice

2.1 Abstract

PPARβ/δ can inhibit pro-inflammatory activities in the liver, which have potential to contribute to hepatocellular carcinoma. Hence, ligand activation of PPARβ/δ in modulating chronic hepatocellular injury-induced hepatocarcinogenesis was examined.

Male hepatitis B virus (HBV) transgenic mice, which develop chronic hepatocellular injury and inflammation around 2-3 months of age, followed by hyperplasia and neoplasia by 9-12 months, and eventually hepatocellular carcinoma by 18 months of age were used for this study. Long term (8 months) ligand (GW0742) activation of

PPARβ/δ in HBV mice showed attenuation in tumor multiplicity, average number of liver foci and hepatic steatosis. These effects were not observed in the control mice. Western blot analysis showed significant attenuation of CYCLIN D1 and c-MYC as well as increased expression of cleaved PARP in livers of ligand activated HBV mice. Short- term (3 weeks) ligand activation of PPARβ/δ in HBV mice attenuated hepatic tumor necrosis factor-alpha (Tnfa) and serum levels of alanine amino transaminase (ALT) compared to that of control. Further, ligand activation of PPARβ/δ attenuated lipopolysaccharide (LPS)-induced expression of Tnfa in primary Kupffer cell cultures from wild-type but not in Kupffer cells from Pparβ/δ-null mice. Similar observations were made in ligand activated KC13-2 Kupffer cells treated with LPS in the absence and presence of PPARβ/δ siRNA. PPARβ/δ-dependent modulation of Tnfa was not seen in primary wild-type hepatocytes. More importantly, ligand activation of PPARβ/δ

46 attenuated LPS-induced expression of Tnfa in Pparβ/δ-DBM (DNA binding mutant form of PPARβ/δ) Kupffer cells. Possible interaction of PPARβ/δ with pro-inflammatory transcription factor NF-κB was confirmed by co-immunoprecipitation of PPARβ/δ with p65 subunit of NF-κB in LPS and ligand activated KC13-2 cells. Combined, results from these studies suggest that ligand activation of PPARβ/δ can attenuate tumor progression in a chronic hepatocellular injury-induced liver cancer model at least in part by modulating anti-inflammatory activities in the Kupffer cells.

2.2 Introduction

Peroxisome proliferator-activated receptor PPARβ/δ (NR1C2) [308] belongs to the nuclear hormone receptor superfamily of ligand inducible transcription factors. PPARβ/δ is highly expressed in skin, colon and mouse liver tissues [117, 309, 310]. Nuclear receptors have been reported to modulate gene expression through dynamic and transient interaction with nuclear chromatin. [27-29, 311]. PPARβ/δ modulates gene expression in a similar manner through interaction with its peroxisome proliferator response elements (PPREs) in the nuclear chromatin. Nucleosome localization and/or chromatin structure play a big role in the binding of PPARβ/δ to its consensus PPREs to modulate transcription of target genes. This binding can be greatly enhanced or diminished by the presence of endogenous and exogenous PPARβ/δ ligands [52].

Some endogenous ligands for PPARβ/δ are arachidonic acid, linoleic acid and oleic acid [312] whereas the synthetic ligands include GW501516 and GW0742 [118].

GW0742 has been reported to be highly selective for PPARβ/δ over other PPARs [313].

PPARβ/δ can also bind to other transcription factors such as NF-κB, STAT3 and ERK5 and modulate their transcriptional activities [150, 314, 315]. Hence, PPARβ/δ can regulate cellular signaling and function on multiple levels, which can have a marked effect on physiology and disease pathogenesis.

PPARβ/δ has been demonstrated to attenuate hepatic inflammation in many different models [140, 160, 164, 298, 316] It was also found to confer protection against environmental toxicants like CCl4 and azoxymethane through anti-inflammatory mechanisms, likely via the modulation of NF-κB in the Kupffer cells [140]. The regulation

48 of inflammation by PPARβ/δ through Kupffer cells is further supported by recent findings, that show innate inflammatory pathways like the Toll-like receptor signaling and cytokine signaling to be highly up regulated in the liver in the absence of PPARβ/δ

[163]. In addition, PPARβ/δ also attenuates hepatic steatosis [165, 167] and fibrosis

[164]. Because of strong evidence of its ability to positively modulate precancerous stages of hepatocellular carcinoma such as hepatic inflammation, lipid accumulation and metabolism, it is tempting to see if PPARβ/δ has a chemopreventive role in the liver.

Hepatocellular carcinoma is the third biggest contributor to mortality worldwide [238] and approximately 600,000 deaths annually are attributed to hepatocellular carcinoma as a result of hepatitis B virus (HBV) infection [317]. Chronic HBV infection induces persistent necro-inflammation of hepatocytes, oxidative DNA damage, uncontrolled cellular proliferation, angiogenesis, fibrosis and cirrhosis, which eventually culminates in hepatocellular carcinoma [272]. It has been shown that the chronic hepatitis and the subsequent inflammatory damage is the driving factor for HBV associated hepatocellular carcinoma. HBV patients have poor prognosis with the median survival being less than 16 months [270]. There is currently only supportive treatment for acute

HBV infection and interferon therapy is thus far the most effective treatment for chronic

HBV, although success rate still lies between 20-50% [318]. Hence, there is a need to develop better therapeutic approaches.

Although a lot of work has been done to investigate the role of PPARβ/δ in conditions

49 pre-disposing to hepatocellular carcinoma, not much has been done to look at the role of PPARβ/δ in hepatocarcinogenesis models in vivo. Limited studies on the subject have conflicting results. For example, studies have shown that PPARβ/δ induces COX-2 expression in human hepatoma cell lines which in turn up regulates PGE2 to drive tumor growth [301, 303]. Contradicting results were found when the same hepatoma cell lines were examined under similar experimental conditions, to display no PPARβ/δ dependent increase in COX-2 expression or cell proliferation. In addition, there was significant inhibition of cellular proliferation in serum starved HepG2 cell line in presence of PPARβ/δ ligand [123].

The present study examined the hypothesis that ligand activation of PPARβ/δ will attenuate chronic inflammatory signaling and subsequent tumorigenesis in a chronic hepatocellular injury-induced liver cancer model in vivo. The Tg(Alb1-HBV)Bri44 (HBV) mice, which overexpress the large envelope protein of HBV resulting in early onset of hepatocellular injury, which subsequently over prolonged period, culminates in hepatocellular carcinoma, were used for these studies. Results from the present study show that ligand activation of PPARβ/δ attenuates hepatic Tnfa at the early stages and tumorigenesis at the later stages of disease pathogenesis.

2.3 Materials and Methods

Animals and treatments

Male C57BL/6 mice and Tg(Alb1-HBV)Bri44 (HBV) mice on a C57BL/6 background obtained from the Jackson Laboratory (Bar Harbor, Maine, USA) were used for this study. The HBV mice contain the large envelope protein of the HBV sequence inserted downstream of the albumin promoter, which allows for over-expression of this protein in the hepatocytes. These mice develop hepatocellular injury at 2-3 months of age, followed by regenerative hyperplasia and subsequent development of liver tumors by

12-15 months. For the HBV short-term ligand activation study, 7-month-old mice were orally administered the highly specific PPARβ/δ ligand GW0742 (provided by

GlaxoSmithKline) at a dose of for 5.0 mg/kg (5 days per week) for 3 weeks. For the

HBV long-term ligand activation study, 7-month-old mice were orally administered

GW0742 at a dose of for 2.5 mg/kg (5 days per week) for 8 months. A recent study has demonstrated that 1 mg/kg and 10 mg/kg doses of GW0742 correspond to plasma concentrations of 440.7 nM and 2270 nM respectively in mice [319] and another study has shown that the EC50 values of GW0742 to activate PPARα and PPARγ are 8900 nM and greater that 10000 nM respectively [147]. Therefore the dose of GW0742 used for these studies are sufficient to specifically activate PPARβ/δ (which has an EC50 value of 50 nM) over its other isoforms. For both studies, mice were dosed with either just a pellet (Bacon-flavored Transgenic Dough Diet, Bioserv, Inc.) in the case of control group, or a pellet mixed with GW0742 powder in the case of treatment group. At the end of the study, mice were euthanized by overexposure to carbon dioxide, followed by cervical dislocation, before whole liver and serum were harvested. Total number of

51 visible tumors was counted for each isolated liver to determine tumor multiplicity. Male wild-type and Pparβ/δ-null mice previously described in [306] as well Pparβ/δ-DBM mice were used for the isolation of primary Kupffer cells and hepatocytes. Pparβ/δ-DBM mice have a mutation in the DNA binding domain that is driven by a CD68 macrophage specific promoter on a Pparβ/δ-null mouse background, which prevents PPARβ/δ from binding its respective PPREs in the Kupffer cells of the liver. Therefore, in these mice,

PPARβ/δ has the ability to physically interact with other proteins such as transcription factors to modulate their activities, but lacks the ability to regulate PPARβ/δ target gene expression via binding to its respective PPREs in the Kupffer cells of the liver. This mouse model differs from that of wild-type mice in which PPARβ/δ is fully functional in all cell types of the liver, and can both bind to its respective PPREs to modulate

PPARβ/δ target gene expression, as well as interfere with signaling pathways of other proteins by physically interacting with them [55]. All animal experiments were conducted in accordance to the Institutional Animal Care and Use Committee (IACUC) at

Pennsylvania State University.

Isolation of primary Kupffer cells and hepatocytes

Kupffer cells and hepatocytes from mice aged 12-16 weeks, were isolated using a method previously described by Smedsrod et al. [320] with minor modifications. Mice were anesthetized by intraperitoneal administration of 250 mg/kg Avertin (2,2,2-

Tribromoethanol, Sigma-Aldrich.) prior to liver perfusion. Briefly, whole livers were perfused with Buffer I (142 mM NaCl, 6.4 mM KCl, 9.6mM HEPES, 30 mM NaOH) followed by Buffer II (Buffer I supplemented with 4.76 mM CaCl2 and collagenase type

IV (Worthington Biochemical Corporation, Lakewood, NJ)) via the inferior vena cava, prior to excision. Perfused livers were then minced and suspended in cold Buffer III

(Buffer I supplemented with 1% BSA) prior to passing through a 70 µm nylon cell strainer. The filtered cells were further purified for cell culture via centrifugation twice at

54 x g for 3 minutes at 4°C to isolate the hepatocyte pellet. The supernatants were further centrifuged at 900 x g for 10 minutes at 4°C. The resulting cell pellet was then re-suspended in Buffer III and passed through a discontinuous gradient of 25% and

50% Percoll (Sigma-Aldrich.) at 4°C. The Percoll fraction containing the purified non- parenchymal cells was then washed once with Buffer III and the Kupffer cell population was enriched by selective adherence to non-treated tissue culture plates. Isolated

Kupffer cells and hepatocytes were cultured in RPMI medium containing L-glutamine

(Mediatech Inc., Manassas, VA) and supplemented with 10% fetal bovine calf serum and 1% penicillin and streptomycin and incubated at 37°C with 5% CO2. The purity of isolated Kupffer cells was determined by their ability to phagocytize latex beads.

Isolated primary Kupffer cells and hepatocytes were cultured and allowed to recover for

48 hours prior to pre-treatment with 1µM GW0742 for 23 hours, followed by 10 ng/ml lipopolysaccharide (LPS from Escherichia coli, 0111:B4, Sigma-Aldrich, St. Louis, MO) for 1 hour.

Cell line

The KC13-2 clonal Kupffer cell line was obtained as a kind gift from a Dr. Pamela A.

Hankey Giblin (Pennsylvania State University, University Park, PA). The cells were cultured in RPMI medium containing L-glutamine and supplemented with 10% fetal

53 bovine calf serum and 10 µg/ml ciprofloxacin hydrochloride and incubated at 37°C with

5% CO2. For PPARβ/δ siRNA knockdown experiments, cells were transfected with either negative control siRNA or PPARβ/δ siRNA (D-001206-13-05 or M-042751-01-

0005; Thermo Scientific, Waltham, MA) for 48 hours prior to treatment with 2µM

GW0742 for 18 hours, followed by 10 ng/ml LPS for 2 hours.

RNA isolation and quantitative polymerase chain reaction (qPCR) analysis

RNA was extracted from whole liver tissue and cells in culture using Ribozol RNA extraction reagent (Amresco Inc., Solon, OH) and 1.25 µg of total RNA was reverse transcribed into cDNA using reverse transcription kit (Promega, WI,). qPCR reactions were carried out using MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories,

Hercules, CA) for adipocyte differentiation-related protein (Adrp), tumor necrosis factor alpha (Tnfa) and angiopoietin-like 4 (Angptl4). Relative mRNA values were normalized to internal control values of glyceraldehyde 3-phosphate dehydrogenase (Gapdh). The primer sets used were ADRP forward, 5’-CACAAATTGCGGTTGCCAAT-3’ and reverse,

5’-ACTGGCAACAATCTCGGACGT-3’, TNFa forward, 5’-

AGGGTCTGGGCCATAGAACT-3’ and reverse, 5’-CCACCACGCTCTTCTGTCTAC-3’,

ANGPTL4 forward, 5’-TTCTCGCCTACCAGAGAAGTTGGG-3’ and reverse 5’-

CATCCACAGCACCTACAACAGCAC-3’, GAPDH forward, 5’-

TCAACAGCAACTCCCACTCTTCCA-3’ and reverse 5’-

ACCCTGTTGCTGTAGCCGTATTCA-3’.

Protein extraction and western blot analysis

Liver tissues were homogenized in lysis buffer containing 50 mM Tris, 150 mM NaCl,

0.1% SDS, and 1% Nonidet P-40 at pH 7.5 supplemented with protease inhibitors, using a chilled Dounce glass homogenizer. Thirty micrograms of protein was loaded per sample and resolved using SDS gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat milk or 0.5% gelatin in Tris buffered saline with Tween-20 (TBST) and subsequently incubated with primary and secondary antibodies. The primary antibodies used were anti-CYCLIN B1 (SC-752), anti-CYCLIN D1 (SC-753), anti-C-MYC (SC-764) and anti-β -

ACTIN (SC-47778) from Santa Cruz Biotechnologies, Santa Cruz, CA. Anti-PARP (CST

#9542S) from Cell Signaling Technology, Danvers, MA and anti-LDH. The secondary antibodies used are biotinylated (Jackson ImmunoResearch Laboratories, West Grove,

PA). The membrane is then incubated with 125I-labeled streptavidin and hybridization signals were quantified by detecting levels of radioactivity. Protein expression levels are presented as fold change from control liver tissues of HBV mice not treated with

PPARβ/δ ligand.

Co-immunoprecipitation

KC13-2 cells were treated as mentioned above and collected by scraping. Nuclear extracts were isolated using the NE-PER nuclear protein extraction Kit (Thermo

Scientific, Rockford, IL) according to manufacturer’s protocol, with lysis buffers supplemented with protease inhibitors. Co-immunoprecipitation was performed using reagents from the Pierce Classic Magnetic IP/Co-IP kit (Thermo Scientific, Rockford, IL)

55 according to manufacturer’s protocol. Nuclear extracts were pre-cleared with magnetic beads for an hour prior to co-immunoprecipitation. Briefly, 2 µg of highly specific

PPARβ/δ (8099) or IgG antibody was added to 150 µg of pre-cleared nuclear extract to form an immune complex overnight. Then using magnetic beads, the immune complex bound to 8099 or IgG antibodies was pulled down and eluted in 44 µl of low-pH elution buffer and neutralized with 4.4 µl of neutralization buffer provided by the kit. 5X Lamelli dye was added to the eluates before they were resolved by SDS gel electrophoresis.

Western blot was performed as described above and expression levels of p65 subunit of NF-κB (SC-372X, Santa Cruz Biotechnologies, Santa Cruz, CA) were detected. All steps for nuclear extraction and co-immunoprecipitation were done at 4°C.

Serum alanine aminotransferase (ALT) analysis

Serum levels of ALT were determined by spectrophotometric analysis utilizing the

RAICHEM ALT assay kit (Cliniqa Corporation, San Marcos, CA) according to manufacturer’s protocol.

Liver histology

Sections of liver tissues were fixed in 10% buffered formalin overnight and transferred to

70% ethanol, prior to being embedded in paraffin for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E). Total number of steatotic regions for each liver tissue section taken from the largest lobe was also counted. A pathologist scored the total number of liver tumor foci for 3 representative sections of each liver. Livers from 5 HBV mice and 5 HBV mice treated with PPARβ/δ ligand were

56 used.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.).

For comparison of two or more groups, one-way analysis of variance using Tukey’s multiple comparison tests were used. Student’s unpaired two-tailed t-test was used for all other statistical analysis. The data are shown as mean ± s.e.m. Differences were considered significant when P ≤ 0.05.

2.4 Results

2.4.1 PPARβ/δ-dependent signaling attenuates tumorigenesis in HBV mice

To determine the chemopreventive role of PPARβ/δ HBV transgenic mice were used.

These mice overexpress the large envelope protein of the HBV genome, to induce liver injury and inflammation at 2-3 months of age followed by hyperplasia, neoplasia and eventually culminating in hepatocellular carcinoma by 12-15 months of age. C57Bl/6J mice were used as control. These mice were fed 2.5 mg/kg of PPARβ/δ ligand GW0742 at 7 months of age to 15 months (5X/Week). At the end of 15 months, in the absence of

PPARβ/δ ligand activation, the HBV livers showed severe hepatomegaly, multiple tumor nodules of varying sizes, depicting the advanced stages of hepatic tumorigenesis. This was not observed in the livers of control C57Bl/6J mice, which looked healthy, retained liver morphology, and had no liver tumor nodules. Ligand activation of PPARβ/δ was confirmed by increase in PPARβ/δ target gene Angptl4 (Figure 2-1A). Histopathological analysis revealed that liver tissue sections of HBV mice treated with GW0742 displayed a significant decrease in the average number of foci, whilst that of C57Bl/6J tissue sections did not have any foci (Figure 2-1B). Tumor multiplicity was also reduced in livers of HBV mice treated with GW0742 (Figure 2-1C). No altered liver foci or tumors were observed in the control C57Bl/6J mice. Overall, the activation of PPARβ/δ with

GW0742 was hepatoprotective, attenuating the progression of hepatocellular carcinoma in HBV transgenic mice in vivo.

Figure 2-1: Ligand activation of PPARβ/δ attenuates liver tumorigenesis in HBV transgenic mice. 7-month-old HBV mice were treated with and without (Control) GW0742 for 8 months (2.5 mg/kg, 5 days per week). Whole liver was isolated at the end of 8 months. Relatively more tumors in control HBV mice compared to HBV mice treated with GW0742. Up-regulation of PPARβ/δ target gene, Angiopoietin-like 4 (Angptl4) seen with ligand activation (A). Values represent the mean ± SEM. N=5. *Significantly different than HBV control by two-tailed Student’s t-test, P ≤ 0.05. Reduced number of foci (B) and tumor multiplicty (C) in ligand activated HBV mice than in control. Values represent the mean ± SEM. N=5. *Significantly different than HBV control by one-way analysis of variance using Tukey’s multiple comparison test, P ≤ 0.05.

2.4.2 PPARβ/δ-dependent signaling attenuates markers of proliferation

and enhances apoptosis in HBV mice

There have been conflicting studies demonstrating that ligand activation of PPARβ/δ, either promotes [301], attenuates [304], or has no effect on proliferation of hepatoma cell lines [123]. To investigate effect of PPARβ/δ ligand on the hepatic proliferative potential of the HBV model, western blot analysis of liver tissues was done to look at markers of proliferation. Ligand activation of PPARβ/δ in HBV liver tissues showed mild attenuation of CYCLIN B1, but significantly reduced expression of CYCLIN D1 and C-

MYC compared to control (Figure 2-2A).

There have also been conflicting reports on the role of PPARβ/δ in modulating apoptosis. Similar to the case of proliferative status, the role of PPARβ/δ in modulating apoptosis in various different types of tumor models is controversial, with studies showing that PPARβ/δ either has no effect on apoptosis [321], promotes apoptosis

[322] or enhances tumorigenesis by inhibition of apoptosis[187, 323]. In this study, the ratio of the expression levels of normalized cleaved PARP to uncleaved PARP was investigated to determine the relative levels of apoptosis in the liver tissues. There were significantly increased levels of cleaved PARP in liver tissues from HBV mice treated with PPARβ/δ ligand compared to the control (Figure 2-2B). Induction of proliferative potential and inhibition of apoptosis are common driving factors of tumorigenesis. Thus, ligand activation of PPARβ/δ attenuates tumorigenesis by inhibiting the proliferative potential and at the same time promoting the apoptotic potential of HBV liver tissues in vivo.

Figure 2-2: Ligand activation of PPARβ/δ attenuates markers of proliferation and increases apoptosis in livers of HBV transgenic mice. Quantitative western blot of CYCLIN B1, CYCLIN D1 and c-MYC (A), and cleaved (C) and uncleaved PARP (U), as well as the ratio of the normalized expression of cleaved PARP to uncleaved PARP (C/U) (B) in liver tissue samples of 7-month-old HBV mice treated with or without GW0742 (2.5 mg/kg, 5 days per week for 8 months). 30 µg of whole cell lysate was loaded per sample. Normalized values were calculated from the independent samples and represent the mean ± SEM. N=3 for HBV Control and N=3 for HBV+GW0742. *Significantly different than HBV control by two-tailed Student’s t-test, P ≤ 0.05.

2.4.3 PPARβ/δ-dependent signaling attenuates hepatic steatosis in HBV

mice

PPARβ/δ is a major regulator of lipid metabolism and has been demonstrated to inhibit hepatic steatosis in diabetic and obese animal models by mechanisms such as attenuation of inflammatory signaling [165], suppression of lipogenesis [167] and induction of fatty acid oxidation [298]. To evaluate the effect of PPARβ/δ in hepatic lipid accumulation, representative liver tissue sections were taken from the largest lobe of

HBV mice treated with and without PPARβ/δ ligand and stained with hematoxylin and eosin. Liver tissues of HBV mice displayed increased hepatic steatosis compared to liver tissues of HBV mice treated with PPARβ/δ ligand GW0742 (Figure 2-3A). For each liver tissue section, the total number steatotic regions were counted and it was determined that HBV mice treated with PPARβ/δ ligand had significantly fewer number of steatotic regions per tissue section compared to HBV mice not treated with PPARβ/δ ligand GW0742 (Figure 2-3B). These results are consistent with some of the previous findings in the field.

Figure 2-3: Ligand activation of PPARβ/δ attenuates liver steatosis in HBV transgenic mice. 7-month-old HBV mice were treated with and without (Control) GW0742 for 8 months (2.5 mg/kg, 5 days per week). Hematoxylin and eosin stained images (10X) of liver sections from each of three HBV (a.1-a.3) and ligand treated HBV (b.1-b.3) mice. Relatively more steatosis in control HBV mice compared to HBV mice treated with GW0742 (A). Reduced number of steatotic regions per liver section (B) in ligand activated HBV mice than in control. Values represent the mean ± SEM. N=5. *Significantly different than HBV control by two-tailed Student’s t-test, P ≤ 0.05.

2.4.4 PPARβ/δ-dependent signaling attenuates hepatotoxicity and hepatic

Tnfa in HBV mice

Earlier studies from our laboratory had demonstrated that ligand activation of PPARβ/δ conferred hepatoprotective effects to CCl4 induced liver injury by down regulation of pro- inflammatory genes [140]. To determine if PPARβ/δ offered similar protective effects in the HBV model, HBV mice were treated for a shorter duration (3 weeks at a dose of 5 mg/kg GW0742, 5X/Week) with or without PPARβ/δ ligand at 7 months of age, where onset of cellular liver injury was already present but prior to development of liver tumors.

Significant attenuation of a marker of hepatocellular injury, serum ALT was observed in

HBV mice treated with PPARβ/δ ligand GW0742 (Figure 2-4A). Ligand activation of

PPARβ/δ also significantly attenuated a marker of hepatic inflammation, Tnfa (Figure

2-4B) in liver tissues of HBV mice. These results are consistent with previous findings from our laboratory. TNFa is an inflammatory cytokine that can promote the severity of liver disease and enhance the development of hepatocellular carcinoma [261, 324].

Early attenuation of hepatocellular injury by inhibition of TNFa could play a role in delaying the progression of tumorigenesis in HBV mice treated with PPARβ/δ ligand.

Figure 2-4: Ligand activation of PPARβ/δ attenuates hepatic Tnfa and a marker of liver damage in HBV transgenic mice. 6-month-old HBV mice were treated with GW0742 for 3 weeks (5 mg/kg, 5 days per week). Serum levels of ALT (A). mRNA was isolated from liver and expression of Tnfa (B) was determined by qPCR. Values represent the mean ± SEM. N=3. *Significantly different than HBV control by two-tailed Student’s t-test, P ≤ 0.05.

2.4.5 PPARβ/δ-dependent attenuation of Tnfa is modulated in the Kupffer

cells and not the hepatocytes

Although previous work from our laboratory found similar attenuation of hepatic Tnfa in response to PPARβ/δ ligand activation in a CCl4 induced liver injury model, it was not concluded as to which cell type was being modulated by PPARβ/δ to attenuate the expression of this pro-inflammatory cytokine [140]. Primary hepatocyte cells (Figure

2-5A) isolated from wild-type mice were treated with LPS to induce Tnfa expression in the absence or presence of PPARβ/δ ligand GW0742. Induction of PPARβ/δ target gene Adrp confirms ligand activation of PPARβ/δ in wild-type hepatocytes (Figure

2-5B). LPS induced Tnfa expression significantly compared to the control and was not attenuated in the presence of PPARβ/δ ligand (Figure 2-5C). This is consistent with previous findings from our laboratory that demonstrated that the anti-inflammatory activity of PPARβ/δ is not likely modulated in hepatocytes of the liver. The Kupffer cells were investigated next, as they are the pro-inflammatory cells of the liver and well known for releasing TNFa in response to hepatocyte damage and pathogen encounter.

Primary Kupffer cells (Figure 2-5D) were isolated from wild-type, Pparβ/δ-null and Ppar

β/δ-DBM mice. Induction of PPARβ/δ target gene Adrp in wild-type but not Pparβ/δ-null

Kupffer cells confirms the activation of PPARβ/δ in wild-type Kupffer cells and the functional absence of PPARβ/δ in Ppar β/δ-null Kupffer cells. LPS significantly induced

Tnfa expression in both wild-type and Ppar β/δ- Kupffer cells, however, only wild-type

Kupffer cells significantly attenuated Tnfa expression in presence of PPARβ/δ ligand. In the Ppar β/δ-DBM Kupffer cells, ligand activation of PPARβ/δ did not induce expression of PPARβ/δ target gene Adrp confirming the inability of PPARβ/δ in these cells to bind

PPREs to induce expression of PPARβ/δ target genes. LPS stimulated Tnfa expression in the Ppar β/δ-DBM Kupffer cells and ligand activation of PPARβ/δ significantly attenuated its expression (Figure 2-5E & F). Combined, these results indicate that the anti-inflammatory effects of PPARβ/δ are mediated in the Kupffer cells of the liver.

These activities are more likely mediated by transrepressive mechanisms involving protein-protein interaction of PPARβ/δ with other pro-inflammatory transcription factors such as NF-κB rather than by PPARβ/δ target genes.

Figure 2-5: Ligand activation of PPARβ/δ inhibits pro-inflammatory cytokine Tnfa mRNA expression in wild-type and PPARβ/δ DNA binding mutant Kupffer cells but not in Pparβ/δ-null Kupffer cells. This effect is not observed in hepatocytes. Primary Kupffer cells (A) and hepatocytes (D) isolated from wild-type liver tissues in culture. Kupffer cells were isolated and plated from liver of mice via collagenase perfusion. Cells were cultured in complete RPMI media and left to recover 48 hours prior to activation with ligand. Hepatocytes from wild-type mice were cultured with the PPARβ/δ ligand GW0742 (1 µM) for 23 hours prior to LPS (10 ng/ml) for 1 hour. mRNA expression of Adrp (B) and Tnfa (C) was measured by qPCR. Kupffer cells from wild-type, Pparβ/δ-null or a Pparβ/δ DNA binding mutant were cultured with the PPARβ/δ ligand GW0742 (1 µM) for 8 hours and mRNA expression of Adrp (E) was measured by qPCR. Kupffer cells from wild-type, Pparβ/δ-null or a Pparβ/δ DNA binding mutant were cultured with the PPARβ/δ ligand GW0742 (1 µM) for 23 hours prior to LPS (10 ng/ml) for 1 hour. mRNA expression of Tnfa (F) was measured by qPCR. Values represent the mean ± SEM with a minimum of N=3 replicates per treatment. *Significantly different than control by two-tailed Student’s t-test, P ≤ 0.05.

2.4.6 PPARβ/δ-dependent attenuation of Tnfa in the KC13-2 Kupffer cell

line is most likely mediated by binding of PPARβ/δ to NF-κB

Combining evidence from the literature that PPARβ/δ can interact with the p-65 subunit of NF-κB to attenuate inflammatory signaling [146, 152] and the current observation that

PPARβ/δ attenuates Tnfa expression independent of target gene expression in primary

Kupffer cells, led to the hypothesis that PPARβ/δ most likely binds to the NF-κB transcription factor in Kupffer cells to attenuate inflammatory signaling. The KC13-2

Kupffer cells were utilized to test this hypothesis. PPARβ/δ ligand enhanced PPARβ/δ target gene Adrp confirming activation of PPARβ/δ (Figure 2-6A). LPS induced Tnfa expression and this was significantly attenuated by PPARβ/δ ligand (Figure 2-6B). To determine if this effect was PPARβ/δ-dependent, the experiment was repeated in these cells with siRNA mediated knockdown of PPARβ/δ (Figure 2-6C). The absence of

PPARβ/δ was confirmed by the lack of induction of target gene Adrp (Figure 2-6D) when these cells were treated with PPARβ/δ ligand. LPS induced Tnfa expression in these cells in the absence of PPARβ/δ, but however, no attenuation with PPARβ/δ ligand was observed (Figure 2-6E). These observations are similar to that observed in wild-type primary Kupffer cells. Further, co-immunoprecipitation experiments followed by western blot analysis revealed that PPARβ/δ was bound to p65 subunit of NF-κB in

LPS treated cells in presence of PPARβ/δ ligand (Figure 2-6F). Collectively, these results suggest that PPARβ/δ mediates anti-inflammatory activities in Kupffer cells likely by transrepression of NF-κB activities via protein-protein interaction with p65 subunit of

NF-κB.

Figure 2-6: Ligand activation of PPARβ/δ inhibits pro-inflammatory cytokine Tnfa in the KC13-2 Kupffer cell line via protein-protein interaction with p65 subunit of NF-κB. The KC13-2 Kupffer cell line was cultured with the PPARβ/δ ligand GW0742 (2 µM) for 18 hours prior to LPS (10ng/ml) for 2 hours. mRNA expression of Adrp (A) and Tnfa (B) was measured by qPCR. KC13-2 Kupffer cells treated with PPARβ/δ siRNA and quantitative western blot of PPARβ/δ expression was measured 48 hours after treatment with PPARβ/δ siRNA (C). mRNA expression of Adrp (D) and Tnfa (E) was measured by qPCR for KC13-2 cells treated with the PPARβ/δ

70 ligand GW0742 (2 µM) for 18 hours prior to LPS (10ng/ml) for 2 hours in the presence of PPARβ/δ siRNA. Quantitative western blot of p65-NF-κB, in co-immunoprecipitation of PPARβ/δ with p65 in nuclear extracts of Control (C), LPS only (L), and LPS and ligand (G+L) activated KC13-2 cells. 150 µg of KC13-2 nuclear lysate was used in co- immunoprecipitation using magnetic beads. 2 µg of 8099 PPARβ/δ antibody was used for immunoprecipitation (F). Values represent the mean ± SEM with a minimum of N=3 replicates per treatment. *Significantly different than control by two-tailed Student’s t- test, P ≤ 0.05.

2.5 Discussion

Results from the present study demonstrate a protective role for PPARβ/δ in the development of chronic hepatocellular injury-induced hepatic tumorigenesis. There are very limited studies investigating the role of PPARβ/δ in development of hepatocellular carcinoma in vivo. Most other studies are limited by using cell lines and xenografts as tumor models and more importantly have conflicting results. This is surprising considering the strong evidence in the literature that demonstrates PPARβ/δ to negatively regulate pre-neoplastic stages such as inflammation, proliferation and steatosis, which contribute to the progression of hepatocellular carcinoma. These results show for the first time, that ligand activation of PPARβ/δ attenuates hepatocarcinogenesis in a chronic hepatocellular injury-induced liver cancer model in vivo by inhibition of early onset of inflammation followed by enhanced apoptosis, reduced proliferative potential and hepatic steatosis at later stages of disease pathogenesis.

The attenuation of serum ALT and Tnfa by ligand activation of PPARβ/δ in the HBV mice is consistent with the hepatoprotective effect of PPARβ/δ shown in a chemically induced liver toxicity model by our laboratory previously [140]. This is interesting as it points to an anti-inflammatory role for PPARβ/δ in the liver despite different etiologies of hepatic injury. In an effort to determine the specific cell type involved, the present study shows that PPARβ/δ does not modulate anti-inflammatory activities in the hepatocytes, which is also consistent with previous findings [140]. Instead, it was found that ligand activation of PPARβ/δ attenuates inflammatory Tnfa expression in the primary Kupffer

72 cells of wild-type mice and this effect is PPARβ/δ-dependent, as it is not observed in

Pparβ/δ-null primary Kupffer cells. These observations were also confirmed in the

KC13-2 Kupffer cell line treated with LPS and PPARβ/δ ligand in the absence and presence of PPARβ/δ. Interestingly, in the KC13-2 Kupffer cells treated with PPARβ/δ siRNA, there was a significant increase in Tnfa in the LPS stimulated Kupffer cells pretreated with PPARβ/δ ligand compared to that of cells not treated with PPARβ/δ ligand.

This difference was not observed in LPS stimulated Pparβ/δ-null primary Kupffer cells pretreated with and without PPARβ/δ ligand. A plausible explanation for the observed discrepancy could be due to the differences in both transcriptome and proteome levels between primary cells and immortalized cell lines. For example, a study utilizing Protein

ANalysis THrough Evolutionary Relationships (PANTHER), showed that primary hepatocytes closely resembled in vivo liver phenotype and function, in terms of differentiation and xenobiotic responses to phenobarbital, whereas these effects were not observed in the immortalized cell lines Huh7 and HepG2 [325]. Similarly, another study demonstrated that the cytochrome P450 family of enzymes is highly expressed in primary hepatocytes compared to the Hepa1-6 cell line [326]. Specifically in the case of macrophages, it has also been shown that primary bone-marrow macrophages exhibit significantly greater expression of CD14 compared to that of the immortalized murine macrophage cell line RAW 264.7 and that there was a significantly higher change in inflammatory cytokine TNFa upon LPS stimulation in primary bone marrow macrophages compared to macrophage cell lines such as RAW 264.7 and J774A.1

[327]. Thus it is possible that variations in expressions of xenobiotic metabolizing enzymes such as cytochrome p450 family of enzymes and macrophage activation

73 surface markers such as CD14 could explain the observed discrepancies between

Pparβ/δ-null primary Kupffer cells and the immortalized KC13-2 Kupffer cell line in response to LPS and PPARβ/δ ligand activation. Further, utilizing Pparβ/δ-DBM Kupffer cells, the results gone on to show that PPARβ/δ can attenuate inflammatory signaling independent of its target gene expression. The supposition that PPARβ/δ mediates anti- inflammatory activities by possibly participating in a protein-protein interaction with an inflammatory transcription factor like NF-κB was confirmed by co-immunoprecipitation of

PPARβ/δ with p65 subunit of NF-κB in KC13-2 Kupffer cells treated with LPS and

PPARβ/δ ligand. These results corroborate numerous studies in the field that show

PPARβ/δ attenuates inflammation in the liver [140, 160, 163, 165, 316, 328] and in other models [143, 151, 329-335] and is well reviewed in [135]. The most commonly described mechanism for the modulation of inflammation by PPARβ/δ shown by multiple independent laboratories has been by suppression of NF-κB activity via direct protein-protein binding of PPARβ/δ to NF-κB [150, 152, 154, 336]. A recent study has also further gone on to show that PPARβ/δ inhibits Tnfa promoter occupancy by NF-κB causing inhibition of its transcriptional activity when treated with increasing doses of

PPARβ/δ ligand [146]. Interestingly, other mechanisms driving the anti-inflammatory activities of PPARβ/δ have also been proposed, such as 1) decreased acetylation status of p65 of NF-κB via increased AMPK activation and SIRT1 expression [159], and 2) attenuation of STAT3 activity by transrepressive mechanisms [314]. Although these mechanisms were not looked at in the present study, it is likely that in this HBV model, ligand activation of PPARβ/δ is attenuating inflammation by a transrepressive mechanism involving NF-κB, which is consistent with other findings in the field.

PPARβ/δ has been demonstrated to interact with NF-κB and this interaction has been shown to interfere with either PPARβ/δ [34] or NF-κB [152, 154] activities. For example in rat heart myotubes, it was observed that activation of NF-κB is accompanied by a strong interaction of NF-κB to PPARβ/δ, as well as a corresponding reduction in

PPARβ/δ DNA binding activity and expression of its target gene, pyruvate dehydrogenase kinase 4 (Pdk4). These effects were found to be prevented by NF-κB inhibitors [34]. In human cardiac AC16 cells, palmitate-induced NF-κB-dependent inflammatory cytokine expression was inhibited by treatment of these cells with

PPARβ/δ ligand GW501516. This observation was also accompanied by increased protein-protein interaction between PPARβ/δ and NF-κB. The anti-inflammatory effects of PPARβ/δ ligand GW501516 and the physical interaction between PPARβ/δ and NF-

κB were inhibited in the presence of PPARβ/δ inhibitor GSK0660 [154]. The present study demonstrated a similar interaction in the nucleus of the KC13-2 Kupffer cell line, which interferes with the expression of NF-κB target gene Tnfa. This transrepressive activity could be occurring by either one of two possible ways; 1) the physical interaction of PPARβ/δ with p65 subunit of NF-κB causes steric hindrance and thus prevents NF-

κB from binding to its response elements to initiate target gene expression, or 2) the nuclear proximity of PPARβ/δ to NF-κB promoter regions could promote binding of

PPARβ/δ to NF-κB that is already residing at its response elements and thus inhibiting transcriptional activity of NF-κB target genes. Although the present study did not investigate between these two possible mechanisms, collectively, these results show that PPARβ/δ plays an anti-inflammatory role in the liver that is most likely mediated by transrepression of NF-κB activity by PPARβ/δ in the Kupffer cells. 75

Although it is not possible to determine the mechanism of tumor suppression from these studies, it is possible that the attenuation of inflammatory cytokine Tnfa by PPARβ/δ at the early stages of cellular injury likely contributed to the overall attenuation of hepatocarcinogenesis in the HBV model. TNFa is usually produced as a hepatic tissue response to injury and its prolonged release during continuous cellular injury, as is the case in the current HBV model, can promote genetic instability and fibrosis, eventually culminating in hepatocellular carcinoma. TNFa has mitogenic potential in the liver [337] and has been strongly linked to the development of hepatocellular carcinoma [261,

338]. TNFa is a potent activator of NF-κB transcription factor. TNFa released from inflammatory Kupffer cells induced NF-κB activation in hepatocytes to promote hepatocellular carcinoma in a cholestatic hepatitis model, and treatment with TNFa neutralizing antibodies prevented tumor progression [243]. Both CYCLIN D1 and C-

MYC are target genes of NF-κB containing NF-κB response elements on their promoters [339, 340]. CYCLIN D1 regulates the G1/S phase transition during cell cycle and is often over-expressed in different tumor models [341]. With respect to the liver,

CYCLIN D1 can promote cellular proliferation in the absence of a mitogen [342].

CYCLIN D1 is also frequently over-expressed in liver tumor models and can contribute to the progression of hepatitis-induced hepatocellular carcinoma [343-347]. C-MYC is a proto-oncogene that is linked to cellular proliferation, termination of differentiation, angiogenesis and cellular transformation, all of which can drive tumor promotion [348].

C-MYC is often over-expressed in hepatocellular carcinoma [349-351] and its inhibition promotes hepatic tumor regression in vivo [352]. The inhibition of both CYCLIN D1 and

C-MYC has been shown to strongly reduce the proliferative potential of human and rat hepatoma cells in vitro [353]. Hence, although it is possible that the PPARβ/δ- dependent attenuation of CYCLIN D1 and C-MYC might have in part contributed to the observed attenuation in tumor progression by reducing the hepatic proliferative potential in the HBV model, further studies are required to investigate the underlying mechanisms.

Hepatic steatosis is a common occurrence in hepatitis infections [354-357] and in some cases a strong risk factor for hepatocellular carcinoma [358, 359]. Histological analysis displayed reduced hepatic steatosis in the HBV mice treated with PPARβ/δ ligand, which is consistent with findings by others that have shown attenuation of hepatic steatosis in in vivo models treated with PPARβ/δ ligand [298, 316, 360]. It would be useful for future experiments to perform Oil Red O staining of liver tissue sections and quantification of hepatic triglycerides in liver tissues of HBV mice treated with and without PPARβ/δ ligand to further confirm the observed PPARβ/δ-dependent reduction in hepatic steatosis. Chronic inflammation is a known precursor to the development of hepatic steatosis and it is possible that the reduced steatosis observed in the HBV model was caused by the anti-inflammatory effect of PPARβ/δ. There is a strong link between TNFa and the development of hepatic steatosis. For example, studies have shown that TNFa induces hepatic fat accumulation in different animal models in vivo and inhibition of TNFa signaling via treatment with anti-TNFa therapy attenuates hepatic steatosis in steatohepatitis and diet induced obesity models [361-363]. Specifically, the

Kupffer cell derived TNFa have been strongly implicated as the major cause of TNFa

77 induced hepatic steatosis and recent studies have shown that the subsequent deletion of Kupffer cell specific TNFa or the inhibition of its activity in Kupffer cells via neutralizing antibodies decreased the incidence of steatosis in vivo [364, 365].

Alteration of lipogenic pathways by up-regulation of SREBP-1c expression [363], inhibition of mitochondrial respiratory chain activity [366] and cholesterol efflux [367] are some of the mechanisms by which TNFa promotes hepatic steatosis. Hence, the

PPARβ/δ-dependent attenuation of TNFa could play a role in the attenuated hepatic steatosis observed in the HBV mice. In support of this a recent study shows evidence that ligand activation of PPARβ/δ attenuates hepatic steatosis which is accompanied by a significant attenuation of TNFa in vivo [165]. Alternatively, another study has shown a mechanism which was not investigated in this study, whereby PPARβ/δ up-regulates a target gene Insig-1 that in turn attenuated processing of lipogenic transcription factor

SREBP-1 into its active form, thereby reducing hepatic steatosis [167].

Evasion of apoptosis is a well-known feature in the progression of tumorigenesis.

Ligand activation of PPARβ/δ significantly increased apoptosis in the HBV model and likely plays a role in the overall reduction in tumorigenesis. It would also be beneficial to further confirm the increase in apoptosis with PPARβ/δ ligand treatment in the livers of

HBV mice via other apoptotic assays as well such as quantification of caspase 3 activities in liver tissues and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays of liver tissue sections in HBV mice treated with and without

PPARβ/δ ligand. In fact, a recent study has demonstrated that ligand activation of

PPARβ/δ inhibits proliferation by inducing early apoptosis in a mouse mammary gland

78 cancer cell line, which subsequently results in reduced clonogenicity [368]. This suggests that PPARβ/δ regulates cell proliferation indirectly by modulating apoptosis, which can subsequently contribute to reduced tumorigenesis. Although not evaluated in the present study, it would be useful to exogenously administer Annexin V (a protein that binds externalized phospatidylserine during apoptosis) to HBV mice treated with and without PPARβ/δ ligand at various time points to detect changes in apoptosis and proliferation side by side via the use of immunohistochemical staining, which will help determine if the attenuation of proliferative potential in livers of HBV mice treated with

PPARβ/δ ligand is preceded by the onset of apoptosis. However, there is currently no consensus on the role of PPARβ/δ in modulating apoptosis. While some studies have shown that PPARβ/δ inhibits apoptosis by various mechanisms [187, 190, 369-371], contradictory studies have shown that this is not the case and that PPARβ/δ either promotes or has no effect on apoptosis [123, 126, 182, 192, 368, 372, 373]. Further studies need to be done to determine the mechanism involved.

In summary, results of the present study not only support the previously published anti- inflammatory, anti-proliferative and anti-steatotic functions of PPARβ/δ in the liver, but have also gone further to show that by modulating these activities, PPARβ/δ-dependent signaling attenuates hepatic tumorigenesis in a chronic hepatocellular injury-induced liver cancer model in vivo. This points to a chemopreventive role for PPARβ/δ in the liver especially in hepatocellular carcinoma models like hepatitis infections, which have chronic inflammation as a precursor to tumor progression. The HBV mice were a suitable model to study the progression of prolonged cellular injury and chronic

79 inflammation, which eventually results in hepatocellular carcinoma. However, the lack of a HBV Pparβ/δ-null model is one of the limitations of this study, which could have further strengthened the conclusion of the present studies. Since PPARβ/δ modulates its anti-inflammatory activities mainly through the Kupffer cells, future studies should also investigate the role of PPARβ/δ in a chronic hepatocellular injury-induced liver cancer model that has a Kupffer cell specific deletion of PPARβ/δ. The role of PPARβ/δ in cancer progression is still very controversial [47, 374, 375] making it difficult to definitively consider it as a target for chemoprevention. As promising as these results are, more work needs to be done to further investigate the potential of PPARβ/δ in preventing hepatic tumor progression.

Chapter 3 PPARβ/δ improves survival of hepatitis C transgenic (HCV)

mice

3.1 Abstract

PPARβ/δ can inhibit pro-inflammatory activities in the liver, which have potential to contribute to liver cancer. Hence, ligand activation of PPARβ/δ has an established hepatoprotective role in the liver by negatively modulating preneoplastic conditions such as oxidative stress, metabolic deregulation and inflammation. Thus, the effects of hepatic PPARβ/δ-dependent signaling in HCV transgenic mice expressing the core protein was examined in male HCV wild-type and HCV Pparβ/δ-null mice in vivo. In preliminary studies utilizing HCV wild-type and HCV Pparβ/δ-null mice treated with

(GW0742) and without (control) PPARβ/δ ligand, a significant increase in survival was observed in HCV wild-type control mice compared to that of HCV Pparβ/δ-null control mice, as well as in HCV wild-type mice treated with GW0742 compared to that of HCV

Pparβ/δ-null mice treated with GW0742. No difference in survival was observed between HCV wild-type control mice and HCV wild-type mice treated with GW0742 as well as between HCV Pparβ/δ-null control mice and HCV Pparβ/δ-null mice treated with

GW0742. Protein microarray displayed a consistent increase in Cofilin 1 and a decrease in SODD, SOCS2 and p27Kip1 expression levels, as well as a decrease in phosphorylation of PKCγ in livers of 18-month-old HCV Pparβ/δ-null mice compared to

HCV wild-type mice. Hematoxylin and eosin stained liver tissue sections revealed no significant difference in hepatic inflammation and steatosis between 24-month-old HCV wild-type and HCV Pparβ/δ-null mice as well as an absence of hepatocellular carcinoma

81 in both genotypes. Taken together, preliminary findings from this study suggest that although the role of PPARβ/δ in HCV core induced hepatic inflammation, steatosis and hepatocellular carcinoma remains in-conclusive, PPARβ/δ does however play a protective role in HCV mice by improving their survival independent of ligand activation.

The lack of an observed effect in survival of HCV wild-type mice treated with PPARβ/δ ligand could possibly be due to saturation of ligand binding sites with endogenous ligands, that might block the action of the exogenous PPARβ/δ ligand, GW0742, or the interference of the HCV core protein with the ligand activation pathway of PPARβ/δ.

Alternatively, it is also possible that a higher dose of GW0742 might be required to see a significant effect.

3.2 Introduction

Peroxisome proliferator-activated receptor, PPARβ/δ (NR1C2) [308] belongs to the nuclear hormone receptor superfamily of ligand inducible transcription factors. Although expressed ubiquitously, its expression has been found to be the highest in liver, colon and skin [117, 309, 310]. Many studies investigating the function of PPARβ/δ in the liver, have demonstrated a hepatoprotective role for this nuclear receptor. PPARβ/δ attenuates hepatic oxidative stress by increasing expression levels of antioxidant enzymes catalase [376] and superoxide dismutase [377]. PPARβ/δ has also been shown to attenuate hepatic steatosis, either by an increase in fatty acid oxidation [162] or by a decrease in fatty acid synthesis [167]. PPARβ/δ also contributes to attenuation of hepatic insulin resistance by improving insulin sensitivity [165, 378]. The anti- inflammatory role of PPARβ/δ in the liver has also been well established [140, 160, 298,

316], and most likely occurs via regulation of the innate immune signaling pathway

[163]. Since prolonged oxidative stress, inflammatory signaling and metabolic deregulations like steatosis and insulin resistance can promote the development of hepatocellular carcinoma, as is in the case of chronic alcoholism, hepatitis infections and non-alcoholic fatty liver disease, it is intuitive to hypothesize that PPARβ/δ might play a preventative role in the development of hepatocellular carcinoma.

Chronic hepatitis C infection has been identified as the leading cause for the development of hepatocellular carcinoma in developed countries and has become a major global health problem [379]. It is estimated that approximately 5.2 million people are infected with hepatitis C (HCV) in the United States alone [380]. HCV is a small,

83 enveloped virus belonging to the Flaviviridae family and consists of a positively polarized single stranded RNA genome encoding structural (core and envelope glycoproteins E1 and E2) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, and

NS5B) proteins [381, 382]. Of these, the HCV core has been heavily implicated in a number of disease pathogenesis. Expression of the HCV core protein in transgenic mice leads to an increase in oxidative stress and mitochondrial DNA damage [383]. The

HCV core protein has also been shown to bind to hepatic mitochondria and interfere with complex I of the electron transport chain in the mitochondria, further contributing to mitochondrial oxidative stress [283]. Studies have demonstrated that the HCV core promotes metabolic deregulations like hepatic steatosis [384, 385] and insulin resistance [386, 387]. The HCV core has also been associated with increasing inflammatory cytokine expression of TNFa and IL-6 as well as activation of innate immune cells [388-390]. All of the above-mentioned modifications by the HCV core can easily lead to the development of hepatocellular carcinoma and have the potential to be prevented by PPARβ/δ-dependent signaling.

The present study aimed to examine the hypothesis that PPARβ/δ plays a protective role in the disease pathogenesis of a HCV core induced hepatocarcinogenesis model in vivo. HCV core transgenic mice, which develop progressive hepatic steatosis and oxidative stress, eventually culminating in the development of hepatocellular carcinoma were used for these studies. These mice were crossed with wild-type and Pparβ/δ-null mice to give HCV wild-type and HCV Pparβ/δ-null mice. Preliminary results from this

84 study demonstrate that PPARβ/δ plays a protective role in these HCV mice, by improving their survival independent of ligand activation.

3.3 Materials and Methods

Animals and treatments

Male HCV mice on a C57BL/6 background, expressing the core protein of HCV genotype 1b, driven by a transcriptional regulatory region from hepatitis B virus, were used for these studies [292]. These mice start to develop hepatic steatosis by 3 months of age with progressive hepatic lipid accumulation, as they get older. They also develop hepatocellular adenomas at 16 months and hepatocellular carcinomas between 19-24 months of age. The HCV mice were crossed with wild-type and Pparβ/δ-null mice on a

C57BL/6 background (previously described in [306]) to give HCV wild-type and HCV

Pparβ/δ-null mice. For studies comparing the phenotypic characteristics, HCV wild-type and HCV Pparβ/δ-null mice were left to age without any treatment until 18 or 24 months of age. At the end of the study, mice were euthanized by overexposure to carbon dioxide, followed by cervical dislocation, before whole liver and serum were collected.

For the ligand activation study, 4 month-old HCV wild-type and HCV Pparβ/δ-null mice were orally administered the highly specific PPARβ/δ ligand GW0742 (provided by

GlaxoSmithKline) at a dose of for 2.5 mg/kg (5 days per week) for 18 months. Mice were dosed with either just a pellet (Bacon-flavored Transgenic Dough Diet, Bioserv,

Inc.) in the case of the control group, or a pellet mixed with GW0742 powder in the case of the treatment group. All animal experiments were conducted in accordance to the

Institutional Animal Care and Use Committee (IACUC) at Pennsylvania State University.

Antibody microarray

Antibody microarrays were done using Kinex™ antibody microarray and subsequent analysis of microarray results were performed by Kinexus (Kinexus Bioinformatics

Corporation, British Columbia, Canada). Liver tissue lysates were prepared from 18 month-old HCV wild-type and HCV Pparβ/δ-null mice. Briefly, liver tissue lysates were homogenized in lysis buffer provided by Kinexus by using a chilled Dounce glass homogenizer. The lysis buffer provided, contained 20 mM MOPS at pH 7.0, 2 mM

EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mM β –glycerophosphate at pH 7.2, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate and 1% Triton X-100 which was supplemented with protease inhibitors (1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 µM pepstatin A, 10 µM leupeptin and 1 mM dithiothreitol). Protein concentrations of homogenized lysates were determined using the BCA protein assay kit (Pierce, Rockford, Illinois) according to the manufacturer's protocol. A total of 2 antibody microarrays were performed. Each microarray contained a 50 µg sample of liver tissue lysates pooled from 2 male mice for HCV wild-type (control) and HCV

Pparβ/δ-null (non-control). Lysates were labeled with a fluorescent dye prior to incubation on microarrays. After incubation, unbound proteins are removed by washing and a laser array scanner detects protein-antibody hybridization signals. Signal intensities were quantified to determine expression levels and phosphorylation status of various proteins.

RNA isolation and quantitative polymerase chain reaction (qPCR) analysis

RNA was extracted from whole liver tissues of 18 month-old HCV wild-type and HCV

Pparβ/δ-null mice using Ribozol RNA extraction reagent (Amresco Inc., Solon, OH) and

1.25 µg of total RNA was reverse transcribed into cDNA using reverse transcription kit

(Promega, WI,). qPCR reactions were carried out using MyiQ Real-Time PCR Detection

System (Bio-Rad Laboratories, Hercules, CA) for tumor necrosis factor alpha (Tnfa), interleukin-6 (Il-6), interleukin-1 beta (Il-1β), and cluster of differentiation 14 (CD14).

Relative mRNA values were normalized to internal control values of Actin. The primer sets used were CD14 forward, 5’-TTCAGAATCTACCGACCATGGAGC-3’ and reverse,

5’-CAATTGAAAGCGCTGGACCAA-3’, TNFa forward, 5’-

AGGGTCTGGGCCATAGAACT-3’ and reverse, 5’-CCACCACGCTCTTCTGTCTAC-3’,

IL-6 forward, 5’-ATCCAGTTGCCTTCTTGGGACTGA-3’ and reverse 5’-

TAAGCCTCCGACTTGTGAAGTGGT-3’, IL-1β forward, 5’-

AAGGGCTGCTTCCAAACCTTTGAC-3’ and reverse 5’-

ATACTGCCTGCCTGAAGCTCTTGT-3’, ACTIN forward, 5’-

CCGTGAAAAGATGACCCAGATC-3’ and reverse 5’-

TGGTACGACCAGAGGCATACAG-3’,

Protein extraction and western blot analysis

Liver tissues of 18 month-old HCV wild-type and HCV Pparβ/δ-null mice were homogenized in lysis buffer containing 50 mM Tris, 150 mM NaCl, 0.1% SDS, and 1%

Nonidet P-40 at pH 7.5 supplemented with protease inhibitors, using a chilled Dounce glass homogenizer. Thirty micrograms of protein was loaded per sample and resolved

88 using SDS gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat milk or 0.5% gelatin in Tris buffered saline with Tween-20 (TBST) and subsequently incubated with primary and secondary antibodies. The primary antibodies used were anti-SOCS2 (2779S) and anti- p27Kip1 (2552S) from Cell Signaling Technology, Danvers, MA as well as anti-β -

ACTIN (SC-47778) from Santa Cruz Biotechnologies, Santa Cruz, CA. The secondary antibodies used were biotinylated (Jackson ImmunoResearch Laboratories, West

Grove, PA). The membranes were then incubated with 125I-labeled streptavidin and hybridization signals were quantified by detecting levels of radioactivity. Protein expression levels are presented as fold change from control liver tissues of HCV wild- type mice.

Liver histology

Liver tissue sections of 24 month-old male mice were fixed in 10% buffered formalin and transferred to 70% ethanol, before being embedded in paraffin for histological analysis.

Tissue sections were stained with hematoxylin and eosin (H&E). The degree of hepatic inflammation and macrovesicular fatty change was analyzed and scored by a pathologist.

Statistical analysis

Statistical analysis for the histopathological analysis and Kaplan-Meier survival curves was performed using GraphPad Prism (GraphPad Software, Inc.). For comparison of two survival curves, the log-rank (Mantel-Cox) test was used. For comparison of

89 percentage of mice with a specific liver pathology, Fisher’s exact test was used. For analysis of statistical difference in quantitative western blots and polymerase chain reactions, an unpaired two-tailed Student’s t-test was used. Statistically significant difference was inferred when P ≤ 0.05. For anti-body microarray analysis, Z scores, Z ratios and % CFC were calculated by Kinexus. Z scores reflect the number of standard deviations a measured protein expression is from the mean protein expression for a given sample (HCV wild-type (control) or HCV Pparβ/δ-null (non-control) liver tissues). Z ratios which were calculated from Z score differences between control and non-control liver tissues, determine significant changes for a given protein expression between the two groups of liver tissues. Briefly, Z normalization was performed after background- corrected raw signal intensity data were logarithmically transformed with base 2. Z- scores for spots of specific antibody-protein hybridization signals from the antibody microarrays were quantified by subtraction of the overall average intensity of all spots within a sample from the raw intensity for each spot and then further dividing this by the standard deviation of all the measured intensities for each sample. Z ratios were then calculated from the Z scores, by dividing the Z score differences in a given comparison by the standard deviation of all the differences for that comparison. Thus, the Z ratio reflects the degree of difference in protein expression and phosphorylation levels between liver tissues from HCV wild-type (control) and HCV Pparβ/δ-null (non-control) mice. A positive Z ratio value indicates an increase in expression/phosphorylation with respect to the control and a negative Z ratio value indicates a decrease in expression/phosphorylation with respect to the control. Z ratio values greater than ± 1.5 are inferred to be significant. % CFC (Percent change from control) reflects the percent

90 change in normalized signal intensity of non-control to control samples. A 100% CFC value corresponds to a 2-fold change in signal intensity from control samples. A positive

%CFC reflects an increase in percent change from control whereas a negative %CFC reflects a decrease.

3.4 Results

3.4.1 PPARβ/δ-dependent signaling improves survival in HCV mice in vivo

HCV wild-type and HCV Pparβ/δ-null mice were treated with and without PPARβ/δ ligand GW0742 and monitored over a period of 22 months to determine survival.

Survival rate of all 4 groups are depicted in Kaplan-Meier curves. PPARβ/δ significantly improved the survival rate of HCV wild-type mice compared to that of the HCV Pparβ/δ- null mice (Figure 3-1A). A significant increase in survival was also observed in HCV wild-type mice treated with PPARβ/δ ligand compared to that of HCV Pparβ/δ-null mice treated with ligand (Figure 3-1B). However, there was no significant difference in survival observed between HCV wild-type mice and HCV wild-type mice treated with

PPARβ/δ ligand (Figure 3-1C). A similar observation was made for HCV Pparβ/δ-null mice and HCV Pparβ/δ-null mice treated with PPARβ/δ ligand (Figure 3-1C). These findings suggest that the presence of PPARβ/δ contributes to improved survival in HCV mice in vivo. The lack of a significant change in survival rate between HCV wild-type mice and HCV wild-type mice treated with PPARβ/δ ligand, suggests that the protective effects of PPARβ/δ in improving survival are likely occurring independent of ligand activation. This observation could be due to the saturation of the ligand-binding pocket of PPARβ/δ with endogenous ligands, thus blocking the targeted action of the exogenous PPARβ/δ ligand, GW0742, or alternatively a higher dose of GW0742 might be needed to see an effect. Another highly possible explanation could be that the HCV core protein is interfering with the ligand activation pathway of PPARβ/δ. A recent study has demonstrated that in these HCV mice, persistent activation of the nuclear receptor

PPARα is required for the HCV core protein to cause hepatic steatosis and hepatocarcinogenesis [391] and an earlier study from the same group demonstrated that the core protein interacts with the DNA binding domain of RXRα which leads to the enhanced DNA binding and transcriptional activity of RXRα-RXRα homodimers and

PPARα-RXRα heterodimers [392]. This raises the possibility that the prolonged sequestration of RXRα by the core protein to favor the RXRα-RXRα homodimerization and PPARα-RXRα heterodimerization, may limit the available pool of RXRα that is needed to bind ligand activated PPARβ/δ.

Figure 3-1: PPARβ/δ promotes survival in HCV transgenic mice. 4-month- old HCV wild-type and HCV Pparβ/δ-null mice were treated with (GW0742) and without (control) PPARβ/δ ligand for 18 months (2.5 mg/kg, 5 days per week). Survival analysis for 22 months is depicted in Kaplan-Meier curves. Significantly improved survival of HCV wild-type control mice compared to HCV Pparβ/δ-null control mice (A). Significantly, improved survival of HCV wild-type mice treated with GW0742 compared to that of HCV Pparβ/δ-null mice treated with GW0742 (B). No significant (N.S.) differences in survival between HCV wild-type control mice and HCV wild-type control mice treated with GW0742 and no significant differences in survival between HCV Pparβ/δ-null control mice and HCV Pparβ/δ-null control mice treated with GW0742 (C). N=10 per group. *Significantly different than control by log-rank (Mantel-Cox) test when P ≤ 0.05.

3.4.2 PPARβ/δ-dependent signaling does not modulate hepatic

inflammatory cell infiltration and steatosis in HCV mice in vivo

Previous findings have demonstrated that the absence of PPARβ/δ contributes to increased lymphocyte infiltration in livers of CCl4 treated mice [140]. To determine if similar findings might be observed in the HCV mice, preliminary studies utilizing histopathological analysis of liver tissues from 24 month-old HCV wild-type and HCV

Pparβ/δ-null mice were performed (Table 3-1). An increase in hepatic inflammatory mononuclear cell infiltration and a decrease in peribiliary inflammatory cell infiltration in

HCV wild-type mice compared to HCV Pparβ/δ-null mice were observed. However, the differences between the two genotypes were not significant. Similarly, although 18 month-old HCV Pparβ/δ-null mice showed a slight increase in mRNA of hepatic inflammatory markers Tnfa and Il-6 as well as a marker of activated macrophages,

CD14 compared to that of HCV wild-type mice, these differences were not significant

(Figure 3-2). The HCV core has been shown to promote hepatic steatosis as part of its disease pathogenesis [384], and because PPARβ/δ has been demonstrated to reduce steatosis in other hepatic models [162, 167], macrovesicular fatty change between the two genotypes was determined. There was a non-significant increase in hepatic lipid accumulation in HCV wild-type mice compared to HCV Pparβ/δ-null mice. Interestingly, histopathological analysis did not reveal any presence of hepatocellular carcinoma in

HCV wild-type or HCV Pparβ/δ-null liver tissues and there were also no differences in hepatocellular centrilobular hypertrophy at different stages of disease severity between both genotypes as well. Taken together, limited findings from the histopathological analysis and mRNA quantification of liver tissues from HCV wild-type and HCV Pparβ/δ-

95 null mice do not conclusively determine a role for PPARβ/δ in modulating hepatic inflammation, steatosis or hepatocellular carcinoma in HCV mice warranting further investigation.

Table 3-1: Histopathological analyses do not confirm a role for PPARβ/δ in modulating hepatic inflammation, steatosis and centrilobular hypertrophy in HCV mice in vivo. Histopathological analysis of hematoxylin and eosin stained liver tissue sections of 24 month-old HCV wild-type and HCV Pparβ/δ-null mice. Fractions and corresponding percentages of mice of each genotype with the absence or presence of varying degrees of hepatic mononuclear and peribiliary inflammatory cell infiltration as well as hepatic macrovesicular fatty change and centrilobular hypertrophy were scored as minimal, slight or moderate. (–) indicates that none of the mice had the corresponding observed pathology. N=8 for HCV wild-type and N=12 for HCV Pparβ/δ-null mice. There were no significant differences between HCV wild-type and HCV Pparβ/δ-null mice as determined by Fisher’s exact test.

Figure 3-2: PPARβ/δ-dependent modulation of hepatic inflammatory cytokines in HCV transgenic mice. Liver tissues were isolated from 18-month-old HCV wild-type and HCV Pparβ/δ-null mice and mRNA levels of Tnf-α (A), Il-6 (B), Il-1β (C) and CD14 (D) was determined by qPCR. Values represent the mean ± SEM. N=7 for HCV wild-type and N=6 for HCV Pparβ/δ-null mice. There were no significant differences between HCV wild-type and HCV Pparβ/δ-null mice as determined by two- tailed Student’s t-test.

3.4.3 Hepatic protein expression and phosphorylation in response to

targeted disruption of PPARβ/δ function in HCV mice in vivo

Preliminary studies using anti-body microarrays of liver tissues from 18 month-old HCV wild-type and HCV Pparβ/δ-null mice were done to determine if differences in expression and phosphorylation status of various signaling molecules between the two genotypes existed. A total of 2 antibody microarrays were performed with liver tissue lysates pooled from 2 HCV wild-type and HCV Pparβ/δ-null mice used for each microarray. A partial list of proteins for each microarray, whose expression and levels of phosphorylation, that show a statistically significant Z ratio, accompanied by minimal percent error range within the HCV wild type and HCV Pparβ/δ-null liver tissues, thus validating them for further quantitative analysis, are shown in Table 3-2 (I & II).

Consistently, between the 2 microarrays, the expression levels of Cofilin 1 was increased and that of SOCS2, p27 Kip1 and SODD were decreased in livers of HCV

Pparβ/δ-null mice compared to that of HCV wild-type mice. Similarly both microarrays displayed decreased phosphorylation of hepatic PKCγ in HCV Pparβ/δ-null mice. The consistency of these findings between the 2 microarray analyses suggests strongly that these proteins are modulated by PPARβ/δ in the livers of HCV mice. Because of the consistent change in expression and phosphorylation patterns of SOCS2, p27Kip1,

Cofilin 1, SODD and PKCγ, the global signal intensities for these proteins from the 2 microarrays were pooled to determine a combined net difference between HCV wild type and HCV Pparβ/δ-null liver tissues as can be seen in Table 3-3. Further validation by quantitative immunoblotting was done for SOCS2 and p27Kip1 (Figure 3-3). Limited information from the immunoblots, show a decrease in SOCS2 expression in HCV wild

98 type liver tissues compared to that of HCV Pparβ/δ-null liver tissues was found, corroborating the results of the microarray analyses, although, the difference was not statistically significant. However, no change in protein expression of p27Kip1 was found between HCV wild type liver tissues compared to that of HCV Pparβ/δ-null, contradicting the results of the microarray analyses. This observed discrepancy could be due to differences in the specificity of the type of antibodies used for microarray analyses, versus that for quantitative immunoblotting. Interestingly, contradicting results between the 2 microarrays were also found with expression levels of NF- κB p65 and p50 subunits being increased in one microarray but not in the other. A similar trend was also observed with phosphorylation levels of PKCδ as well. In addition, a number of proteins found to be significantly modulated by PPARβ/δ in one microarray analysis were not modulated in the other and vice-versa. The discrepancies in these observations could be due to reactivity of antibodies with off target proteins giving rise to false positives, or the result of experimental error during microarray analysis which results in changes between hepatic protein modulation between HCV wild-type and

HCV Pparβ/δ-null to be identified in one microarray, but not in the other. Nevertheless, the results of the anti-body microarray serve as a qualitative and semi quantitative analysis and a quantitative western blot analysis of promising targets should be done to further validate findings.

Antibody Array (I) Target Protein Fold Full Target Protein Name Phospho Site % CFC Name Change Bax Apoptosis regulator Bcl2-associated X protein Pan-specific 355 7.1 STAT4 Signal transducer and activator of transcription 4 (acute phase responsePan-specific factor) 273 5.5 Catenin b1 Catenin (cadherin-associated protein) beta 1 Pan-specific 284 5.7 IkBb Inhibitor of NF-kappa-B beta (thyroid receptor interacting protein 9) Pan-specific 244 4.9 NFkappaB p65 # NF-kappa-B p65 nuclear transcription factor Pan-specific 265 5.3 LAR LCA antigen-related (LAR) receptor tyrosine phosphatase Pan-specific 186 3.7 STAT6 Signal transducer and activator of transcription 6 Pan-specific 186 3.7 STAT5A Signal transducer and activator of transcription 5A Pan-specific 186 3.7 STAT2 Signal transducer and activator of transcription 2 Pan-specific 178 3.6 Bcl-xS/L Bcl2-like protein 1 Pan-specific 161 3.2 Fos Fos-c FBJ murine osteosarcoma oncoprotein-related transcription factorPan-specific 164 3.3 IkBa Inhibitor of NF-kappa-B alpha (MAD3) Pan-specific 171 3.4 Dok2 Docking protein 2 Y139 139 2.8 STAT5B Signal transducer and activator of transcription 5B Pan-specific 118 2.4 Trail Tumor necrosis factor-related apoptosis-inducing ligand Pan-specific 142 2.8 NFkappaB p50 # NF-kappa-B p50 nuclear transcription factor Pan-specific 109 2.2 STAT1a Signal transducer and activator of transcription 1 alpha Pan-specific 140 2.8 p107 Retinoblastoma (Rb) protein-related p107 (PRB1) Pan-specific 110 2.2 Chk1 Checkpoint protein-serine kinase 1 S280 93 1.9 MDM2 double minute 2 S166 105 2.1 Chk2 Checkpoint protein-serine kinase 2 T68 88 1.8 CD45 Leukocyte common antigen CD45 receptor-tyrosine phosphatase (LCA,Pan-specific T200) 86 1.7 IRS1 Insulin receptor substrate 1 S639 77 1.5 CytoC Cytochrome C Pan-specific 90 1.8 B23 (NPM) B23 (nucleophosmin, numatrin, nucleolar protein NO38) T234/T237 81 1.6 Histone H2A.X Histone H2A variant X S140 80 1.6 PKCq Protein-serine kinase C theta S695 67 1.3 CK2a Casein protein-serine kinase 2 alpha/ alpha prime T360/S362 90 1.8 IGF1R Insulin-like growth factor 1 receptor protein-tyrosine kinase Y1280 72 1.4 PAK1 p21-activated kinase 1 (alpha) (serine/threonine-protein kinase PAK 1) T212 72 1.4 ErbB3 Tyrosine kinase-type cell surface receptor HER3 Y1328 70 1.4 Hsc70 Heat shock 70 kDa protein 8 Pan-specific 67 1.3 Cofilin 1 * Cofilin 1 Pan-specific 72 1.4 Dok2 Docking protein 2 Y139 71 1.4 EFNA5 Ephrin-A5 Pan-specific 51 1 IRS1 Insulin receptor substrate 1 Y1179 72 1.4 IKKa Inhibitor of NF-kappa-B protein-serine kinase alpha (CHUK) T23 49 1 4G10 4G10 pTyr 63 1.3 CDK1/2 Cyclin-dependent protein-serine kinase 1/2 Y15 52 1 Rac1 Ras-related C3 botulinum toxin substrate 1 Pan-specific -36 -0.7 Integrin a4 Integrin alpha 4 (VLA4) S1027 -26 -0.5 Rac1 Ras-related C3 botulinum toxin substrate 1 S71 -43 -0.9 Tau Microtubule-associated protein tau S721 -43 -0.9 TBK1 Serine/threonine-protein kinase TBK1 Pan-specific -46 -0.9 PP2Cd Protein-serine phosphatase 2C - catalytic subunit - delta isoform Pan-specific -38 -0.8 PKCg * Protein-serine kinase C gamma T655 -40 -0.8 SODD * Silencer of death domains (Bcl2 associated athanogene 4 (BAG4)) Pan-specific -55 -1.1 p27 Kip1 * p27 cyclin-dependent kinase inhibitor 1B Pan-specific -57 -1.1 SOCS2 * Suppressor of cytokine signaling 2 Pan-specific -52 -1 PKCd # Protein-serine kinase C delta Y313 -56 -1.1 PP2A/Bb Protein-serine phosphatase 2A - B regulatory subunit - beta isoform Pan-specific -61 -1.2

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Antibody Array (II) Target Protein Fold Full Target Protein Name Phospho Site % CFC Name Change Cofilin 1 * Cofilin 1 Pan-specific 181 3.6 IR (INSR) Insulin receptor beta chain Pan-specific 134 2.7 PKCd # Protein-serine kinase C delta Y313 126 2.5 FasL Tumor necrosis factor ligand, member 6 Pan-specific 125 2.5 Cellular inhibitor of apoptosis protein 1 (baculoviral IAP repeat- c-IAP1 containing protein 3, apoptosis inhibitor 2 (API2)) Pan-specific 76 1.5 Lck Lymphocyte-specific protein-tyrosine kinase Y192 68 1.4 RONa Macrophage-stimulating protein receptor alpha chain Pan-specific 68 1.4 ATF2 Activating transcription factor 2 (CRE-BP1) T69 + T71 64 1.3 Cdc34 Cell division cycle 34 (ubiquitin-conjugating ligase) Pan-specific 62 1.2 PKCe Protein-serine kinase C epsilon Pan-specific 60 1.2 eIF4G Eukaryotic translation initiation factor 4 gamma 1 S1107 60 1.2 DAXX Death-associated protein 6 (BING2) Pan-specific 59 1.2 Kit Kit/Steel factor receptor-tyrosine kinase Y703 59 1.2 NIK (MAP3K14) NF-kappa beta-inducing kinase Pan-specific -35 -0.7 Cyclin B1 Cyclin B1 Pan-specific -36 -0.7 RafA (Araf) A-Raf proto-oncogene serine/threonine-protein kinase Pan-specific -37 -0.7 Smad2 Mothers against decapentaplegic homolog 2 S467 -37 -0.7 PKCg * Protein-serine kinase C gamma T514 -37 -0.7 Nek2 NIMA (never-in-mitosis)-related protein-serine kinase 2 Pan-specific -39 -0.8 NFkappaB p65 # NF-kappa-B p65 nuclear transcription factor Pan-specific -39 -0.8 p27 Kip1 * p27 cyclin-dependent kinase inhibitor 1B Pan-specific -45 -0.9 NFkappaB p65 NF-kappa-B p65 nuclear transcription factor S529 -45 -0.9

SODD * Silencer of death domains (Bcl2 associated athanogene 4 (BAG4)) Pan-specific -47 -0.9 p25 CDK5 regulatory subunit p25 Pan-specific -46 -0.9 p21 CDKI1 cyclin-dependent kinase inhibitor 1 (MDA6) Pan-specific -48 -1 SOCS2 Suppressor of cytokine signaling 2 Pan-specific -48 -1 p73 Tumor suppressor protein p73 Pan-specific -48 -1 SOD (Mn) Superoxide dismutase [Mn] Pan-specific -49 -1 NFkappaB p50 # NF-kappa-B p50 nuclear transcription factor Pan-specific -50 -1 NBS1 Nijmegen breakage syndrome protein 1 S343 -50 -1 MST1 Mammalian STE20-like protein-serine kinase 1 (KRS2) Pan-specific -50 -1 Protein-tyrosine phosphatase 1D (SHP2, SHPTP2, Syp, PTP2C, PTP1D PTPN11) S580 -51 -1 RafB (Braf) RafB proto-oncogene-encoded protein-serine kinase Pan-specific -51 -1 ROKb (ROCK1) RhoA protein-serine kinase beta Pan-specific -60 -1.2

Table 3-2: Partial lists from 2 anti-body microarrays of target proteins significantly modulated by PPARβ/δ as identified by KinexTM anti-body microarray analysis. Liver tissue protein lysates prepared from 18 month-old HCV wild-type and HCV Pparβ/δ-null mice were used for protein microarray analysis. Each microarray contained a sample of lysate pooled from 2 HCV wild-type (Control) mice and 2 HCV Pparβ/δ-null (Non-Control) mice. Two antibody microarrays in total were done (Array I & II). % CFC indicates percent change from control. Fold change indicates degree of increase or decrease in fold of expression with respect to control. Pan- specific antibodies detected protein expression levels whereas, phosphorylation status was determined by using antibodies to corresponding phosphorylation sites. Positive values indicate increased expression/phosphorylation with respect to Control. Negative values indicate decreased expression/phosphorylation with respect to Control. Target

101 proteins with (*) indicate consistent expression patterns between the two microarrays. Target proteins with (#) indicate opposite expression patterns between the two microarrays. *All proteins shown in the partial lists have a significant Z ratio greater than ± 1.5.

Table 3-3: List of combined fold changes in expression and phosphorylation of proteins with consistent expression patterns between antibody microarray I and II. Liver tissue protein lysates prepared from two 18 month-old HCV wild-type (Control) and two HCV Pparβ/δ-null (Non-control) mice were used for each protein microarray analysis. Global signal intensities of protein expression and phosphorylation patterns that are consistent between microarray I and II were pooled. % CFC indicates percent change from control. Fold change indicates degree of increase or decrease in fold with respect to control. Pan-specific antibodies detected protein expression levels whereas, phosphorylation status was determined by using antibodies to corresponding phosphorylation sites. Positive values indicate increased expression/phosphorylation with respect to Control. Negative values indicate decreased expression/phosphorylation with respect to Control. N=4 for HCV wild-type (Control) and N=4 HCV Pparβ/δ-null (Non-control). *List of proteins shown in the table have a significant Z ratio of greater than ± 1.5.

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Figure 3-3: Western blot quantification of changes in microarray analysis of SOCS2 and p27Kip1. Quantitative western blot of SOCS2 and p27Kip1 in liver tissue samples of 18 month-old HCV wild-type and HCV Pparβ/δ-null mice. 30 µg of whole cell lysate was loaded per sample. Normalized values were calculated from the independent samples and represent the mean ± SEM. N=6 for HCV wild-type and N=6 for HCV Pparβ/δ-null. There were no significant differences between HCV wild-type and HCV Pparβ/δ-null mice as determined by two-tailed Student’s t-test.

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3.5 Discussion

The hepatoprotective effects of PPARβ/δ in the literature prompted the investigation of the role of PPARβ/δ in HCV core-induced hepatitis C transgenic mice. Preliminary findings from the current study show a significant decrease in survival of HCV mice lacking PPARβ/δ expression. Further, this protective effect of PPARβ/δ seems to be modulated in a manner independent of ligand activation. The ubiquitous expression pattern and the numerous crucial physiological functions that PPARβ/δ regulates would suggest that the absence of PPARβ/δ could possibly derail several important signaling pathways, contributing to the observed reduction in survival in HCV Pparβ/δ-null mice.

However, the lack of a difference in survival between wild-type and Pparβ/δ-null mice, suggests that the HCV core protein is contributing to the increase in mortality and

PPARβ/δ is preventing this occurrence in these HCV mice. Although the underlying mechanisms of this effect were not investigated in the present study, a possible pathway by which PPARβ/δ could be promoting survival is by attenuation of oxidative stress. The induction of oxidative stress in chronically infected hepatitis C patients has been well documented. Liver tissues of hepatitis C infected patients display increased reactive oxygen species [393, 394], decreased expression and activity of anti-oxidant enzymes glutathione [395-397] and Cu, Zn- and Mn-superoxide dismutase [398, 399].

Similar observations have been seen in mice expressing the core protein of HCV. In these mice, the core protein has been demonstrated to increase lipid peroxidation, reduce glutathione activity and increase mitochondrial [383, 400] and DNA damage

[401, 402]. Additionally, the HCV core has also been demonstrated to increase oxidative stress by promoting other pathologies, such as mitochondrial dysfunction [282, 283,

104

403, 404] and iron overload [405-407]. Studies have also shown that reducing oxidative stress by over-expressing anti-oxidant enzymes or mimicking their activity can result in a direct decrease in mortality in in vivo models [408-410]. Contradicting the function of

HCV core, PPARβ/δ on the other hand, plays a role in attenuating oxidative stress in different models by modulating expression levels of anti-oxidant enzymes, catalase and

Cu, Zn- and Mn-superoxide dismutase [376, 377, 411]. In support of this notion, reduced expression of Mn- superoxide dismutase is observed in one of the protein microarray of HCV Pparβ/δ-null liver. Thus, it is possible that PPARβ/δ could be promoting survival by attenuating oxidative stress in the HCV mice in vivo likely via up- regulation of anti-oxidant enzymes.

Previous studies had demonstrated that the absence of PPARβ/δ expression [140] or lack of its activation [164] enhances hepatic inflammatory cell infiltrates in CCl4 treated mice. These observations are in agreement with the established anti-inflammatory role of PPARβ/δ in the liver [162, 163, 165]. In the current study, preliminary histopathological analysis of hepatic inflammatory cell infiltration revealed no significant differences between HCV wild-type and HCV Pparβ/δ-null liver tissues. This is surprising, as it was expected that the lack of PPARβ/δ expression in these HCV mice might enhance recruitment of inflammatory cells to the liver. However, quantification of mRNA levels of hepatic inflammatory cytokines revealed that there was a non- significant increase in inflammatory cytokines Tnfa and Il-6 as well as a maker of macrophage activation, CD14, in HCV Pparβ/δ-null liver tissues, compared to that of

HCV wild-type liver tissues. Evaluation of these inflammatory cytokines in the pro-

105 inflammatory Kupffer cells instead of in whole liver tissue might give a better understanding of the inflammatory status between these two genotypes as it is possible that PPARβ/δ modulates inflammatory cytokine signaling in the Kupffer cells and not in the hepatocytes of the liver. Therefore even if the Kupffer cells in the livers of HCV

Pparβ/δ-null mice exhibit a significantly greater degree of inflammatory signaling compared to that of HCV wild-type mice, this effect could be masked because of the relatively small population of Kupffer cells in the liver compared to that of hepatocytes.

Similarly there are studies pointing to an anti-steatotic role for PPARβ/δ in the liver [165,

167, 412] albeit with different mechanisms proposed. However, histopathological quantification of macrovesicular fatty changes do not display a statistically significant difference in hepatic steatosis between HCV wild-type and HCV Pparβ/δ-null mice, indicating that PPARβ/δ expression alone does not seem to modulate hepatic steatosis in these mice. However, it could be reasoned that ligand activation of PPARβ/δ might be required to observe a PPARβ/δ-dependent anti-inflammatory or anti-steatotic effect between the two HCV genotypes. Interestingly, histopathological analyses did not reveal any presence of hepatocellular carcinoma in both HCV wild-type and HCV

Pparβ/δ-null mice, which is surprising considering approximately twenty-five percent of these HCV mice have been demonstrated to develop hepatocellular carcinoma by 19 months of age [292]. Increasing the sample size of the mice might be required to increase the chances of seeing the development of liver tumors at 24 months of age.

Although the results of the anti-body microarray need to be further verified by immunoblotting, the consistence in the hepatic expression patterns of Cofilin 1, SODD,

106 p27 Kip1 and SOCS2 as well as the phosphorylation status of PKCγ between the two antibody microarrays strongly suggests that these proteins are modulated by PPARβ/δ in these HCV mice and are worth further investigation. PKCγ is an isozyme of the protein kinase C family that can be activated by diacylglycerol and Ca2+ [413] and is found to be highly localized in the brain [414]. The expression of this protein has also been detected in Hep3B hepatoma cells [415], however, not much is known about its function in the liver, thus prompting further investigation. SODD is an anti-apoptotic protein shown to be increased in pancreatic tumors, but no difference in expression was observed between liver tumors and corresponding non-tumorous liver tissues [416].

Similar to PKCγ, not much is known about the role of SODD in the liver. Although not much is known about the expression of SODD and PKCγ activity in the liver, it would be worthwhile to investigate the PPARβ/δ-dependent modulation of their function in HCV mice in vivo. Cofilin 1 plays a role in depolymerization of actin [417] and has been identified as a marker of hepatic portal myofibroblasts [418]. The increased expression of Cofilin 1 in HCV Pparβ/δ-null liver tissues suggests that it could contribute to an increase in number of portal myofibroblasts in livers of these animals. Hepatic portal myofibroblasts are strongly linked to the development of fibrosis [419, 420], thus, it is might be worthwhile to investigate if HCV Pparβ/δ-null animals display increased hepatic fibrosis. p27 Kip1 is an inhibitor of cell cycle progression and its reduced expression has been associated with the progression of several tumors [421].

Specifically in hepatocellular carcinoma, increase in p27 Kip1 expression has been associated with a reduced rate of hepatic tumorigenesis and improved survival [422,

423]. Similarly, reduced expression of SOCS2 has also been correlated with enhanced

107 tumorigenesis in breast [424], prostate [425] and pulmonary adenocarcinoma [426]. It has also been demonstrated that reduced SOCS2 expression correlates with increased hepatic tumorigenesis and vascular invasion as well as a significant reduction in survival

[427]. The reduced expression of p27 Kip1 and SOCS2 in HCV Pparβ/δ-null livers compared to that of HCV wild-type in the microarray analyses suggests a possible heptoprotective role for PPARβ/δ in these mice and might also explain the significant increase in survival observed in HCV wild-type mice. Whilst quantitative immunoblotting for hepatic SOCS2 showed a non-significant decrease in expression in HCV Pparβ/δ- null mice, this trend was not observed for hepatic expression of p27Kip1. A plausible explanation for this could be that antibodies with different degrees of specificities for

SOCS2 or p27Kip1 were used in immunoblotting compared to that used in the microarray analyses. Hence the expression of these proteins in HCV wild-type mice and

HCV Pparβ/δ-null liver tissues should be re-verified by the same antibodies that were used for microarray analyses. Alternatively, another plausible explanation is that antibody microarrays have a much higher sensitivity compared to immunoblotting. Also, in the microarray analyses, proteins are quantified when they are in their functional native form, whereas in immunoblotting, the quantified proteins are denatured preventing distinction between native and non-native proteins. Quantification of functional native proteins is more accurate compared to that of traditional immunoblots that utilize reducing conditions to detect proteins. Therefore, it might also be useful to do a native polyacrylamide gel electrophoresis.

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Taken together, although the results of the present study are limited, and do not conclusively determine a role for PPARβ/δ in modulating hepatocellular carcinoma in

HCV mice in vivo, there is clearly a protective role for PPARβ/δ in promoting survival in these mice. It would be useful for future studies to try to decipher the mechanism by which PPARβ/δ increases the life span of these HCV mice.

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Chapter 4 Conclusions and Future Directions

The goal of this work was to determine the role of PPARβ/δ in regulating the progression of chronic hepatocellular injury-induced hepatic tumorigenesis. The regulation of the various physiological processes by PPARβ/δ in the liver prompted the investigation of its signaling in chronic hepatocellular injury-induced liver cancer models in vivo, which have not been shown before. Results of these studies show for the first time that PPARβ/δ-dependent signaling contributes to attenuated tumorigenesis in HBV and improved survival of HCV transgenic mice in vivo. Hepatic tumorigenesis in both the HBV and HCV mouse models used in these studies are driven by different viral proteins resulting in different mechanisms that promote hepatic tumor progression, and therefore, it is not surprising to see differences in the regulation of PPARβ/δ-dependent signaling between the two models. In this section, the modulation of tumorigenesis in

HBV mice and that of survival in HCV mice by PPARβ/δ along with potential future directions for each model will be discussed separately.

PPARβ/δ-dependent regulation of tumorigenesis in HBV mice

Hepatic tumorigenesis in the HBV model is driven by chronic cellular injury and inflammation. The rationale for targeting PPARβ/δ in this model was the strong evidence in the literature that PPARβ/δ has potent anti-inflammatory activities. As hypothesized, ligand activation of PPARβ/δ attenuated the early onset of inflammatory cytokine Tnfa production in this model. This was accompanied by subsequent enhancement of apoptosis and inhibition of hepatic proliferative potential and attenuation of steatosis, leading to an overall reduction in tumorigenesis. There have

110 been many mechanisms proposed for the PPARβ/δ-dependent attenuation of TNFa expression and they have all involved the modulation of NF-κB activity. Based on the results of the present study, it is likely that PPARβ/δ binds NF-κB to repress its activities by preventing its binding to NF-κB response elements. Although co-immunoprecipitation of PPARβ/δ with p65 subunit of NF-κB in the presence of LPS and PPARβ/δ ligand in these studies supports this notion, co-immunoprecipitation of PPARβ/δ with p65 subunit of NF-κB should also be done in KC13-2 cells treated with PPARβ/δ siRNA to show that this effect is dependent on PPARβ/δ. Further verification for the occupancy of NF-κB at the promoter region of TNFa can also be done via chromatin-immunoprecipitation

(ChIP) assays.

The sequence-specific co-repressor BCL6 adds another dimension to the PPARβ/δ- dependent modulation of NF-κB activity. BCL6 is a repressor of several NF-κB dependent inflammatory genes [428]. Some studies have demonstrated that ligand activation of PPARβ/δ releases the BCL6 co-repressor to which it was bound thus allowing BCL6 to relocate to promoter regions of genes to inhibit transcription [157,

158]. It has also been demonstrated that in the absence of PPARβ/δ ligand, the

PPARβ/δ-BCL6 complex prevents BCL6 from binding its response elements [157, 429] and that the absence of BCL6 prevents PPARβ/δ-dependent anti-inflammatory effects in pancreatic beta cells [156]. Whereas, another more recent study demonstrated that ligand activation of PPARβ/δ enhances association of BCL6 and PPARβ/δ at the promoter regions of Tnfa genes, and prevents the binding of NF-κB to its response elements. The lack of a PPRE in the Tnfa promoter, suggests that in this case,

111

PPARβ/δ could have been bound to BCL6, which in turn recruited PPARβ/δ to the promoter of Tnfa [146]. Because PPARβ/δ has also been shown to directly bind p65 subunit of NF-κB as well to suppress its activity, it will be useful for future studies to investigate if BCL6 is also a critical player in contributing to the PPARβ/δ-dependent modulation of NF-κB activity and the mechanism involved. It could be possible that upon ligand activation PPARβ/δ interacts with NF-κB and prevents it from binding its response elements due to steric hindrance, or the release of BCL6 upon ligand activation could bind promoters of inflammatory genes to repress transcriptional activity.

Hence it could be two separate pathways that PPARβ/δ utilizes to modulate its anti- inflammatory activities. Another possibility is that upon ligand activation PPARβ/δ could form a complex with NF-κB and BCL6, such that only the BCL6 and PPARβ/δ portion of the complex can bind promoter regions of inflammatory genes, preventing NF-κB from binding due to stearic hindrance and keeping it in a sequestered state. A simple way to determine if the three proteins form a complex would be to treat Kupffer cells with LPS and PPARβ/δ ligand, and then to immunoprecipitate PPARβ/δ and perform western blot analysis for the presence of PPARβ/δ, NF-κB and BCL6. If all three proteins are found to indeed form a complex together, ChIP-sequencing can be done to determine the

DNA binding sequence of the complex. This will shed light on which of the three proteins in the complex is interacting with DNA.

Previous studies from our laboratory had shown that anti-inflammatory effects of

PPARβ/δ were not modulated in the hepatocytes [140]. This was confirmed in the current study and further it was observed that PPARβ/δ is modulating its anti-

112 inflammatory effects through the Kupffer cells. It is worthwhile to investigate why this is the case. A recent study utilizing transcriptome analysis revealed that livers of Pparβ/δ- null mice increased TLR signaling, antigen processing and presentation, expression of

Kupffer cell marker genes in addition to induction of NF-κB activity [163]. Hence it is likely that PPARβ/δ mediates anti-inflammatory effects in the absence of PPARβ/δ ligand by modulating expression of genes involved in innate immunity, independent of ligand binding. This could explain the reason behind the anti-inflammatory effects observed in the Kupffer cells and not the hepatocytes. In addition, histopathological analysis of F4/80 expression levels in liver tissue sections of HBV and HBV mice treated with PPARβ/δ ligand would help determine if the number of Kupffer cells in the liver is decreased with PPARβ/δ ligand treatment as this could also contribute to a reduction in inflammatory signaling. Although not investigated in the present studies, it would have been interesting to determine the inflammatory status of tumor-associated macrophages (TAMs) in the liver tumors of HBV and HBV mice treated with PPARβ/δ ligand. Recruited monocytes have the ability to differentiate into macrophages that can polarize into two different phenotypes based on environmental stimuli known as the M1 phenotype (classically activated) or the M2 phenotype (alternatively activated). Toll like receptor ligands and IFN-gamma stimulate an M1 phenotype. The M1 phenotype, characterized as IL-12high, IL-23high, IL-10low, plays a role in mediating resistance against infection and tumors by producing inflammatory cytokines (TNFa, IL-1β and IL-6), reactive oxygen species, nitrogen intermediates and are involved in T helper 1 responses. The M2 phenotype on the other hand, is generally characterized as IL-12low,

IL-23low and IL-10high and is known to induce T helper 2 responses involved in anti-

113 inflammatory and wound healing activities. There are currently four known subdivisions of the M2 phenotype depending on the nature of the stimuli, namely M2a, M2b, M2c and M2d. Whilst IL-4 and IL-13 stimulate a M2a phenotype, immune complexes in combination with either toll-like receptor ligands or Il-1receptor ligands stimulate a M2b phenotype. The M2c phenotype has been found to be induced by IL-10 and glucocorticoids [430]. The M2d phenotype was the most recent subset to be identified and was found to be stimulated by IL-6 and LIF (leukemia inhibitory factor) found in ovarian tumor ascites. The M2d phenotype was not found to be induced by stimuli that differentiate M2a-c subsets such as IL-4 (or IL-13), IL-1 or IL-10, making this a new subset of M2 cells [431]. It has been shown that TAMs resemble a M2d phenotype and play a role in regulating the tumor microenvironment by dampening inflammation and promoting tumorigenesis and chemoresistance and are well reviewed in [432]. Among the four M2 subsets, the M2d macrophage subset was found to exhibit pro-tumorigenic and immunosuppressive potential that are associated with TAMs. For example, this macrophage subset upon stimulation with IL-6 or LIF led to a significant reduction in inflammatory factors TNFa and PTX3 (anti-angiogenic factor long pentraxin 3), as well as a significant induction in VEGF (vascular endothelial growth factor), which promotes angiogenesis and metastasis. This expression profile very closely mimics that of TAMs and is distinct from that of the M2a-c phenotypes [431]. Due to the limitations in human macrophage models, most macrophage polarization studies have utilized murine models. Interestingly it has been shown that characterization of gene expression markers for M1 and M2 polarized macrophages differs between human and murine models. [433]. For example, Ym1 (Chitinase 3-like 3), Fizz1 (Found in inflammatory

114 zone 1) and Arg1 (Arginase 1) are markers of alternatively activated M2 macrophages in mice, but not in that of human macrophages [433]. More recently, TGM2

(transglutaminase 2) was identified as a novel conserved marker for alternatively activated macrophages in both humans and mice [434]. Although an M2d phenotype in

TAMs can promote tumor aggression, especially in later stages of tumorigenesis, the

M2a-c anti-inflammatory phenotype is actually desirable in chronic inflammatory conditions that can predispose to metabolic syndromes such as insulin resistance and steatosis, both of which in turn can subsequently lead to the development of tumorigenesis. It has been demonstrated that PPARβ/δ can promote M2a alternative activation of macrophages. For example, it was found that adipocyte derived T helper 2 cytokines induce PPARβ/δ expression in mouse adipose tissue macrophages and that

PPARβ/δ is required for M1 to M2a phenotypic switch in these macrophages, as these effects were ablated in Pparβ/δ-null macrophages. The same study further went on to show that in mice with macrophage specific deletion of PPARβ/δ, an increased expression of inflammatory markers (TNFa, MCP-1 and IL-6) in the white adipose tissue was observed.[166]. Another study demonstrated that PPARβ/δ is required for IL-4 induced alternative M2a activation of bone-marrow derived macrophages. This study further went on to show that the PPARβ/δ ligand GW501516 could stimulate markers of alternative M2a gene expression in wild-type bone-marrow derived macrophages and could also cause a further significant induction in these markers when co-incubated with both GW501516 and IL-4. Utilizing wild-type and Pparβ/δ-null mice the same study also showed that PPARβ/δ was required for alternative M2a activation of resident macrophages such as the Kupffer cells and white adipose tissue macrophages [435].

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These studies demonstrate the anti-inflammatory properties of PPARβ/δ by modulating macrophage polarization, which can prevent inflammatory metabolic disorders such as insulin resistance and hepatic steatosis in mice. However, at present, not much is known about the role of PPARβ/δ in M2 alternatively activated macrophages, in particular the M2d pro-tumorigenic macrophages. However, the aim of the present studies was to determine if ligand activation of PPARβ/δ could inhibit early onset of hepatic inflammation by reducing the expression of pro-inflammatory M1 gene expression marker such as Tnfa by interfering with inflammatory signaling (instead of macrophage polarization) in the Kupffer cells of the liver, which will subsequently lead to reduced overall hepatic tumorigenesis. At the later stages of disease progression, the cells in the tumor microenvironment do not behave in a normal regulated fashion thus making it difficult to predict and target potential avenues for therapeutic approaches.

In this study, it was hypothesized that the attenuation of TNFa in the Kupffer cells possibly led to the reduced expression of CYCLIN D1 and C-MYC in the hepatocytes, contributing to a reduction in overall proliferative potential. Further experiments are needed to determine if the reduction in CYCLIN D1 and C-MYC directly lead to the inhibition of cellular proliferation in livers of HBV mice treated with PPARβ/δ ligand.

Immunohistochemical staining for markers of cellular proliferation such as Ki-67 and

PCNA (proliferating cell nuclear antigen) in liver tissue sections of HBV mice treated with and without PPARβ/δ ligand may further confirm that the PPARβ/δ-dependent inhibition of CYCLIN D1 and C-MYC does directly contribute to reduced hepatic proliferation in the HBV mice. Alternatively, intraperitoneal injection of the HBV mice

116

(treated with and without PPARβ/δ ligand) with bromodeoxyuridine (BrdU) and quantification of the percentage of BrdU labeled nuclei can also give insight on

PPARβ/δ-dependent regulation of hepatocyte proliferation in these mice in vivo.

Treating primary HBV hepatocytes with conditioned media from LPS and PPARβ/δ ligand treated Kupffer cells from wild-type, Pparβ/δ-null and Pparβ/δ-DBM mice in the absence or presence of TNFa neutralizing antibodies will determine the direct effect of paracrine TNFa signaling from Kupffer cells to increase CYCLIN D1 and C-MYC expression in hepatocytes in vitro. Alternatively, HBV hepatocytes can be cultured on transwell permeable cell culture plate inserts, and transferred on top of wild-type,

Pparβ/δ-null and Pparβ/δ-DBM Kupffer cells in culture that have been treated with LPS and PPARβ/δ ligand in the absence and presence of TNFa neutralizing antibodies, such that there is free exchange of cytokine signaling between Kupffer cells and hepatocytes.

The HBV hepatocytes can then be harvested to monitor expression levels of CYCLIN

D1 and C-MYC. These experiments will determine if 1) PPARβ/δ-dependent attenuation of TNFa in the Kupffer cells has the ability to decrease hepatocyte proliferative potential via paracrine signaling and that 2) this occurs independent of PPARβ/δ target gene expression.

Chronic TNFa signaling can contribute to hepatic steatosis [361-363] and in the current studies, it was hypothesized that PPARβ/δ-dependent attenuation of TNFa possibly attenuated hepatic steatosis in the HBV mice. Utilizing the HBV model and crossing them with TNFa deficient mice and treating these mice with and without PPARβ/δ ligand will confirm the development of hepatic steatosis promoted by TNFa in these HBV mice

117 and also further confirm if attenuation of hepatic steatosis in these mice by PPARβ/δ- dependent signaling occurs via modulation of TNFa. Alternatively, the HBV mice could be treated with and without TNFa inhibitors, in the absence and presence of PPARβ/δ ligand. Further experiments should also examine the possibility that PPARβ/δ could have contributed to the reduced steatosis by other pathways as well such as increasing fatty acid oxidation or decreasing fatty acid synthesis. For example PPARβ/δ has been shown to inhibit hepatic steatosis by up-regulation of its target gene Insig-1, which prevents processing of SREBP-1, a transcription factor that regulates fatty acid synthesis [167]. Another important contributor to hepatic steatosis is deregulation of metabolic signaling. It will be useful for future studies to compare the metabolic profile of

HBV liver tissues with that of HBV liver tissues treated with PPARβ/δ ligand, to gain insight on other potential metabolic targets modulated by PPARβ/δ-dependent signaling. A recent study compared the metabolic profile of human hepatoma cell line

HepG2 with HepG2.2.15 (HepG2 cell line with stable expression and replication of HBV) and showed that HBV can contribute to a significant alteration of metabolic profiles

[436]. Knocking down PPARβ/δ expression in these cell lines and treating them with and without PPARβ/δ ligand will allow for the determination of PPARβ/δ-dependent modulations of altered metabolic profiles during HBV expression and replication.

Unfortunately, the lack of a HBV Pparβ/δ-null mouse model is one of the limitations of the present study as these mice had a very short life expectancy of less than 6 months.

This indicates the importance of PPARβ/δ in this HBV mouse model. Using more than one highly specific PPARβ/δ agonists such as GW501516 or KD3010 in addition to

118

GW0742 would have further strengthened the conclusion that the anti-tumorigenic effects observed in the HBV mice treated with PPARβ/δ ligand are PPARβ/δ-dependent and not due to non-specific effects of PPARβ/δ ligand GW0742. Also, future studies should try to utilize a Kupffer cell specific deletion of PPARβ/δ utilizing Cre-lox recombination in HBV mice. In these mice, treatment with and without PPARβ/δ ligand will shed light on the direct role of PPARβ/δ-dependent signaling in the Kupffer cells on hepatic CYCLIN D1 and C-MYC expression and subsequent tumor progression in vivo.

This mouse model will also determine if the observed attenuation of hepatic steatosis and enhanced apoptosis with PPARβ/δ ligand treatment was a result of PPARβ/δ- dependent anti-inflammatory signaling from the Kupffer cells of the liver. Although

PPARβ/δ-dependent anti-inflammatory activities can contribute to attenuation of hepatic steatosis, proliferative potential and increased apoptosis, other pathways by which

PPARβ/δ could additionally modulate these activities should also be investigated in future studies.

A potential pathway by which PPARβ/δ could modulate proliferation and apoptosis is the protein kinase C alpha (PKCα) pathway. Activation of the PKCα pathway can enhance proliferation and promote resistance to apoptosis. Studies have shown that over-expression of the large envelope protein, as is the case of the HBV model, can increase PKCα activity to drive hepatic tumorigenesis [437]. PKCα promotes hepatocellular carcinoma progression [438-440] and high levels of PKCα expression have been found in human hepatocellular carcinoma samples that correspond with poor prognosis in patients [441]. Hence, it will be useful to know if PPARβ/δ-dependent

119 signaling can modulate PKCα activity in the HBV model. Previous findings from our laboratory have demonstrated that PPARβ/δ can attenuate PKCα activity by increasing ubiquitin dependent turnover, leading to reduced epidermal cell proliferation [127].

However, it is also possible that PPARβ/δ can modulate PKCα activity by other mechanisms. PKCα activity is governed by upstream activity of diacylglycerol kinase zeta (DGKζ) [442] as seen in Figure 4-1. Increased DGKζ expression leads to increases in diacylglycerol (DAG), which subsequently increases PKCα activity. Recent studies have shown that ligand activation of PPARβ/δ increases DGKζ expression [443,

444] in cardiac myocytes. Hence, it will be useful to investigate PPARβ/δ-dependent regulation of DGKζ expression in the liver and subsequent PKCα activity. Although the promoter region of DGKζ does not contain PPREs, it does contain Sp1 sites and there is in fact evidence that PPARβ/δ can regulate gene expression via Sp1 [445]. A proposed mechanism has been that PPARβ/δ could form a complex with Sp1 to bind to

DGKζ promoter to enhance transcriptional activity. It will be useful for future studies to determine the mechanism behind PPARβ/δ/ DGKζ signaling to modulate PKCα activity in hepatocellular carcinoma models. Hepatoma cell lines like HepG2 in the absence and presence of PPARβ/δ expression could be stably transfected with HBV viral proteins and treated with and without PPARβ/δ ligand to determine the mechanism behind

PPARβ/δ/DGKζ signaling. Co-immunoprecipitation of PPARβ/δ with Sp1 would determine their interaction with each other. Further, a ChIP assay, immunoprecipitating

Sp1 with its bound DNA and further enrichment of the DNA sequence to determine promoter occupancy of DGKζ will confirm if PPARβ/δ has the potential to modulate

DGKζ via Sp1 binding sites. Western blot analysis can also be done to compare the 120 correlation between increasing PKCα activity and proliferative status and apoptosis resistance in these cells in absence and presence of PPARβ/δ ligand.

Figure 4-1: Schematic of potential pathway by which PPARβ/δ could modulate PKCα activity. Ligand activated PPARβ/δ binds Sp1 transcription factor and the complex is recruited to promoter region of DGKζ to increase its expression. This leads to a subsequent decrease in diacylglycerol accumulation followed by a reduction in PKCα activity.

In summary, this study shows for the first time that PPARβ/δ-dependent signaling attenuates hepatocarcinogenesis in a hepatitis viral protein-induced HBV mouse model that is driven by chronic cellular injury and inflammatory signaling. It can be seen that all the preneoplastic factors that PPARβ/δ has been shown to modulate, contribute to the overall attenuation of tumorigenesis, such as reduced inflammatory signaling, steatosis, proliferation and enhanced apoptosis. These results show strong evidence that this is

121 most likely the result of PPARβ/δ-dependent attenuation of inflammatory signaling in the

Kupffer cells of the liver. However, other unexplored mechanisms modulated by

PPARβ/δ should also be considered. At present, there is no cure for chronic HBV infections. Current treatment strategies include the use of pegylated interferons that have low efficacy or nucleoside/nucleotide inhibitors to viral replication that need to be taken life-long. These treatments also come with their own list of side effects [446].

Based on the findings of the current study a Kupffer cell-type specific treatment approach to targeting PPARβ/δ signaling looks like a promising therapeutic avenue. The present study utilized synthetic ligands and it would be interesting to screen for endogenous ligands that are highly specific for PPARβ/δ and to determine if similar effects are observed in this HBV model. For example, although most dietary fatty acids have the ability to activate all three PPAR isoforms, the monounsaturated fatty acid, oleic acid is an endogenous ligand found to be more specific in activation of PPARβ/δ over PPARα and PPARγ [447]. Recent studies have demonstrated that during dietary intake of oleic acid, PPARβ/δ regulates the oleic acid-induced upregulation of the well- known PPARβ/δ target gene Angptl4 in the hearts of mice and subsequently protects against fatty acid-induced oxidative stress by inhibiting fatty acid uptake [448]. It would be beneficial to see if dietary intake of natural ligands such as oleic acid can be utilized in a similar manner to mediate PPARβ/δ-dependent anti-tumorigenic properties, especially in obese individuals as obesity has been linked to inflammatory conditions that drive tumor promotion, and PPARβ/δ has been demonstrated to prevent both obesity and inflammatory signaling in multiple models. If natural ligands like oleic acid also show similar promising effects as synthetic PPARβ/δ agonists, they can be easily

122 incorporated into everyday diets as potential anti-cancer therapy.

PPARβ/δ-dependent improvement of survival in HCV mice

The inability to generate a HBV Pparβ/δ-null mouse with a long enough survival duration, led to the investigation of the role of PPARβ/δ in another hepatitis model, the

HCV mouse model [292]. These HCV mice express the HCV core protein and will go on to develop hepatic tumors over time, by 16-18 months of age and progress from thereon to form hepatocellular carcinoma at later stages (~18-24 months). Interestingly, it has been observed that in these mice, tumorigenesis is accompanied by hepatic steatosis but in the absence of an inflammatory background. For these studies, the HCV transgenic mice were crossed with wild-type and Pparβ/δ-null mice to generate HCV

Pparβ/δ-null mice. The goal was to determine if PPARβ/δ would contribute to the same modulation of preneoplastic activities and tumor progression in the HCV mice, that it did in the HBV mice.

Although these HCV mice do not develop chronic inflammation with disease pathogenesis, it was expected that the lack of PPARβ/δ expression in the HCV Pparβ/δ- null mice would enhance inflammatory responses. However, preliminary findings from histopathological quantification of hepatic inflammatory infiltrates between HCV wild- type and HCV Pparβ/δ-null mice did not yield any conclusive results. However, isolation of primary Kupffer cells and hepatocytes from HCV wild-type and HCV Pparβ/δ-null mice and quantification of the expression of inflammatory cytokines such as TNFa and

IL-6 might give more insight into PPARβ/δ-dependent expression of inflammatory

123 cytokines between the two genotypes and within the two liver cell types. Analyzing inflammatory cytokine expression between HCV and HCV Pparβ/δ-null livers based on cell type might give a more significant result. Future studies could also look to determine the effect of a Kupffer cell specific deletion of PPARβ/δ in these mice to observe if the presence of PPARβ/δ in the Kupffer cells offers protection against inflammatory cytokine signaling. This would demonstrate, that whilst inflammatory signaling may not be a significant contributor to HCV induced tumorigenesis in this mouse model, the underlying reason could be due to the protective anti-inflammatory effects of PPARβ/δ, as the absence of PPARβ/δ might induce an inflammatory response that would aggravate the disease pathogenesis and accelerate hepatic tumorigenesis. Further, it would also be important to perform these studies in the absence and presence of

PPARβ/δ ligand as well, because this would allow for the investigation of an additional level of anti-inflammatory modulation by PPARβ/δ as discussed above.

PPARβ/δ did not seem to protect against hepatic steatosis in the HCV mice as HCV

Pparβ/δ-null mice had slightly reduced (although not significant) amounts of hepatic macrovesicular changes. However evidence from the literature and in the current studies have demonstrated that PPARβ/δ can attenuate hepatic steatosis in presence of ligand. Hence, it is possible that activation of PPARβ/δ by ligand is required for attenuation of steatosis in these HCV mice. HCV core protein in these mice induces progressive steatosis, which is an important risk factor for the development of hepatocellular carcinoma, especially in patients infected with chronic hepatitis C. Also, like HBV infections, HCV infections also contribute to a variety of metabolic disorders

124 like insulin resistance and subsequent type II diabetes mellitus [449-451]. The HCV core protein has been demonstrated to decrease insulin sensitivity [387], whereas PPARβ/δ on the other hand, has been shown to improve insulin resistance in several models

[452-455]. For example the HCV core protein has been demonstrated to down-regulate glucose transporter 4 (GLUT4) expression a protein required for glucose uptake [456].

The reduced expression of this protein leads to enhanced plasma glucose and subsequent development of insulin resistance. On the other hand, over-expression of

PPARβ/δ has been shown to increase GLUT4 expression and subsequent glucose uptake and utilization. This effect was further enhanced by ligand activation of PPARβ/δ

[457, 458]. This is consistent with liver profile analysis that shows reduced expression of genes involved in carbohydrate metabolism in livers of Pparβ/δ-null mice [163].

Additionally, HCV core has also been shown to induce insulin resistance by increasing

SOCS3 expression, which attenuates insulin receptor substrates 1 and 2 (IRS 1&2) via ubiquitin mediated proteasomal degradation [386]. HCV has also been shown to promote inflammatory cytokine, TNFa expression and that of its soluble receptor, which have also been linked to development of insulin resistance [459, 460]. Studies have shown that PPARβ/δ has the potential to attenuate both SOCS3 [452, 461] and TNFa

[165] to promote improved insulin signaling as well. Thus, it will be worthwhile for future studies to determine the mechanism behind the regulation of glucose uptake in the current HCV model. Like enhanced steatosis, insulin resistance is also a risk factor for hepatocellular carcinoma [462]. Insulin resistance has been shown to promote liver fibrosis, which can accelerate the process of hepatocarcinogenesis [463]. Thus, it would be worthwhile for future studies to utilize the HCV wild-type and HCV Pparβ/δ-null mice

125 treated with and without PPARβ/δ ligand to determine if PPARβ/δ-dependent signaling can positively modulate insulin sensitivity in this HCV mouse model.

PPARβ/δ provided a survival advantage in HCV mice compared to HCV Pparβ/δ-null mice pointing to an important role that PPARβ/δ plays in the longevity of these mice.

Based on the protein microarray data, it is possible to identify potential targets that are being modulated by PPARβ/δ to contribute to the improvement of survival and possible attenuation of hepatocarcinogenesis progression. Oxidative stress is a major contributor to the progression of hepatocellular carcinoma and studies have demonstrated that reducing oxidative stress by over-expressing anti-oxidant enzymes or mimicking their activity can result in a direct decrease in mortality in in vivo models [408-410]. Redox homeostasis occurs when there is a balance between reactive oxygen species production and expression of anti-oxidant enzymes. Disruption of this balance occurs when the production of reactive oxygen species overrides the expression of anti-oxidant enzymes, resulting in oxidative stress. The HCV core protein is a potent inducer of oxidative stress and interestingly, in this HCV model, the core protein of the virus has been demonstrated to promote hepatic oxidative stress in the absence of inflammation

[383]. It was found that with age, the core protein caused an increase in lipid peroxidation products, disruption of the balance of antioxidant activity as well as mitochondrial DNA damage in these mice. There are also plenty of other studies that describe the induction of oxidative stress by the HCV core protein all of which result in enhanced mitochondrial stress [282, 283, 464]. In addition, oxidative stress is a common occurrence in patients with chronic hepatitis C infection [393, 465, 466].

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Studies have demonstrated that expression and ligand activation of PPARβ/δ can attenuate oxidative stress by up-regulation of anti-oxidant enzymes catalase and superoxide dismutase 1 & 2 (SOD 1&2) [158, 377, 467]. Interestingly, it was observed from the protein microarray data that there was a significant decrease in the expression of SOD2 in liver tissues of HCV Pparβ/δ-null mice, which is consistent with other findings in the field, and could potentially increase mitochondrial stress and DNA damage that is known to be associated with the HCV core protein. The promoter regions of all three anti-oxidant enzymes catalase [468], SOD1 [469] and SOD2 [470] contain functional PPREs to which PPARβ/δ can potentially bind to and increase transcription. Hence, it will be useful to determine if there are any differences in hepatic oxidative stress between HCV and HCV Pparβ/δ-null mice treated with and without

PPARβ/δ ligand at age > 18 months by measuring levels of hydroperoxides or malondialdehyde. In addition, to see if oxidative stress observed translates into DNA damage, immunohistochemical analysis of liver tissues can be done to observe localization of 8-hydroxydeoxyguanosine (8-OHdG). Since mitochondrial DNA damage was also a feature of oxidative stress in these HCV mice, quantification of mitochondrial

DNA deletion present between direct repeats of mitochondrial DNA (corresponding to

979-5650 of mouse mitochondrial DNA. Damage at this region observed in HCV mice to increase with age from 2 months on [383]) by PCR can also be determined. Also, reporter assays can be used to determine if over-expression or ligand activation of

PPARβ/δ directly increases transcriptional activity of catalase, SOD1 and SOD2 (Figure

4-2). Further, Chip assays in the presence and absence of ligand can be used to determine the promoter occupancy of these anti-oxidant enzymes by PPARβ/δ. Taking

127 this a step further, it would be interesting to see how PPARβ/δ would modulate oxidative stress in the current HCV model in the absence and presence of alcohol. Many studies have shown that in patients with HCV infection and alcohol abuse, both factors can act synergistically to enhance the severity of liver disease [471] mainly through the mediation of oxidative stress [472]. There is evidence that ligand activation of PPARβ/δ can attenuate oxidative stress in rats chronically fed with an ethanol liquid diet [473].

Hence, it will be useful to know if PPARβ/δ exerts protective effects against the oxidative stress induced by HCV and alcohol synergism.

Figure 4-2: Schematic of potential pathway by which PPARβ/δ could modulate oxidative stress. Ligand activated PPARβ/δ binds PPREs of promoter regions of anti-oxidant enzymes catalase (CAT), superoxide dismutase 1 and 2 (SOD1 and SOD2) to up-regulate their transcription and expression, which can lead to attenuation of oxidative stress.

In summary, it can be seen that PPARβ/δ has the potential to promote survival and attenuate potential development of hepatic tumorigenesis in the HCV mouse model as

128 well. The mechanisms underlying the prolonged survival mediated by PPARβ/δ need further investigation as mentioned above. Further, the protein microarray data of the liver tissues of HCV wild-type and HCV Pparβ/δ-null liver tissues reveal several promising molecular targets that PPARβ/δ may be modulating such as SOCS2 and p27Kip1, which may also contribute to the observed reduction in mortality in HCV mice.

In addition, it has been recently demonstrated that the absence and presence of

PPARβ/δ in combination with or without ligand activation can give rise to a number of different gene expression responses [52], which are also worth exploring in these hepatitis models as they would give insight into the dynamic nature of PPARβ/δ dependent regulation of cell signaling in the livers of hepatitis models. At the same time, one should also take into consideration the effect of species difference between humans and mice. The use of humanized hepatitis mouse models might prove useful in comparing between the two species to determine any variations in hepatic responses to

PPARβ/δ-dependent signaling.

Taken together, although limited, and require further investigation, results of the present study provide evidence for the first time that PPARβ/δ plays a protective role in viral protein-induced mouse models of liver cancer. In the case of the HCV mice, although the role of PPARβ/δ in modulating tumorigenesis could not be concluded, it is clear that

PPARβ/δ-dependent signaling is playing a protective role in these mice by significantly improving their life span and further studies are needed to investigate the underlying mechanisms. As for the HBV mice, the anti-inflammatory role of PPARβ/δ clearly

129 contributes in part to the observed chemopreventive characteristics. However, it is also possible that PPARβ/δ could modulate tumorigenesis by other mechanisms such as inhibition of proliferation or promotion of apoptosis. Although further studies need to be done to decipher if PPARβ/δ modulates these activities by attenuating inflammatory signaling alone or by modulation of other signaling pathways as well, it is clear that

PPARβ/δ-dependent signaling can attenuate the progression to hepatocellular carcinoma and holds strong promise as a potential therapeutic approach to treat liver cancers of inflammatory hepatitis etiologies.

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VITA

Gayathri Balandaram

Education

Pennsylvania State University, University Park, PA Aug 2010 – Dec 2015 Doctor of Philosophy in Molecular Medicine (Thesis)

Washington State University, Pullman, WA Aug 2007 – Dec 2009 Master of Science in Biochemistry (Thesis)

Western Michigan University, Kalamazoo, MI Aug 2000 – Dec 2004 Bachelor of Science in Biochemistry and Biomedical Sciences (Double Major)

Publications

Koga T, Yao P, Balandaram G, Goudarzi M, Fornace AJ, Gonzalez FJ and Peters JM  Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) regulates ethanol- induced cytocrome P450 (CYP) 2B10 expression. (Manuscript in preparation)

Balandaram G, Kramer LR, Kang B, Murray IA, Perdew GH, Gonzalez FJ and Peters JM  PPARβ/δ attenuates hepatic tumorigenesis in hepatitis B transgenic (HBV) mice. Toxicology (2015) (Manuscript in preparation)

Yu S, Allen JN, Dey A, Balandaram G, Kennett MJ, Xia M, Xiong N, Peters JM, Hankey-Giblin PA  The Ron receptor tyrosine kinase attenuates obesity-associated diseases by regulating macrophage heterogeneity. Journal of Immunology (2015) (Manuscript submitted)

Significant Honors and Awards

• NIH/NIAID graduate student fellowship in “Animal models of Inflammation” (2012 – 2015).

• 2014 First place Carcinogenesis Specialty Section, graduate student award, Society of Toxicology Conference (2014).