DR. BHARAT BHUSHAN (Orcid ID : 0000-0002-1716-9764) DR ......Title: Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Non‐alcoholic Fatty Liver Disease in Murine

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/hep.30696 This article is protected by copyright. All rights reserved.

DR. BHARAT BHUSHAN (Orcid ID : 0000-0002-1716-9764)

DR. WENDY M MARS (Orcid ID : 0000-0003-4495-1054)

DR. JOSEPH LOCKER (Orcid ID : 0000-0002-4190-188X)

Article type : Original

Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses

Non-alcoholic Fatty Liver Disease in Murine Fast-food Diet Model

Bharat Bhushan, Swati Banerjee, Shirish Paranjpe, Kelly Koral, Wendy M. Mars,

John W. Stoops, Anne Orr, William C. Bowen, Joseph Locker and George K.

Michalopoulos

Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh,

PA

Bharat Bhushan: [email protected]

Swati Banerjee: [email protected]

Shirish Paranjpe: [email protected]

Kelly Koral: [email protected]

Wendy M. Mars: [email protected]

John Stoops: [email protected]

Anne Orr: [email protected]

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This article is protected by copyright. All rights reserved.

William C. Bowen: [email protected]

Joseph Locker: [email protected]

George K. Michalopoulos: [email protected]

Keywords: Epidermal Growth Factor Receptor (EGFR), Fatty acid Synthase, Sterol

regulatory element-binding transcription factor 1 (SREBF1), Peroxisome proliferator-

activated receptor gamma (PPARγ), Hepatocyte nuclear factor 4 alpha (HNF4α) and

c-MET

Corresponding author:

George K. Michalopoulos, M.D., Ph.D.

Department of Pathology (Chair)

School of Medicine, University of Pittsburgh

200 Lothrop St., South BST S410, Pittsburgh, PA 15261

E-mail: [email protected]; Fax: 1-412-648-9846

List of Abbreviations:

EGFR: Epidermal growth factor receptor; NAFLD: Non-alcoholic fatty liver disease;

NASH: Non-alcoholic steatohepatitis; HCC: Hepatocellular carcinoma; HCV:

Hepatitis C virus; PHx: Partial hepatectomy; EGFRi: EGFR inhibitor (Canertinib);

FFD: Fast-food diet; HNF4α: Hepatocyte nuclear factor 4 alpha; SREBF1: Sterol

regulatory element-binding transcription factor 1, PPARγ: Peroxisome proliferator-

activated receptor gamma; PCNA: Proliferating cell nuclear antigen; IPA: Ingenuity

pathway analysis

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Financial Support:

Support for this study was provided by the Cleveland Foundation and the Menten

Endowment Foundation of the University of Pittsburgh.

Abstract

EGFR is a critical regulator of hepatocyte proliferation and liver regeneration. Our

recent work indicated EGFR can also regulate lipid metabolism during liver

regeneration after partial-hepatectomy. Based on these findings, we investigated role

of EGFR in a mouse model of NAFLD utilizing a pharmacological inhibition strategy.

C57BL6/J mice were fed chow-diet, or fast-food diet with/without EGFR inhibitor

(Canertinib) for 2-months. EGFR inhibition completely prevented development of

steatosis and liver injury in this model. In order to study if EGFR inhibition can

reverse NAFLD progression, mice were fed fast-food diet for 5-months, with/without

Canertinib-treatment for the last 5-weeks of the study. EGFR-inhibition remarkably

decreased steatosis, liver injury, fibrosis and improved glucose tolerance. Microarray

analysis revealed ~40% of genes altered by fast-food diet were differentially

expressed after EGFR-inhibition, and thus, are potentially regulated by EGFR.

Several genes and enzymes related to lipid metabolism (particularly fatty-acid

synthesis and lipolysis), which were disrupted by fast-food diet, were found to be

modulated by EGFR. Several crucial transcription factors that play a central role in

regulating these lipid metabolism genes during NAFLD, including PPARγ, SREBF1,

ChREBP and HNF4α, were also found to be modulated by EGFR. In fact, ChIP-

analysis revealed PPARγ binding to several crucial lipid metabolisms genes (Fasn,

Scd1 and Plin2) was drastically reduced by EGFR inhibition. Further upstream,

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EGFR-inhibition suppressed AKT signaling, which is known to control these

transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the

effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor-

tyrosine-kinase MET, as investigated using MET-KO mice. In-conclusion, our study

revealed a role of EGFR in NAFLD and the potential of EGFR-inhibition as a

treatment strategy for NAFLD.

INTRODUCTION

Non Alcoholic Fatty Liver Disease (NAFLD) has become the most common cause of

chronic liver disease worldwide, affecting around 25% of world population and about

60 -80 million individuals in the United States itself (1). NAFLD comprises both

benign steatosis and also non-alcoholic steatohepatitis (NASH) with chronic

inflammation, which can progress to various stages of hepatic fibrosis, cirrhosis and

hepatocellular carcinoma (HCC). With the recent progress made in treatment for

HCV, NAFLD/NASH is now realized as one of the major contributors for

development of cirrhosis and its progression to HCC, which has enhanced the need

to develop effective therapeutic approaches for NAFLD/NASH treatment. Alarmingly,

there is currently no accepted pharmacologic treatment for NAFLD (2).

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase highly

expressed in liver, which is known to play an important role in hepatocellular

carcinogenesis (HCC) and hepatocyte proliferation during liver regeneration after

partial-hepatectomy (PHx) and acute liver injury (3, 4). It is known that hepatocyte

proliferation during normal liver regeneration is associated with transient

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accumulation of lipid droplets in preparation for DNA synthesis. Apart from its

conventional role in the above processes, interestingly, our recent study indicated an

important role for EGFR in transient steatosis accompanying liver regeneration after

PHx (5). Treatment with potent EGFR inhibitor, Canertinib, resulted in complete

absence of lipid accumulation in hepatocytes during liver regeneration along with

decreased expression of fatty acid biosynthesis genes, without major impact on

hepatocyte proliferation itself due to compensatory activation of other proliferative

pathways (5). An independent study published concomitantly also reported severe

inhibition of PHx-associated lipid accumulation and impaired induction of fatty acid

synthase gene (encodes rate limiting enzyme for fatty acid synthesis) in mice lacking

catalytic activity of EGFR in liver (6). Further, a study utilizing a gain of function

mutation for EGFR in its kinase domain indicated that EGFR can also regulate lipid

metabolism in quiescent mouse liver with mutant mice showing higher HMG-CoA

reductase, fatty acid synthase and SREBF1/2 (major transcription factors regulating

lipid synthesis) expression (7). EGFR is also reported to play an important role in

drug-induced acute liver injury and liver fibrogenesis/stellate cell activation in a CCl4

–induced chronic liver injury model with EGFR KO and/or EGFR inhibitor treated

mice showing attenuated liver injury and fibrosis (3, 8, 9). Increased gene expression

of EGFR has been also observed in NAFLD/NASH patients and correlated with

NAFLD progression (10). Based on all these studies, we hypothesized that inhibition

of EGFR can be hepato-protective in a NAFLD model and EGFR can be a potential

therapeutic target for NAFLD. Here we report, using a fast food diet (FFD) model in

mice (11), that EGFR inhibition not only completely prevents development of

steatosis and liver injury in a two months study, when administered from beginning of

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the study, but also reverses steatosis and to certain extent fibrosis in a 5 months

study, where EGFR inhibitor was administered only for the last 5 weeks of the study.

Materials and Methods

Animals and treatments

EGFR inhibition studies: For the two months study, 6-8 weeks old male C57BL6/J

mice were fed ad libitum (i) chow diet, (ii) fast-food diet [western diet - high saturated

fats (21% by weight; 42% kcal from fat), high cholesterol (0.2%) and high

carbohydrates (sucrose: 34% by weight) in diet (TD.88137, Teklad) - along with

high-fructose-glucose solution (d-glucose: 18.9g/L and d-fructose: 23.1g/L) in

drinking water] as described previously (11) or (iii) Canertinib (a highly potent and

selective EGFR inhibitor/ EGFRi) added in the same fast-food diet for 2-months.

Canertinib was administered to mice in diet for estimated dose of 80 mg/kg/day as

described previously (5). For the five months study, 6-8 weeks old male C57BL6/J

mice were fed ad libitum (i) chow diet, (ii) and (iii) fast-food diet for 5-months. Group

(ii) and (iii) were administered vehicle (PBS) and Canertinib (80mg/kg; i.p.),

respectively, 5 days/week for the last 5-weeks of 5-months period (schematics

describing design for both 2 and 5-months studies is shown in Fig. 1A).

MET deletion study: METfl/fl mice (a gift from Dr. Snorri Thorgeirsson, currently

available at the Jackson Laboratories; Stock # 016974) were crossed with mice

expressing AFP (enhancer)-albumin (promoter) driven Cre recombinase to obtain

mice with liver specific deletion of exon 16 of MET gene (MET KO mice). MET KO or

wild-type littermates (age- and body weight-matched) were fed FFD for 5-months

period (schematics describing study design is shown in Suppl. Fig. 12A).

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Other methodological details are provided in supplementary information.

RESULTS

EGFR inhibition prevents and reverses steatosis and improves serum

parameters in fast food diet-fed mice

In order to study the role of EGFR in the development of diet-induced steatosis, mice

were fed fast-food diet (FFD) with or without Canertinib, a potent EGFR inhibitor

(EGFRi), for 2-months. Canertinib is highly selective for EGFR (IC50 : 0.8 nM) with

very weak activity against only two other non-ErbB family kinases (EphA6 - IC50 : 72

nM; GAK - IC50 : 44 nM) (5). Livers of FFD-fed animals developed extensive

steatosis (mainly macrovesicular), which was almost completely prevented by EGFR

inhibition (Fig. 1B). FFD feeding also increased liver to body weight ratio compared

to chow, which was normalized by EGFRi treatment (Fig. 1C). Next, we investigated

if EGFRi treatment can reverse already developed diet-induced steatosis. Mice were

fed FFD for 5 months and administered either EGFRi (5 days/week) or PBS (i.p.) for

the final 5 weeks of the 5 months period. As expected, FFD-fed mice showed

extensive macrovesicular steatosis that was dramatically reversed in EGFRi-treated

mice, which displayed some residual microvesicular steatosis (Fig. 1D). LW/BW ratio

was increased by 60% in FFD-fed mice compared to chow and was significantly

decreased after EGFRi treatment (Fig. 1E).

Serum ALT, AST and cholesterol levels were also increased in FFD-fed mice at both

2 and 5-months (with a greater increase at 5-months) and were lowered after EGFR

inhibition (Fig. 2A-B). However, serum triglyceride levels were not significantly

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altered either by FFD feeding or by EGFRi intervention (Fig. 2A-B). In order to

assess the effect of EGFRi-treatment on glucose sensitivity, glucose tolerance test

was performed for the 5-months study. Blood glucose levels remained remarkably

elevated after glucose administration in FFD-fed mice compared to basal levels, but

were normalized quickly after initial elevation in EGFRi group (Fig. 2C). Further,

serum insulin levels increased ~2 fold in FFD group at 5-months, consistent with

previous report on FFD model (11), and were normalized by EGFR inhibition (Fig.

2D). Overall, the data indicate that EGFRi-treatment can effectively attenuate FFD-

induced steatosis, liver injury, hypercholesterolemia and glucose intolerance.

In order to study effects of EGFR inhibition per se (without FFD), Canertinib or PBS

(i.p.) was administered to chow-fed mice for 5-weeks similar to 5-months FFD study.

EGFRi-treated mice appear to be completely normal with no apparent signs of

toxicity or effects on body weight and liver to body weight ratios (Suppl. Fig. 1A-C).

Liver histology and serum parameters (ALT, AST, cholesterol and triglycerides) were

completely normal after EGFR inhibition (Suppl. Fig. 1A and D). Further, no

difference was observed in Oil Red O staining, glucose tolerance test and insulin

tolerance test between chow-fed controls and EGFRi-treated mice indicating effects

of EGFRi were specific to FFD model (Suppl. Fig. 1A, E and F).

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Effects of EGFR inhibition on fibrosis-associated parameters in 5 months

study

At 5-months, FFD-fed mice showed significant pericellular fibrosis, which was

decreased after EGFRi-treatment (Fig. 3A). This was corroborated by a remarkable

increase in gene expression of Col1A1, Col3A1 and α-SMA along with elevation of

α-SMA protein levels in FFD-fed mice, which were significantly lowered by EGFRi-

treatment (Fig. 3B-E). Similar pattern in expression of these genes was also

observed at 2-months (Suppl. Fig. 2A-C), but fibrosis was not observed (data not

shown). The effect on fibrosis-associated genes was also demonstrated by analysis

of microarray data (described in next section), which showed hepatic fibrosis/stellate

cell activation, collagen fibril organization and wound healing among the top

biological processes/signaling pathways altered by EGFR inhibition specifically in the

5 months study but not in the 2-months study (Suppl. Fig. 3B-C and Suppl. Fig. 4B).

Global changes in gene expression profile after EGFR inhibition

Next, we investigated changes in the global gene expression profile using

microarray. Total of 1733 and 1458 genes were differentially altered (at least 2-fold

up- or down-regulation) in FFD + EGFRi vs FFD group in the 2- and 5-months

studies, respectively. To filter out genes relevant to FFD, we specifically looked at

genes which were altered (upregulated or downregulated at least 2-fold) in FFD-fed

mice compared to Chow-fed mice and were, at the same time, also differentially

expressed (at least 2-fold) in FFD + EGFRi vs FFD group. 988 and 1348 genes were

differentially expressed (at-least 2-fold up/down-regulation) in FFD-fed mice

compared to Chow at 2 and 5-months, respectively. Interestingly, out of these FFD-

altered genes, 454 genes (46%) in the 2 months study and 486 genes (36%) in the

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5-months study (shown as overlap in Fig. 5A and Suppl. Fig. 3A) were also altered

(at least 2-fold) by EGFRi treatment (i.e. differentially expressed in FFD + EGFRi

group vs FFD group). These included both genes, which were induced by FFD and

downregulated by EGFRi (110 and 229 genes in the 2 and 5-months studies,

respectively; top 25 genes listed in Suppl. Table 1 and 2, respectively) along with

genes, which were repressed by FFD but upregulated by EGFRi (277 and 204 genes

in the 2 and 5-months studies, respectively). Analysis of this specific subset of genes

using DAVID revealed significant enrichment of several relevant biological process

including lipid metabolism, fatty acid biosynthesis, unsaturated fatty acid

biosynthesis commonly in both the 2 and 5-months studies (Fig. 4B and Suppl. Fig.

3B) (individual genes in some of these bio-processes are listed in Suppl. Table 3-4).

Analysis of these genes using IPA, taking directionality of change into account,

predicted several canonical pathways related to fatty acid, cholesterol, triglyceride

and glycogen biosynthesis, fatty acid oxidation and glycolysis to be significantly

altered by EGFRi treatment (Fig. 4C and Suppl. Fig. 3C). Further analysis of

diseases and functions associated with these genes using IPA predicted similar

alteration of functions related to lipid metabolism, carbohydrate metabolism, liver

fibrosis, necrosis/apoptosis/cell death and HCC (Suppl. Fig. 4A-B). Based on

downstream gene signatures, IPA predicted alteration of several transcriptional

factors related to lipid metabolisms by EGFRi-treatment, including SREBF1,

SREBF2 and PPARγ consistently in both the 2 and 5-months studies (Fig. 4D and

Suppl. Fig. 3D, respectively). Interestingly, based on the directionality of changes in

downstream genes expression, PPARγ was predicted to be significantly inhibited in

both the 2 and 5-months studies by EGFRi-treatment (effected downstream gene

network for PPARγ is shown in Fig. 4E and Suppl. Fig. 3E, respectively). Since

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pooled samples were used for microarray analysis, it is difficult to make mechanistic

conclusions based on these data. Nevertheless, several of the microarray data

analysis was further validated in following sections.

Downregulation of fatty acid biosynthesis pathway by EGFR inhibition

In order to investigate the mechanisms by which EGFR regulates lipid metabolism,

based on our microarray data analysis, we first looked into fatty acid synthesis

pathways. The first committed and rate limiting step in the synthesis of fatty acids is

conversion of acetyl-CoA to malonyl CoA by acetyl CoA carboxylase (ACC) in

cytosol. ACC protein expression was increased in FFD-fed mice, which was

decreased by EGFRi treatment (Fig. 5A-B). Subsequent synthesis of saturated fatty

acid (palmitate) from malonyl CoA and acetyl CoA is carried in series of reactions

catalyzed by the single multi-enzyme protein, fatty acid synthase. Protein expression

of fatty acid synthase was elevated in FFD-fed mice and normalized by EGFRi-

treatment (Fig. 5A-B). A similar pattern was also observed for protein expression of

Stearoyl-CoA desaturase (SCD-1), which is an important rate-limiting enzyme

regulating formation of major monounsaturated fatty acids from saturated fatty acids

(Fig. 5A-B). Carbohydrates both in drinking water (glucose and fructose) and diet

(sucrose and corn starch) are major source of over-nutrition in the FFD model as

opposed to the HFD model. ATP citrate lyase is a key enzyme that links

carbohydrate metabolism to fatty acid biosynthesis by converting TCA cycle

intermediate citrate into acetyl CoA, which then feeds into the fatty acid synthesis

pathway. Similar to enzymes involved in fatty acid synthesis, expression of ATP

citrate lyase increased in FFD-fed mice and decreased after EGFRi-treatment

consistently in both the 2 and 5-months studies (Fig. 5A and B). Fatty acid synthase

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(encoded by gene FASN) and SCD-1, but not other fatty acid synthesis enzymes

(data not shown), were found to be regulated at the transcriptional level by EGFR

inhibition (Fig. 5C-F). Additionally, we also investigated effect of EGFR inhibition on

expression of several important genes involved in fatty acid oxidation, but none of

them were significantly altered in both 2 and 5-months studies (Suppl. Fig. 6A-B).

Modulation of lipolysis pathway by EGFR inhibition

Several lipid-droplet associated proteins, which are negative regulators of lipolysis

and among other functions exert inhibitory effects on lipases to control lipid

homeostasis, including perilipins, CIDEC, CIDEA and G0/G1 switch-2 protein, have

recently emerged as important players in pathogenesis of NAFLD (12, 13). Analysis

of our microarray data indicated striking downregulation of expression of some

specific lipid droplet-associated proteins (perilipin2, perilipin4, CIDEC or FSP27,

CIDEA and G0/G1 switch-2) by EGFRi-treatment. All of these were among the top

25 FFD-inducible genes, which were downregulated by EGFRi in the 2 and/or 5-

months study (Suppl. Table 1-2). This was further validated by real-time PCR. There

was a significant induction of perilipin2, perilipin4, FSP27, CIDEA and G0/G1 switch2

genes in FFD-fed mice in the 2 months study and all of them except perilipin4 were

remarkably downregulated in the EGFRi group (Fig. 6A). In the 5-months study,

perilipin4, FSP27 and CIDEA were induced at the gene level in FFD-fed mice, but

not perilipin2 and G0/G1 switch 2; however, all of them were significantly

downregulated in EGFRi-treated FFD-fed mice (Fig. 6B). Perilipin2, perilipin4 and

FSP27 protein expression was also increased in FFD-fed mice at both 2 and 5-

months with the most prominent effect on perilipin2; expression of all three proteins

was decreased by EGFRi-treatment (Fig. 6C-D). Next, we investigated expression of

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several lipases which are reported to be important for triacylglycerol degradation in

hepatocytes (14). mRNA levels of Pnpla2 (ATGL; adipose triglyceride lipase),

carboxylesterase 1b (Ces1b) and Ces1c were decreased in FFD-fed mice and

remarkably increased by EGFR inhibition, especially in 2-months study (Fig. 6A-B).

ATGL protein expression also appears to be increased by EGFR inhibition at 2-

months (Fig. 6C and D). Ces1d gene expression was suppressed in FFD-fed mice

and remained suppressed after EGFR inhibition (Suppl. Fig. 7A-B). No remarkable

changes were observed in hormone sensitive lipase (HSL) at both gene and protein

level after EGFR inhibition (Suppl. Fig. 7A-D). Lipoprotein lipase (LPL) is another

major lipase that is secreted by parenchymal cells and localized on endothelial cells

for hydrolysis of triglycerides in VLDL and chylomicrons. It is normally expressed at

very low level in liver, but a recent study indicated an important role of LPL produced

from hepatocytes in determining serum LPL levels and maintaining systemic lipid

homeostasis (15). Consistent with previous literature (16), expression of LPL was

very low at both mRNA and protein level in Chow-fed mice and not much altered in

FFD-fed mice. Interestingly, LPL mRNA and protein levels were increased

remarkably only in the EGFRi group in both the 2 and 5-months study (Fig. 6A-D).

EGFR inhibition modulates major transcription factors regulating lipid

metabolism

SREBF1, a major transcription factor controlling fatty acid synthesis genes, has been

shown to be regulated by EGFR (7). Our microarray data predicted a significant

alteration of the SREBF1 downstream gene network by EGFR inhibition and

SREBF1 was among top 10 transcription factors predicted to be altered by EGFR

inhibition (Fig. 4D and Suppl. Fig. 3D). Consistent with microarray analysis,

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SREBF1 gene expression, total protein levels and nuclear levels were increased in

FFD-fed mice, and were significantly reduced by EGFR inhibition (Fig. 7A-D and

Suppl. Fig. 8A). ChREBP is another important transcription factor that regulates

lipogenic gene expression, especially in response to carbohydrate-rich diet as

utilized in our study. ChREBP gene expression was significantly suppressed by

EGFR inhibition specifically in 2 months study (Fig. 7A and C). Further, protein levels

of ChREBP appear to be downregulated by EGFR inhibition at both 2 and 5-months,

which were induced in FFD-fed mice compared to chow-fed mice (Fig. 7B and D).

SREBF1 and several fatty acid synthesis/lipolysis genes are regulated by PPARγ in

liver and hepatocyte-specific PPARγ KO mice are protected from HFD-induced

steatosis, similar to the phenotype observed by EGFR inhibition in this study (12, 17,

18). Apart from this, PPARγ also regulates lipid droplet associated proteins such as

perilipin-2, perilipin-4 and CIDEC (12), which control lipolysis and were found to be

altered by EGFRi treatment in this study. Our microarray data analysis predicted

inhibition of PPARγ transcriptional activity by EGFRi consistently at both 2 and 5-

months (Fig. 4D-E and Suppl. Fig. 3D-E). In line with previous studies utilizing a HFD

model (17), PPARγ mRNA and protein levels were remarkably increased in FFD-fed

mice at both 2 and 5 months (Fig. 7A-D). Interestingly, EGFRi-treatment strikingly

decreased PPARγ expression even below basal (Chow-diet) levels consistently at

both 2 and 5-months (Fig. 7A-D). Importantly, the increase in nuclear levels of

PPARγ was reversed by 5 weeks of EGFRi treatment in the 5-months study (Suppl.

Fig. 8A). Further, PPARγ DNA binding activity to a specific PPARγ response element

was significantly reduced by EGFRi treatment in the 5-months study (Suppl. Fig. 8B).

Interestingly, PPARα expression was not altered at mRNA or protein level in both 2

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and 5-months studies (Fig. 7A-D). HNF4α is a master regulator of transcription in

liver, which maintains the quiescent state of differentiated hepatocytes and is also

reported to inhibit PPARγ transcriptionally in liver (19). HNF4α KO mice display a

fatty liver phenotype (20) and forced expression of HNF4α in a fibrosis model

reverses the fibrotic phenotype (21). Further, HNF4α expression is downregulated in

patients with NASH and murine NAFLD models, which is reported to be important for

VLDL secretion and lipid mobilization from liver (22). Here we report that HNF4α

expression drastically decreased in FFD-fed mice both at 2 and 5-months (Fig. 7B

and D). Interestingly, HNF4α expression was restored by EGFRi-treatment to chow-

fed mice levels (Fig. 7B and D).

Next, utilizing ChIP assay, we investigated if EGFR inhibition is altering direct

binding of these transcription factors (namely PPARγ and HNF4α) to crucial lipid

metabolism genes, which were altered in our study. Fasn, Scd1 and Plin2 genes

were selected for ChIP analysis. Based on analysis of previously published ChIP-seq

data for H4K5Ac, PPARα and HNF4α, we first characterized PPAR and HNF4α

binding enhancers near these three genes (Supplementary methods and Suppl. Fig.

8C). As shown in Suppl. Fig. 8C, the chromatin modification H4K5Ac

characteristically surrounded nuclear receptor-binding enhancers. For each of these

genes, a few enhancers were located within 30 kb of the promoters. Of these, we

chose the three strongest PPARα-binding enhancers for PPARγ ChIP analysis.

Since PPARα has identical DNA binding, it is a good surrogate for detection of

PPARγ, which has not been characterized in mouse liver. Indeed, the ChIP detection

of PPARγ showed an excellent correspondence to these sites (Fig. 7E and Suppl.

Fig. 8C). EGFR inhibition dramatically reduced PPARγ binding to all three selected

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enhancer sites for each of the three investigated genes (Fig. 7E). Further, in-silico

analysis of previously published ChIP-seq data also revealed that several of these

PPAR-binding enhancers also have HNF4α binding sites (indicated by arrows in

Suppl. Fig. 8C). In contrast to PPARγ binding, which displayed drastic reduction,

ChIP analysis for HNF4α revealed that binding of HNF4α either remained unaltered

(for Fasn enhancer site) or showed consistent pattern of increase (for Scd1 and

Plin2 enhancer sites) after EGFR inhibition, although, could not achieve statistical

significance (Fig. 7F). Compared to previously published ChIP-seq data in Suppl.

Fig. 8C, there is an excellent correspondence of relative peak strength, and of

positive or negative binding of HNF4α in our ChIP analysis (Fig. 7E-F).

Effects of EGFR inhibition on hepatocyte proliferation and cell cycle

Chronic proliferation secondary to liver injury is a characteristic feature of NAFLD,

which is considered a contributory factor for development of HCC, but its role in fatty-

liver pathogenesis in largely unknown. It is known that hepatocyte proliferation during

normal liver regeneration is associated with transient accumulation of lipid droplets in

preparation to enter into DNA synthesis (5). This proliferation-associated lipid

accumulation may have a compounding effect on an already steatotic liver during

NAFLD and may contribute to progression of the disease. Recent studies indicate a

role of key cell cycle protein, CyclinD1, which governs entry into cell cycle, in

regeneration-associated steatosis and lipid metabolism, in general (23, 24). Here we

show that FFD feeding for 5-months induces robust hepatocyte proliferation as

evidenced by an increase in PCNA-positive hepatocytes, which was dramatically

reduced by EGFR inhibition (Fig. 8A-B). Further, gene expression of several cyclins

(CyclinD1, CyclinA2 and CyclinB1), which sequentially control progression through

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the cell cycle, was increased by FFD and lowered by EGFRi-treatment (Fig. 8C-D).

Interestingly, major cyclin-dependent kinase inhibitor, p21, was induced at the mRNA

level in FFD-fed mice (around 10 and 20-fold at 2 and 5-months, respectively).

However, induction of p21 was dramatically increased by EGFRi-treatment (around

40 and 100-fold at 2 and 5-months, respectively) (Fig. 8C-D). Similar changes in

cyclinD1 and p21 expression were also observed at the protein level (Fig. 8E-F).

Effects of FFD diet on EGFR ligands

Next, we investigated if expressions of any of the relevant EGFR ligands are altered

by FFD feeding. While, EGF is constantly available to liver from Brunner’s glands via

systemic circulation, amphiregulin and TGF-α are known to be produced locally and

regulated in liver (4). Expression of amphiregulin has previously reported to increase

remarkably (6-fold) in patients with severe NASH (grade-4 fibrosis) and in hepatic

stellate cells of murine model of NASH (25). Further, amphiregulin can directly cause

proliferation and activation of human stellate cells inducing collagen production (25).

Indeed, amphiregulin expression was remarkably increased in livers of FFD-fed mice

compared to chow-fed controls at both mRNA and protein levels (consistently in both

2 and 5 months studies) and remain elevated after EGFR inhibition (Suppl. Fig. 9A-

D). TGF-α is known to be produced by hepatocytes after various insults, but its

protein expression was not altered by FFD feeding in our model (Suppl. Fig. 9C-D).

Interesting, TGF-α protein expression in liver increased specifically after EGFRi

treatment, which might be a compensatory response to inhibition of EGFR (Suppl.

Fig. 9C-D).

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Effects of EGFR inhibition on various signaling pathways

Further, we investigated activation status of several signaling pathways downstream

of EGFR (including STAT3, MAPKs and PI3K-AKT), which were predicted to be

altered based on microarray analysis (Fig. 4B and Suppl. Fig. 3C and 5B) and can

potentially regulate lipid metabolism. STAT3 did not appear to be altered by EGFR

inhibition in a consistent manner (data not shown). ERK1/2, displayed increased

phosphorylation (i.e. activation) after FFD-feeding and decreased activation after

EGFR inhibition in both the 2 and 5-months studies (Fig. 8E-F), which is consistent

with alteration of proliferative signaling. AKT signaling is an important downstream

pathway for EGFR and other tyrosine kinase receptors such as insulin receptor and

known to be required for hepatic lipogenesis in NAFLD models by regulating

lipogenic transcriptional factors, including SREBF1 and PPARγ (26). Indeed, AKT

phosphorylation (both Ser473 and Thr 308) was strikingly increased in FFD group

compared to chow group and remarkably decreased by EGFR inhibition consistently

in both 2 and 5-months studies, which correlated with PPARγ expression (Suppl. Fig.

10A and B). Further, GSK3β-Ser9 phosphorylation which is downstream target of

AKT showed similar pattern with striking decrease after EGFR inhibition (Suppl. Fig.

10A and B). Interestingly, expression of total insulin receptor-β and IGF-I receptor-β

proteins was remarkably increased specifically in FFD + EGFRi group, which was

consistent in both 2 and 5-months studies (Suppl. Fig. 10A and B).

Further, considering alteration of FFD-activated AKT signaling by EGFR inhibition,

which is also a downstream mediator for insulin signaling, we investigated if EGFR

inhibition per se can alter insulin signaling in liver (a crucial pathway regulating lipid

metabolism in liver). Insulin was administered to EGFR-inhibited or control mice and

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insulin signaling was studied (Suppl. Fig. 11). As expected, insulin stimulation

dramatically increased phosphorylation of insulin receptor and downstream

phosphorylation of AKT (both Ser473 and Thr308) in liver. However, insulin signaling

pathway remains activated even after EGFR inhibition (Suppl. Fig. 11).

Liver-specific MET deletion did not prevent fatty liver development in mice fed

fast-food diet for 5 months

Previous studies reported that MET (receptor for HGF), which is another mitogenic

receptor tyrosine kinase in hepatocytes and shares major functionalities with EGFR,

can interact with insulin receptor and alters insulin signaling (27). Both MET and

EGFR are known to compensate for each other for proliferative functions in liver (5).

Our previous study showed that inhibitory effects on proliferation-associated lipid

accumulation after PHx, were specific to EGFR inhibition and were not observed in

MET-KO mice (5). In order to test if this differential effect of receptor tyrosine kinases

on lipid accumulation holds even in a diet-induced steatosis model, we investigated

the effect of MET deletion on the FFD-induced fatty liver phenotype. Wild-type (WT)

or hepatocyte-specific MET-KO mice (utilizing Albumin-Cre system) were fed FFD

for 5-months (Suppl. Fig. 12A). WT-mice displayed extensive lipid accumulation,

which was not prevented in the MET-KO mice (Suppl. Fig. 12B). Serum ALT, AST,

and cholesterol levels remain similarly elevated over normal range in both WT and

MET-KO mice (Suppl. Fig. 12C). Further, both the groups developed remarkable and

comparable glucose intolerance (Suppl. Fig. 12D). All of these data indicate the

inhibitory effects on FFD-induced fatty liver phenotype were specific to EGFR

inhibition and were not shared with MET.

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DISCUSSION

Our study demonstrates that EGFR and the multiplicity of genes subject to its control

are important regulators and enablers in development of NAFLD resulting from

nutritionally inappropriate diet with content which is prevalent in the Western world.

The details presented in our study demonstrate that EGFR inhibition affects

expression of enzymes involved in a coordinated manner as component of serial

pathways involved in fat metabolism, including fatty acid synthesis and lipolysis. The

mechanisms by which EGFR controls these pathways are undoubtedly complex and

involve multiple signaling by a variety of transcription factors, including PPARγ,

ChREBP, SREBF1 and HNF4α. These transcription factors are known to regulate

expression of each other in liver leading to coordinated regulation of lipid metabolism

enzymes. For instance, PPARγ expression is increased during steatosis, which can

induce SREBF1 and its downstream target genes involved in fatty acid synthesis

(17). Further, HNF4α keeps PPARγ levels in check in quiescent liver by inhibiting its

gene expression (19). While EGFR inhibition lowered FFD-induced PPARγ and

SREBF1 expression, it restored HNF4α levels, which were decreased by FFD

feeding in our study. Further, our ChIP analysis revealed that EGFR inhibition

dramatically decreased PPARγ binding to enhancer sites of important lipid

metabolism genes. Some of these sites were also shared by HNF4α, binding of

which either showed consistent pattern of increase or remain unaltered by EGFR

inhibition in a site specific manner. The signaling pathways downstream of EGFR

which control these transcription factors are not completely clear, but our data

indicated AKT signaling may play an important role. Indeed, AKT signaling is

previously reported to be required for de novo hepatic lipogenesis in NAFLD models

by regulating lipogenic transcriptional factors, including SREBF1 and PPARγ (liver

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specific AKT deletion in ob/ob mice downregulated PPARγ/SREBF1 and prevented

steatosis) (26).Nevertheless, the net result is a complex but effective regulation of

lipid metabolism by EGFR, resulting in dramatic suppression of lipid accumulation by

EGFR inhibition. It should be noted that this effect of EGFR inhibition on lipid

metabolism is not likely shared with other receptor tyrosine kinases. While inhibition

of EGFR in our previous study prevented lipid accumulation in regenerating

hepatocytes after PHx, inhibition of HGF/MET signaling had no such effect (5).

Similar results were obtained in this study, where FFD-induced fatty liver phenotype

was specifically attenuated by EGFR inhibition, but not by elimination of MET

signaling in mice.

Our study also reveals an often overlooked effect, that of compensatory proliferation

of hepatocytes to preserve liver/body weight ratio (hepatostat), in conditions causing

injury and death of hepatocytes (4). The robust increase in PCNA and cyclins in the

FFD diet (Fig. 8) indicates that this process is occurring in mice fed the FFD diet.

This proliferative response was dramatically reduced in EGFR-treated mice.

Previous studies have shown that in situations associated with continuous

hepatocyte proliferation, there is a decrease in hepatocyte ploidy (4, 28).

Hepatocytes in rodent and human liver at the state of polyploidy are aneuploid and

randomly miss chromosomes. An increased conversion of polyploid/aneuploid

hepatocytes to diploid/aneuploid hepatocytes is likely to expose hepatocytes to

situations of allelic imbalance with otherwise recessive mutations, occurring in the

complex environment of NAFLD, associated with genotoxicty from lipid-peroxidation,

reactive oxygen radicals, etc. Such changes are well documented in NAFLD (29).

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These changes open pathways for hepatocyte initiation towards a neoplastic

phenotype.

An additional consideration of the effects of compensatory proliferation relates to the

very accumulation of fat in hepatocytes. Proliferating hepatocytes enter into a

transitory accumulation of lipid droplets, as part of a metabolic adaptation related to

preparation for hepatocyte mitosis (4). This change in hepatocyte metabolism aimed

to accumulate fat in cytoplasm, is likely to generate an enhanced tendency for

accumulation of lipids in the context of a nutritionally imbalanced fatty diet. This is

creating a vicious cycle in which the mandate of the “hepatostat” to maintain liver

mass creates conditions which further aggravate hepatocyte injury and loss of liver

mass. Though the diet used in the study causes an apparent increase in liver to body

weight ratio this probably does not reflect a “true” liver mass increase, since much of

the weight increase is due to inert material (stored lipid) which does not contribute to

the multiple and complex homeostatic functions controlled by the liver.

The effects of EGFR inhibition on NAFLD open potential therapeutic possibilities.

There are several well characterized EGFR inhibitors used primarily for cancer

therapeutics (30). While it would be rather inadvisable to use EGFR inhibitors as a

long term treatment for NAFLD, it would, however, be rather advisable to consider

using a short term EGFR inhibitor treatment for advanced NAFLD or NASH

situations. Ours and previous studies have shown that EGFR inhibition can also

improve already developed fibrosis (8, 9). Whatever the details of the therapeutic

modalities that can be based on EGFR inhibition to reverse NAFLD/NASH, our

findings and the intensity of the effects provide a rational basis for future therapeutic

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considerations. Further, future studies can also be directed to target druggable

downstream mediators of EGFR signaling to investigate their therapeutic potential

for NAFLD.

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FIGURE LEGENDS

Figure 1. EGFR inhibition prevents and reverses steatosis in fast food diet-fed

mice. (A) Schematics showing 2 and 5 months study design. In the 2 months study,

EGFRi (Canertinib 80 mg/kg) was given in diet from the beginning of the study. In

the 5 months study, EGFRi (or vehicle) was administered (i.p.) only for the final 5

weeks (after the development of NAFLD) of the study. Representative

photomicrographs of Oil Red O and H&E stained liver sections at (B) 2 and (D) 5

months. Bar graphs showing liver to body weight ratio in various groups at (C) 2 and

(E) 5 months. * and # represent significant difference w.r.t chow and FFD groups,

respectively, at P < 0.05.

Figure 2. Improvement in serum parameters and glucose tolerance after EGFR

inhibition. Bar graphs showing ALT, AST, cholesterol and triglyceride levels in

serum at (A) 2 months and (B) 5 months. (C) Glucose tolerance test data showing

blood glucose levels at various time points after glucose administration (i.p.) in

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various groups at 5 months. (D) Bar graphs showing insulin levels in serum at the

end of 5 months study. * and # represent significant difference w.r.t chow and FFD

groups, respectively, at P < 0.05.

Figure 3. Effects of EGFR inhibition on fibrosis-associated parameters in 5

months study. (A) Representative photomicrographs of Sirius red stained liver

section at 5 months. mRNA expression of (B) Col1A1 and (C) Col 3a1 and (D) α-

SMA at 5 months. (E) Western blot analysis showing protein expression of α-SMA at

5 months. * and # represent significant difference w.r.t chow and FFD groups,

respectively, at P < 0.05.

Figure 4. Global changes in gene expression profile after EGFR inhibition at 2

months. (A) Venn diagram with overlap showing subset of genes altered (at least 2

fold) in FFD vs Chow group and also differentially expressed in FFD+EGFRi vs FFD

group at 2 months. Enrichment analysis using DAVID showing (B) biological

processes (GO terms) predicted to be altered in FFD+EGFRi vs FFD group based

on set of genes shown as overlap in (A). (C) Canonical signaling pathways and (D)

transcription factors predicted to be altered in FFD+EGFRi vs FFD group based on

set of genes shown as overlap in (A), analyzed using IPA (Ingenuity Pathway

Analysis). (E) IPA analysis of microarray data showing inhibition of PPARγ

downstream gene network in FFD+EGFRi vs FFD group. Negative z-score in (D)

represents predicted inhibition of transcription factor activity and positive z-score

represents predicted activation (absolute z-score > 2 considered as significant)

based on expression profile of downstream genes. p-values signifies extent of

overlap between set of downstream target genes of a given transcription factor in

dataset compared to all known downstream target genes of that transcription factor

in the reference genome.

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Figure 5. Downregulation of fatty acid synthesis pathway by EGFR inhibition.

Western blot analysis showing protein expression of fatty acid synthase, acetyl Co-A

carboxylase (ACC), ATP citrate lyase, stearoyl-CoA desaturase (SCD-1) at (A) 2

months and (B) 5 months. mRNA expression of (C) fatty acid synthase (FASN) and

(D) SCD1 at 2 months. mRNA expression of (E) FASN and (F) SCD1 at 5 months. *

and # represent significant difference w.r.t chow and FFD groups, respectively, at P

< 0.05.

Figure 6. Modulation of lipolysis pathway by EGFR inhibition. mRNA expression

of perilipin 2, perilipin 4, fat-specific protein 27 (CIDEC), CIDEA, G0/G1 switch 2,

lipoprotein lipase, Pnpla2 (adipose triglyceride lipase), carboxylesterase 1b (Ces1b)

and Ces1c at (A) 2 months and (B) 5 months. Western blot analysis showing protein

expression of perilipin 2, perilipin 4, fat-specific protein 27 (CIDEC), lipoprotein lipase

and adipose triglyceride lipase (ATGL) at (C) 2 months and (D) 5 months. * and #

represent significant difference w.r.t chow and FFD groups, respectively, at P < 0.05.

Figure 7. EGFR inhibition modulates major transcription factors regulating

lipid metabolism. mRNA expression of SREBF1, PPARγ, ChREBP and PPARα at

(A) 2 months and (C) 5 months. Western blot analysis showing protein expression of

PPARγ, SREBF1, HNF4α, ChREBP and PPARα at (B) 2 months and (D) 5 months.

(E) ChIP analysis showing PPARγ binding to three different enhancer sites (selected

based on analysis of previously published ChIP-seq data shown in Suppl. Fig. 8C) in

the regulatory regions for Fasn, Scd1 and Plin2 genes in FFD and FFD + Canertinib

groups at 5 months. Sites are labelled as distance from transcription start site. (F)

ChIP analysis showing HNF4α binding to same sites as in (E). Several of the PPAR-

binding enhancers also have previously reported HNF4α binding sites (indicated by

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arrows in Suppl. Fig. 8C). * and # represent significant difference w.r.t chow and FFD

groups, respectively, at P < 0.05.

Figure 8. Effects of EGFR inhibition on hepatocyte proliferation and cell cycle.

A) Representative photomicrographs of PCNA stained liver section at 5 months with

quantification of PCNA positive hepatocytes (brown stained nuclei) shown in (B).

mRNA expression of cyclin D1, cyclin A2, Cyclin B1 and p21 at (C) 2 months and (D)

5 months. Western blot analysis showing protein expression of Cyclin D1, phospho

ERK 1/2 and p21 at (E) 2 months and (F) 5 months. * and # represent significant

difference w.r.t chow and FFD groups, respectively, at P < 0.05.

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Documents

DR. BHARAT BHUSHAN (Orcid ID : 0000-0002-1716-9764) DR ......Title: Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Non‐alcoholic Fatty Liver Disease in Murine