Forkhead Box M1 Transcription Factor Drives Liver Inflammation Linking to Hepatocarcinogenesis in Mice

Journal Pre-proof Forkhead Box M1 transcription factor drives liver inflammation linking to hepatocarcinogenesis in mice Tomohide Kurahashi, Yuichi Yoshida, Satoshi Ogura, Mayumi Egawa, Kunimaro Furuta, Hayato Hikita, Takahiro Kodama, Ryotaro Sakamori, Shinichi Kiso, Yoshihiro Kamada, I-Ching Wang, Hidetoshi Eguchi, Eiichi Morii, Yuichiro Doki, Masaki Mori, Vladimir V. Kalinichenko, Tomohide Tatsumi, Tetsuo Takehara

PII: DOI: Reference:

S2352-345X(19)30144-4 https://doi.org/10.1016/j.jcmgh.2019.10.008 JCMGH 537

To appear in: Cellular and Molecular Gastroenterology and Hepatology Accepted Date: 15 October 2019 Please cite this article as: Kurahashi T, Yoshida Y, Ogura S, Egawa M, Furuta K, Hikita H, Kodama T, Sakamori R, Kiso S, Kamada Y, Wang I-C, Eguchi H, Morii E, Doki Y, Mori M, Kalinichenko VV, Tatsumi T, Takehara T, Forkhead Box M1 transcription factor drives liver inflammation linking to hepatocarcinogenesis in mice, Cellular and Molecular Gastroenterology and Hepatology (2019), doi: https://doi.org/10.1016/j.jcmgh.2019.10.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute.

FoxM1

Forkhead Box M1 transcription factor drives liver inflammation linking to hepatocarcinogenesis in mice CCL2

Hepatocytes

Macrophage Recruitment

Normal Liver

Liver Inflammation

Chronic hepatitis

Liver Fibrosis

Cirrhosis

Liver Cancer

HCC

1

Forkhead Box M1 transcription factor drives liver inflammation linking to

2

hepatocarcinogenesis in mice

3

Tomohide Kurahashi1#, Yuichi Yoshida1#*, Satoshi Ogura1, Mayumi Egawa1, Kunimaro Furuta1,

4

Hayato Hikita1, Takahiro Kodama1, Ryotaro Sakamori1, Shinichi Kiso1, Yoshihiro Kamada1, 2,

5

I-Ching Wang3,4, Hidetoshi Eguchi5, Eiichi Morii6, Yuichiro Doki5, Masaki Mori5, Vladimir V

6

Kalinichenko3, Tomohide Tatsumi1, Tetsuo Takehara1*

7

8

9

10

11

12

1

Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University,

Suita, Osaka, Japan 2

Department of Molecular Biochemistry and Clinical Investigation, Graduate School of Medicine,

Osaka University, Suita, Osaka, Japan 3

Center for Lung Regenerative Medicine, Divisions of Pulmonary Biology and Developmental

13

Biology, Cincinnati Children’s Hospital Medical Center, College of Medicine of the University of

14

Cincinnati, Cincinnati, Ohio, USA

15

16

17

18

19

4

Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan.

5

Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University,

Suita, Osaka, Japan 6

Department of Pathology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan

#

Tomohide Kurahashi and Yuichi Yoshida contributed equally to this work. 1

20

*Co-corresponding authors:

21

Tetsuo Takehara, MD, PhD, Department of Gastroenterology and Hepatology, Osaka University

22

Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan. e-mail:

23

[email protected]; tel: (81) 6-6879-3621, fax: (81) 6-6879-3629.

24

Yuichi Yoshida, MD, PhD, Department of Gastroenterology and Hepatology, Osaka University

25

Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan. e-mail:

26

[email protected]; tel: (81) 6-6879-3621, fax: (81) 6-6879-3629.

27

Short Title

28

Hepatocyte FoxM1 promotes liver inflammation.

29

Abbreviations

30

ALT, alanine aminotransferase; AS, antisense; CCL2, chemokine (C-C motif) ligand 2; CCl4, carbon

31

tetrachloride; DOX, doxycycline; FoxM1, Forkhead Box M1 transcription factor; GAPDH,

32

glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; HFHC,

33

high-fat/high-cholesterol; RT-PCR, reverse transcription polymerase chain reaction; S, sense; Tet,

34

tetracycline; TG, transgenic; WT, wild-type.

35

Acknowledgments

36

The authors thank Dr. Pradip Raychaudhuri (University of Illinois at Chicago) for providing

37

CMV-T7-FoxM1 and 6×CDX2-Luc plasmids.

38

Author Contributions 2

39

Study concept and design: T. Kurahashi, Y. Yoshida, S. Ogura, and T. Takehara. Acquisition of data:

40

T. Kurahashi, S. Ogura, M. Egawa, K. Furuta. Analysis and interpretation of data: T. Kurahashi, Y.

41

Yoshida, S. Ogura, M. Egawa, K. Furuta, H. Hikita, T. Kodama, R. Sakamori, S. Kiso, Y. Kamada, I.

42

C. Wang, H. Eguchi, E Morii, Y. Doki, M. Mori, V. V. Kalinichenko, T. Tatsumi, and T. Takehara.

43

Writing, review, or revision of the manuscript: T. Kurahashi, Y. Yoshida, and T. Takehara.

44

Conflicts of interest

45

The authors disclose no conflicts.

46

Funding

47

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for

48

the Promotion of Science.

49

Word count (abstract): 244

50

Word count (excluding title page, methods, tables/figures, or references): 3207

51

Number of Tables and Figures 1 table and 11 figures

52

3

53

SYNOPSIS

54

Hepatocyte-specific FoxM1 transgenic (TG) mice develop spontaneous liver inflammation,

55

fibrosis and carcinogenesis. Spontaneous liver inflammation in TG mice was associated with

56

increased expression of chemokine CCL2. FoxM1-specific inhibitor effectively reduces liver

57

inflammation and CCL2 expression in models of liver injury.

58

4

59

ABSTRACT

60

BACKGROUND & AIMS: Liver inflammation has been recognized as a hallmark of

61

hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic

62

transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver

63

inflammation has never been explored.

64

METHODS: We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice using the

65

Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and

66

time-dependent manner.

67

RESULTS: After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice

68

exhibited spontaneous development of hepatocyte death with elevated serum ALT levels and hepatic

69

infiltration of macrophages. The removal of DOX in TG mice completely removed this effect,

70

suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent

71

manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic

72

and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that

73

CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since

74

birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic

75

expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacological

76

inhibition of FoxM1 reduced liver inflammation in models of liver injury.

77

CONCLUSIONS: Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation 5

78

linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the

79

treatment of liver injury but also for the prevention toward HCC.

80

Keywords: FoxM1; Liver inflammation; CCL2

81

6

82

Introduction

83

Hepatocellular carcinoma (HCC) is one of the most common types of malignant neoplasms

84

among primary liver cancers and a major cause of cancer-related deaths worldwide.1 It is well known

85

that HCC occurs in patients with chronic liver diseases, including hepatitis virus infections, alcoholic

86

or nonalcoholic fatty liver diseases, and other liver diseases.2 Irrespective of the etiology, chronic

87

liver inflammation promotes subsequent hepatic wound-healing responses, eventually leading to the

88

development of liver fibrosis and HCC.3, 4 Thus, the molecular mechanisms driving liver

89

inflammation to HCC need to be better understood to identify a therapeutic strategy to prevent HCC

90

in patients with chronic liver diseases.

91

The Forkhead box M1 (FoxM1) transcription factor is a member of the Fox family of

92

proteins that share a highly conserved structure in the winged helix DNA-binding domain.5-7 Several

93

studies have shown that FoxM1 acts as an important regulator for cell proliferation and is frequently

94

expressed at a higher level than normal in a variety of human cancers.7-12 Previous animal studies

95

have shown that FoxM1 is critical for cancer cell proliferation of HCC.13, 14 We previously reported

96

that a high FoxM1 expression defines poor prognosis in patients with HCC after surgical resection15

97

and that FoxM1 is regulated via the mevalonate pathway of lipid metabolism in hepatoma cell

98

lines.16 Consistent with our observations, FoxM1 was shown to be overexpressed in tumor tissues of

99

hepatitis B virus (HBV)-related HCC compared with the surrounded non-tumor tissues.17 In addition,

100

FoxM1 expression has been also shown to be increased in the livers of chronic hepatitis or liver 7

101

cirrhosis patients with HBV infection compared with the normal liver samples,17 suggesting that

102

FoxM1 may be involved in the initial step of hepatocarcinogenesis during the development of liver

103

injury.

104

In adult human organs, FoxM1 is abundantly expressed in testis, thymus, intestine, and

105

colon.18 By contrast, no FoxM1 expression is present in adult murine livers under normal

106

conditions;18 however, it is induced in the livers during liver regeneration after partial hepatectomy19

107

and during toxin-induced liver injury.20 These results suggest that FoxM1 may be involved in not

108

only hepatocyte proliferation but also liver inflammation. Consistent with these results, a previous

109

study demonstrated that FoxM1 is required for allergen-mediated goblet cell proliferation and

110

pulmonary inflammation through the induction of inflammatory cytokines or chemokines in a murine

111

model of chronic lung disorder.21 However, to date, the role of hepatocyte FoxM1 in liver

112

inflammation during the development of liver injury has remained unclear.

113

In this study, we hypothesized that hepatocyte FoxM1 affects the inflammatory

114

microenvironment of the liver. To clarify this issue, we developed a new murine model in which

115

ectopic overexpression of FoxM1 in hepatocytes is controlled in a time-dependent manner.

116

8

117

Results

118

Overexpression of FoxM1 in Murine Livers Causes Spontaneous Liver Injury and Fibrosis

119

To test whether increased FoxM1 expression is sufficient to drive liver injury, we

120

established hepatocyte-specific FoxM1 conditional TG (TetO7-FoxM1 tg/tg/

121

Rosa26-LSL-rtTA/Alb-Cretg/-) mice using the Tet-on and Cre-loxP systems (Figure 1A). To induce

122

FoxM1 expression, TG and WT (TetO7-FoxM1 tg/tg /Rosa26-LSL-rtTA/Alb-Cre-/-) mice were treated

123

with DOX. Western blot and immunohistochemical analyses using antibody against FoxM1

124

confirmed that ectopic overexpression of FoxM1 was induced in a liver- and DOX-specific manner

125

(Figure 1B-D). At 13 weeks of age, TG mice showed spontaneous elevations of serum ALT levels

126

and hepatocyte death, confirmed by an increased number of TUNEL-positive hepatocytes (Figure

127

1E-G). Furthermore, TG mice showed spontaneous liver fibrosis, confirmed by collagen-specific

128

picrosirius red staining or immunostaining for α-smooth muscle actin, a marker of activated hepatic

129

stellate cells (Figure 1H and I). This spontaneous liver fibrosis in TG mice was further assessed by

130

the increased expression of fibrosis-related genes in the livers (Figure 1J). Taken together,

131

overexpression of FoxM1 in the livers induced spontaneous liver injury and fibrosis.

132

Short-Term Overexpression of FoxM1 Induces Reversible Liver Inflammation With Macrophage

133

Recruitment

9

134

To investigate whether FoxM1 itself has a direct effect on spontaneous liver injury in TG

135

mice, we introduced transient overexpression of FoxM1 at 8 weeks of age for 3 days and repressed

136

its expression by removing DOX (Figure 2A and B). After 3 days of DOX treatment, TG mice

137

exhibited elevated serum ALT levels and hepatocyte death, confirmed by TUNEL staining; this

138

spontaneous liver injury was completely reversible by removing DOX (Figure 2C-E). Furthermore,

139

the increased liver injury in TG mice was associated with hepatic infiltration of inflammatory cells,

140

consisting of F4/80 positive macrophages; this spontaneous liver inflammation was also completely

141

reversed by removing DOX (Figure 2F). In addition, the fluorescence-activated cell sorting analysis

142

confirmed that the number of CD11b+F4/80+ macrophages was increased in the livers of TG mice in

143

a DOX-dependent manner (Figure 2G). These data indicate that short-term overexpression of FoxM1

144

is sufficient to induce liver injury and inflammation with macrophage recruitment.

145

To investigate the molecular mechanisms underlying the spontaneous recruitment of

146

hepatic macrophages in TG mice, we examined the gene expression of macrophage-related markers

147

in the livers of TG mice. TG mice showed a significant increase in the hepatic gene expression of

148

macrophage-related markers, including F4/80, Cd68, Nos2, Tnfα, Ccl2, Ccr2, Ccl5, Arg1, and Cd206

149

(Figure 2H). Among these markers, Ccl2 showed a 145-fold higher hepatic expression in TG mice

150

than in WT mice (Figure 2H). Consistently, 3-day DOX treatment resulted in increased serum CCL2

151

levels in TG mice, and this increase was completely reversed by removing DOX (Figure 2I).

10

152

To rule out the possibility that CCL2 induction results from increased liver injury in TG

153

mice, we overexpressed FoxM1 for 1 day with DOX (Figure 2J). There was no apparent increase in

154

serum ALT levels and no difference in serum ALT levels in WT and TG mice after 1 day of DOX

155

treatment (Figure 2K). However, there was a significant increase in hepatic gene expression of Ccl2

156

compared with WT mice at this earlier, 1 day time point (Figure 2L), suggesting that the increased

157

expression of hepatic Ccl2 gene in TG mice might occur prior to induction of liver injury.

158

Next, we titrated the levels of DOX in the drinking water and developed TG mice with

159

lower FoxM1 expression using low dose of DOX (DOX low dose: 0.01 mg/mL) (Figure 2M). Even

160

DOX low dose induced the moderate expression of FoxM1 (27.4%; Figure 2N) and an increase in

161

TUNEL-positive hepatocytes (Figure 2O) and serum ALT levels (Figure 2P) as was observed with

162

the higher dose of DOX (DOX on: 0.2 mg/mL). Furthermore, DOX low dose increased Ccl2 gene

163

expression in livers of TG mice as observed with DOX on (Figure 2Q).

164

CCL2 as a Direct Target of FoxM1 in Hepatocytes

165

To investigate whether hepatocyte FoxM1 regulates the expression of CCL2, we next

166

performed in vitro experiments using murine hepatocyte cell lines. si-RNA-mediated knock down of

167

FoxM1 resulted in a significant decrease in the gene expression of Ccl2 and protein expression of

168

CCL2 in murine hepatocytes cell lines BNL-CL2 (Figure 3A and B) and AML12 (Figure 3C and D).

11

169

We then performed in vitro transcriptional analysis to investigate whether CCL2 is a direct

170

target of FoxM1. One potential FoxM1 binding site was identified in the −2468/+67 bp promoter

171

region of the murine Ccl2 gene at −1343/−1338 bp (Figure 3E). Co-transfection of FoxM1

172

expression vector resulted in a significant increased activity of the −1401/+67 bp Ccl2 luciferase

173

reporter, and the deletion of the FoxM1 binding site in the Ccl2 promoter region −1136/+67 bp

174

abolished the capacity of FoxM1 to stimulate this activity, indicating that −1343/−1338 bp in the

175

Ccl2 promoter region functions a FoxM1 binding site (Figure 3E). To further confirm the binding of

176

FoxM1 to the promoter region of the Ccl2 gene, the chromatin immunoprecipitation assay was

177

performed in murine hepatocyte BNL-CL2 cells using two antibodies against FoxM1. This assay

178

showed the specific binding of FoxM1 protein to the Ccl2 promoter DNA (Figure 3F). Collectively,

179

the data indicate that CCL2 is a direct target of FoxM1 in hepatocytes and suggest the involvement

180

of hepatic CCL2 induction in the spontaneous macrophage recruitment of TG livers.

181

Hepatocytes Are the Major Source of CCL2 Production in TG Mice

182

To investigate which cell population produces CCL2 in TG mice, we examined the gene

183

expression of Ccl2 and the protein expression of CCL2 in hepatocytes and non-parenchymal cells

184

(NPCs) isolated from livers of WT and TG mice after 3 days of DOX treatment. Hepatocytes of TG

185

mice showed a significant increase in Ccl2 gene expression compared to those of WT mice, whereas

186

that in NPCs was comparable between the two groups (Figure 4A). Western blot analysis showed the

12

187

increased expression of CCL2 protein in hepatocytes of TG mice compared with those of WT mice

188

(Figure 4B). These data suggest that the major source of CCL2 is hepatocytes rather than NPCs in

189

TG mice.

190

FoxM1 Induction Has a Modest Effect on Cell Death in Cultured Hepatocytes in vitro

191

We investigated the effect of FoxM1 induction on hepatocyte cell viability and death in

192

vitro using primary cultured hepatocytes isolated from WT and TG mice. WT and TG hepatocytes

193

were cultured in vitro and were treated with 100 ng/ml of DOX for 24 hours. Western blot analysis

194

confirmed that FoxM1 protein was induced in TG hepatocytes treated with DOX (Figure 5A).

195

Consistent with in vivo data, in vitro treatment with DOX increased in Ccl2 gene expression and

196

CCL2 protein expression in the supernatant of TG hepatocytes but not in the supernatants of WT

197

hepatocytes (Figure 5B-E). The WST assay showed that DOX treatment resulted in a modest

198

reduction (0.95-fold) in cell viability of TG hepatocytes (Figure 5F and G) and the caspase 3/7

199

activity assay showed a modest increase (2.03-fold) in cell death of TG hepatocyte (Figure 5H and I).

200

Among cell cycle-related genes, gene expression of Ccnb1 and Skp2, known FoxM1-target genes,

201

increased in TG hepatocytes treated with DOX (Figure 5J-O). These in vitro data suggest that FoxM1

202

induction has a modest effect on hepatocyte death.

203

Hepatocyte-Specific CCL2 Inhibition Reduces Liver Injury in TG Mice

13

204

To investigate whether hepatocyte-derived CCL2 plays a role on the development of

205

spontaneous liver injury in TG mice, we inhibited CCL2 expression using hepatocyte-specific

206

N-acetylgalactosamine (GalNAc)-siRNA system.22 Subcutaneous administration of

207

GalNAc-conjugated siRNA against murine Ccl2 (GalNAc-siCcl2) reduced gene expression of

208

hepatic Ccl2 and serum levels of CCL2 compared with administration of GalNAc-siControl (control)

209

in TG mice after 3 days of DOX treatment, confirming that hepatocytes are the major source of

210

CCL2 production in TG mice (Figure 6A). Subcutaneous administration of GalNAc-siCcl2 also

211

reduced liver injury (Figure 6B) as shown by a reduced serum ALT levels and a reduced number of

212

TUNEL-positive hepatocytes compared with the control in TG mice after 3 days of DOX treatment

213

(Figure 6C-E). Furthermore, subcutaneous administration of GalNAc-siCcl2 reduced infiltration of

214

F4/80 positive macrophages compared with control in TG mice after 3 days of DOX treatment

215

(Figure 6F). Although TG mice did not show apparent liver fibrosis assessed by picrosirius red

216

staining after 3 days of DOX treatment (Figure 6G and H), subcutaneous administration of

217

GalNAc-siCcl2 resulted in a significant reduction of gene expression of hepatic Col1α2 compared

218

with control in TG mice after 3 days of DOX treatment (Figure 6I). Collectively, these data suggest

219

that hepatocyte-derived CCL2 contributes to liver injury in TG mice.

220

Spontaneous Liver Injury in TG Mice Occurs Through Hepatic Macrophage Recruitment Induced

221

by FoxM1 Expression

14

222

To investigate the consequence of hepatic macrophage recruitment in liver injury of TG

223

mice, we depleted the macrophages of these mice. Immunohistochemical or real-time RT-PCR

224

analysis showed that the administration of clodronate liposomes resulted in the successful depletion

225

of F4/80 positive macrophages in the livers of WT and TG mice (Figure 7A and B).

226

Macrophage-depleted TG mice exhibited lower serum ALT levels and TUNEL-positive hepatocytes

227

than PBS liposomes-treated TG mice (Figure 7C-E). These data suggest that spontaneous hepatocyte

228

death in TG mice is associated with hepatic macrophage recruitment induced by FoxM1 expression.

229

FoxM1-Dependent Liver Inflammation Leads to Hepatocarcinogenesis

230

In patients with chronic liver diseases, continuous liver inflammation and fibrosis is known

231

to lead to tumorigenesis. Therefore, we investigated whether long-term overexpression of FoxM1

232

affects hepatocarcinogenesis. At 48 weeks of age, although all TG mice treated with DOX after birth

233

were found to develop liver tumors, WT mice did not develop any tumors (Figure 8A-C).

234

Histological analysis showed that liver tumors in TG mice were hepatocellular carcinoma (HCC),

235

because tumor cells were large with hyperchromatic nuclei in compact growth pattern and did not

236

form any tubular structure (Figure 8B). In addition, liver tumors in TG mice showed increased

237

expression of Arginase-1 and Glypican-3, known makers of HCC, 23, 24 confirming that these tumors

238

are HCC (Figure 8B). The Ki-67 immunohistochemistry confirmed that liver tumors in TG mice

239

showed higher cell proliferation than the non-tumor regions of TG mice or the livers of WT mice

15

240

(Figure 8D, E). Furthermore, picrosirius red staining and increased hepatic expression of

241

fibrosis-related genes showed that the non-tumor regions of TG mice developed liver fibrosis (Figure

242

8F-H). Our data indicate that overexpression of FoxM1 in hepatocytes results in liver injury and

243

subsequent liver inflammation and fibrosis, leading to hepatocarcinogenesis.

244

Hepatic FoxM1 Is Induced in Humans and Mice With Chronic Liver Injury

245

To confirm the significance of FoxM1 in liver injury, we examined the expression of

246

FoxM1 in the liver tissues of patients with chronic liver disease, such as chronic hepatitis B, chronic

247

hepatitis C, nonalcoholic steatohepatitis, and liver cirrhosis. As we reported previously,15, 16 an

248

immunohistochemical analysis confirmed increased FoxM1expression in HCC tissues (Figure 9A).

249

This analysis also showed higher FoxM1 expression in liver tissues of chronic liver diseases

250

compared with normal liver tissues (Figure 9A). To confirm an increase in hepatic FoxM1 expression

251

during the development of chronic liver injury, we next used two murine models of chronic liver

252

injury induced by a high-fat/high-cholesterol (HFHC) diet or chronic administration of CCl4. The

253

gene expression of Foxm1 was increased as serum ALT levels were elevated in both models (Figure

254

9B). Immunohistochemical analysis also confirmed a higher hepatic expression of FoxM1 in chronic

255

liver disease tissues than in normal liver tissues (Figure 9C). Collectively, these data suggest a role of

256

FoxM1 in liver injury.

257

FoxM1 Inhibitor Reduces Liver Inflammation in a Liver Injury Model 16

258

We finally investigated whether the pharmacological inhibition of FoxM1 reduces liver

259

inflammation during the development of liver injury. For this purpose, we used a murine model of

260

liver injury fed a HFHC diet, which induced hepatic FoxM1 expression (Figure 9B and C). Mice fed

261

a HFHC diet were treated with a known FoxM1 inhibitor, thiostrepton.25, 26 The thiostrepton-treated

262

mice showed significantly lower serum ALT levels and fewer TUNEL-positive hepatocytes than the

263

control mice (Figure 10A-D). The thiostrepton-treated mice also showed significantly fewer F4/80

264

positive macrophages in the livers than the control mice (Figure 10E). Furthermore, thiostrepton

265

treatment resulted in a decrease of hepatic Ccl2 expression or serum CCL2 levels in mice with

266

HFHC diet (Figure 10F, G). On the other hand, thiostrepton treatment did not affect hepatocyte

267

proliferation in HFHC diet-fed mice (Figure 10H and I). The anti-inflammatory effect of thiostrepton

268

was also confirmed in TG mice, showing that thiostrepton treatment effectively reduced spontaneous

269

liver inflammation without significant anti-proliferation effect on hepatocytes (Figure 11A-I). These

270

findings suggest that FoxM1 inhibition may represent a therapeutic approach against liver injury.

17

271

272

DISCUSSION

Targeting liver inflammation is crucial for the development of new treatments against

273

chronic liver diseases.27, 28 Here, we identified hepatocyte FoxM1 as a key driver to trigger chronic

274

liver inflammation. Similar to the clinical course of patients with chronic liver disease,

275

hepatocyte-specific FoxM1 transgenic mice developed spontaneous liver inflammation and fibrosis,

276

leading to hepatocarcinogenesis. This FoxM1-dependent liver inflammation with hepatic

277

macrophage recruitment was associated with hepatocyte-derived chemokine CCL2 that was

278

identified as a novel direct target of FoxM1. Finally, we showed that inhibiting FoxM1 with its

279

specific inhibitor effectively reduced liver inflammation in a model of liver injury induced by HFHC

280

diet. Thus, our current study raised a possibility that targeting FoxM1 could be a novel therapeutic

281

approach for liver injury toward HCC.

282

FoxM1 has a cell-autonomous role in forcing the cell proliferation of a variety of cancers,

283

including HCC.29 Several lines of evidence demonstrated that FoxM1 is overexpressed in cancer

284

tissues compared with surrounding non-cancer tissues.7-12 However, little is known whether chronic

285

tissue damage might affect FoxM1 expression in the damaged tissues. According to previous reports,

286

FoxM1 is more expressed in the damaged lung tissues of patients with idiopathic pulmonary fibrosis

287

than those of normal patients30 and that FoxM1 expression is also increased in human gastritis tissues

288

with Helicobacter pylori infection compared with normal gastric tissues.31 In this study, we showed

289

that FoxM1 expression was induced in the damaged livers of murine models and was implicated in 18

290

human specimens of chronic liver diseases. These data indicate that FoxM1 might be involved in the

291

initial process of liver damage and inflammation prior to HCC development.

292

Chronic inflammation is a key characteristic of chronic liver disease.27, 28 The interaction

293

between hepatocytes and non-parenchymal cells via a variety of mediators such as cytokines or

294

chemokines32 has been shown to be crucial for driving liver inflammation; however, the signaling

295

pathways in hepatocytes are not yet fully understood. In this study, we showed that increased FoxM1

296

in murine livers resulted in enhanced liver inflammation. Among inflammatory mediators that were

297

increased in TG livers, CCL2 was found to have a putative FoxM1 binding site in its promoter region,

298

and in vitro experiments confirmed that FoxM1 is able to regulate CCL2 directly in hepatocytes.

299

CCL2 is a well-defined chemokine that recruits CCR2 positive monocytes/macrophages to trigger

300

liver inflammation and promote hepatocarcinogenesis.33, 34 Consistently, spontaneous liver

301

inflammation in TG mice was associated with an increased macrophage recruitment in the injured

302

livers, suggesting a novel cell-nonautonomous role of hepatocyte FoxM1 to trigger chronic liver

303

inflammation via direct regulation of inflammatory mediators and recruitment of inflammatory cells.

304

Therapeutic strategies to target FoxM1 in liver diseases, especially in HCC, has been

305

investigated with in vivo and in vitro experiments so far.14, 16 It has been shown that the inhibition of

306

FoxM1 activity effectively reduces liver tumors in a murine model of HCC.14 We recently

307

demonstrated that statins, well known cholesterol-lowering drugs, also reduce FoxM1 expression and

308

thereby induce cell death in human HCC cells.16 It has been reported that hepatocyte-specific FoxM1 19

309

knockout mice exhibit impaired liver regeneration after partial hepatectomy19 and

310

macrophage-specific FoxM1 knockout mice show delayed wound-healing response and worsened

311

liver function in a liver injury model induced by CCl4,35 implying that FoxM1 inhibition may worsen

312

liver damage and inflammation. In this study, however, we showed that thiostrepton, a

313

well-established FoxM1 inhibitor,25, 26 effectively improved liver damage and reduced liver

314

inflammation with macrophage recruitment and CCL2 expression in a murine liver injury model

315

induced by HFHC diet. Consistent with our data, a recent publication demonstrated that a FoxM1

316

inhibitor effectively reduces lung inflammation in response to house dust mite allergens in mice.36

317

These findings suggest that FoxM1 inhibitors target the cell-nonautonomous function of FoxM1

318

rather than its cell-autonomous function in vivo. Further studies will be needed to elucidate this issue.

319

In conclusion, we have demonstrated that hepatocyte FoxM1 drives inflammatory response

320

linking liver fibrosis and carcinogenesis via induction of inflammatory mediators. Because HCC

321

arises in a background of liver inflammation and fibrosis, targeting hepatocyte FoxM1 would be a

322

potential application not only for the treatment of liver injury but also for early intervention or

323

prevention toward HCC.

324

20

325

Materials and Methods All authors had access to the study data and had reviewed and approved the final

326

327

manuscript.

328

Mice

329

Albumin promoter-driven Cre recombinase (Alb-Cre) transgenic C57BL/6 mice (The

330

Jackson Laboratory, Bar Harbor, ME, USA) were crossed with TetO-GFP-FoxM1-∆N mice

331

(TetO7-FoxM1)

332

(rtTA)

333

Rosa26-LSL-rtTA/Alb-Cretg/-) and wild-type (WT, TetO7-FoxM1

334

mice were treated with low dose (DOX low dose: 0.01 mg/dL) or regular dose (DOX on: 0.2 mg/dL)

335

doxycycline hyclate (catalog D9891: Sigma-Aldrich, Saint Louis, MO, USA) dissolved in 5%

336

sucrose (catalog 30403-55, Nacalai Tesque, Kyoto, Japan) and supplied as drinking water to induce

337

hepatocyte-specific FoxM1 expression. The Alb-Cre transgene was detected using the following

338

sense (S) and antisense (AS) primers: 5′-GCGGCATGGTGCAAGTTGAAT-3′ (S) and

339

5′-CGTTCACCGGCATCAACGTTT-3′ (AS). PCR analysis was performed to detect for the

340

TetO-GFP-FoxM1-∆N

341

5′-CGACAAGCAGAAGAACGGCATC-3′ (S) and 5′-AGTAGGGAAAGTGGTCCTCAATCC-3′

342

(AS). The primers for detection of the ROSA26-LSL-rtTA transgene were as follows:

343

5′-GAGTTCTCTGCTGCCTCCTG-3′ (S) and 5′-AAGACCGCGAAGAGTTTGTC-3′ (AS). The

mice

37, 38

and Rosa26-loxP-STOP-loxP-reverse tetracycline transcriptional activator

(Rosa26-LSL-rtTA)

(Figure

transgene

1A).

with

21

Transgenic

the

(TG,

TetO7-FoxM1

tg/tg

/

tg/tg

/Rosa26-LSL-rtTA/Alb-Cre-/-)

following

primers:

344

wild-type

ROSA26

allele

was

detected

using

the

following

primers:

345

5′-GAGTTCTCTGCTGCCTCCTG-3′ (S) and 5′-CGAGGCGGATACAAGCAATA-3′ (AS). All

346

mice were maintained under specific pathogen-free conditions in automated watered and ventilated

347

cages on a 12-h light/dark cycle. All animal experiments of this study were performed with humane

348

care under approval from the Animal Care and Use Committee of Osaka University Medical School.

349

Real-Time Reverse Transcription Polymerase Chain Reaction

350

Total RNA was prepared using the QIAshredder and the RNeasy Mini kit (catalog 79656

351

and 74106, Qiagen, Hilden, Germany), and quantitative real-time reverse transcription polymerase

352

chain reaction (RT-PCR) was performed using the ReverTra Ace qPCR RT Master Mix (catalog

353

FSQ-201, Toyobo, Osaka, Japan) according to the manufacturer’s protocol. Quantitative real-time

354

RT-PCR reaction was performed with a THUNDERBIRD SYBR qPCR Mix (catalog

355

QPS-201,Toyobo) on a Quant Studio 6 Flex Real-Time PCR system (Thermo Fisher Scientific,

356

Waltham, MA, USA). The following primers used in this study were purchased from Qiagen: murine

357

Foxm1 (QT00120498), Gapdh (QT00199388), ColIα1 (QT00162204), ColIα2 (QT01055572), F4/80

358

(QT00099617),

359

(QT00167832), Ccl5 (QT01747165), Arg1 (QT00134288), Ccna2 (QT00102151), Ccnb1

360

(QT01757007) and Skp2 (QT00118573).

361

Sigma-Genosys:

362

5′-CCAAAGGTAACTGTCCTGGC-3′

Cd68

(QT00254051),

Nos2

(QT00100275),

Tnfα

(QT00104006),

Ccl2

The following primers were synthesized

5′-TCTGGGCTCACTATGCTGCA-3′ (AS) 22

for

(S) murine

by and

Ccr2;

363

5′-CAAAAACTGACTGGGCTTCC-3′ (S) and 5′-GCCCTTGATTCCAAAGAGTG-3′ (AS) for

364

Cd206. The expression values of the murine genes were normalized to the glyceraldehyde

365

3-phosphate dehydrogenase (Gapdh) level.

366

Serum Analysis

367

Blood samples were harvested from the inferior vena cava and centrifuged at 700 g for 10

368

minutes. Serum alanine aminotransferase (ALT) levels were measured using a colorimetric assay kit

369

(catalog 431-30901, Wako, Kyoto, Japan) according to the manufacturer’s protocol.

370

Immunohistochemical Analysis

371

Liver tissues were fixed overnight with 10% formaldehyde neutral buffer solution (catalog

372

37152-51, Nacalai Tesque), embedded in paraffin, and sectioned (4-µm thick). Tissue sections were

373

subjected to Mayer’s hematoxylin and eosin and immunohistochemical staining. We used the

374

following antibodies: rabbit anti-mouse FoxM1 (A-11, 1:500, catalog sc-271746, Santa Cruz

375

Biotechnology, Santa Cruz, CA, USA), rat anti-mouse F4/80 (1:100, catalog MCA497GA, Serotec,

376

Oxford, UK), rabbit anti-mouse α-smooth muscle actin (1:200, catalog ab5694, Abcam, Cambridge,

377

MA, USA), rabbit anti-mouse Ki-67(D3B5) (1:400, catalog 12202, Cell Signaling Technology,

378

Danvers, MA, USA) , goat anti-mouse arginase-1 (M-20,1:100, catalog sc-18355, Santa Cruz

379

Biotechnology), or mouse glypican-3 (F-3, 1:100, catalog sc-390587, Santa Cruz Biotechnology).

380

Overexpression of FoxM1 in hepatocytes of TG mice were assessed by immunohistochemical

381

staining with FoxM1 (D12D5) (1:200, catalog 5436, Cell Signaling Technology). Liver fibrosis was 23

382

quantified by measuring the picrosirius red (catalog 24901-500, Polysciences Inc., Warrington, PA,

383

USA) stained area using ImageJ (version 1.80, US National Institute of Health, Bethesda, MD, USA).

384

FoxM1 expression was quantified by measuring the area stained with anti-FoxM1 antibodies using

385

ImageJ software (version 1.80). Cells with nuclear DNA fragmentation were detected using a

386

transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining kit (catalog

387

S7100; Millipore, Molsheim, France) according to the manufacturer’s protocol. The numbers of

388

TUNEL or Ki-67 positive hepatocytes per view field were counted at 100× magnification.

389

Western Blot Analysis

390

Murine liver samples were homogenized in RIPA buffer containing 50 mM Tris (pH 7.5),

391

0.15 M NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 1% Nonidet P-40, phosphatase

392

inhibitor cocktail (catalog 0574-61, Nacalai Tesque), and protease inhibitor cocktail (catalog

393

25955-11, Nacalai Tesque). For Western blot analysis, the primary antibodies anti-human FoxM1

394

(D12D5) (1:1000, catalog 5436, Cell Signaling Technology) and GAPDH (1:1000, catalog 5174,

395

Cell Signaling Technology) were used. The donkey anti-rabbit immunoglobulin G–horseradish

396

peroxidase (1:3000, catalog sc-2357, Santa Cruz Biotechnology) was used as a secondary antibody.

397

Preparation of Mononuclear Cells

398

The livers of WT or TG mice were passed through a 70-µm nylon mesh to create single

399

cell suspensions. The cell suspension was collected and parenchymal cells were isolated from

400

mononuclear cells by centrifugation at 50g for 5 min. The mononuclear cells were washed in PBS 24

401

and suspended in 40% Percol (catalog 17089101, GE Healthcare Biosciences AB, Uppsala, Sweden).

402

The suspended cells were gently overlaid onto 70% Percol (GE Healthcare Biosciences AB) and

403

centrifuged at 750g for 20 min. The mononuclear cells were collected from the interface, washed

404

twice in PBS, and used for flow cytometry.

405

Flow Cytometric Analysis

406

Prepared liver mononuclear cells were suspended in a solution of PBS, 0.3% w/v bovine

407

serum albumin, and 0.1% w/v sodium azide. To avoid nonspecific bindings of antibodies to Fc

408

receptors, the cells were pretreated with anti-CD16/32 antibody (clone 2.4G2, catalog 553141, BD

409

Biosciences, Franklin Lakes, NJ, USA) for 15 minutes. Then, the cells were stained for 20 min at

410

4°C with the following antibodies: allophyocyanin-conjugated anti-mouse CD11b (clone M1/70,

411

catalog 553312, BD Biosciences) and phycoerythrin-conjugated anti-mouse F4/80 (clone T45-2342,

412

catalog 565410, BD Biosciences) for macrophages. To label dead cells, 7-amino-actinomycin D

413

(7-AAD, catalog 559925, BD Biosciences) were used. These samples were subjected to flow

414

cytometric analysis using a fluorescence-activated cell sorting Canto II system (BD Biosciences).

415

Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The number of

416

CD11b+F4/80+ macrophages in a cell subset was determined using the following calculation: total

417

liver mononuclear cell number × corresponding cell subset proportion to the total cells.

418

Enzyme-Linked Immunosorbent Assay

419

The levels of serum CCL2 and secreted CCL2 protein from cell lines were measured using 25

420

MCP-1/CCL2 mouse uncoated ELISA kit (catalog 88-7391-86, Thermo Fisher Scientific) in

421

accordance with the manufacturer’s instructions.

422

Cell Culture

423

Murine primary hepatocytes and NPCs were isolated from WT and TG mice by two-step

424

collagenase-pronase perfusion as previously described.39 Briefly, the murine liver was perfused with

425

Liver Perfusion Medium (catalog 17701038; Thermo Fisher Scientific) for 5 minutes to remove

426

blood. For digestion of murine liver, the tissue was perfused with 53% pronase solution and 0.27%

427

collagenase solution for 1 and 5 minutes. All of the solutions were warmed to 37°C before use and

428

perfused at a flow rate of 4ml/min. The perfused murine liver was transferred into Dulbecco’s

429

modified Eagle’s medium containing 10% fetal calf serum and minced to release the hepatocytes.

430

The suspension was filtered through a cell strainer with a pore size of 70µm (catalog 352350,

431

Corning, NY, USA) and was washed three times with centrifugation at 50g for 1 minutes at 4°C.

432

After collecting the supernatant, the cell pellet was resuspended in William’s medium E with 10%

433

fetal calf serum. In a select experiment, the NPCs in the supernatant were pelleted at 400g for 5

434

minutes and subjected to Western blot and real-time RT-PCR analysis. Hepatocytes (25000 cells/cm2)

435

were seeded on type I collagen-coated microplate (catalog 4820-010, Iwaki Glass, Shizuoka, Japan)

436

in William’s medium E with 10% fetal calf serum, 2 mM L-glutamine, 10-7 M insulin, 10-7 M

437

dexamethasone, 100U/ml penicillin and 100U/ml streptomycin. Isolated hepatocytes with >90%

438

viability, determined by trypan blue exclusion, were cultured overnight. To induce FoxM1 protein 26

439

expression, primary hepatocytes isolated from WT and TG mice were treated with DOX for 24h.

440

After treating with DOX for 24 hours, WST assay and caspase-3/7 activity were measured in primary

441

hepatocytes of WT and TG mice using Cell count Reagent SF (catalog 07553-44, Nacalai Tesque)

442

and a luminescent substrate assay (Caspase-Glo assay, catalog G8093, Promega, Madison, WI, USA)

443

according to the manufacturer’s instructions. The murine non-transformed hepatocyte cell lines,

444

BNL-CL2 and AML12 were obtained from the American Type Culture Collection (Manassas, VA,

445

USA). For the small RNA interference assay, cells were transfected with either 20 nM murine Foxm1

446

siRNA (catalog MSS204338, Thermo Fisher Scientific) or control siRNA (catalog 12935112,

447

Thermo Fisher Scientific) using Lipofectamine RNAiMAX (catalog 13778150, Invitrogen

448

Corporation, Carlsbad, CA, USA) according to the manufacturer’s instructions.

449

Dual Luciferase Assay

450

The promoter sequences for murine Ccl2 genes were obtained from the National Center for

451

Biotechnology Information database. Murine Ccl2 promoter fragments were amplified by PCR of

452

murine

453

5′-TCCGGCCCATGAGAGAACTGCTT-3′

454

5′-ACTATGCCTGGCTCCTGGTA-3′ (S2, -1401/-1381), 5′-GAAGACTCCGCTCAGCCAC-3′ (S3,

455

-1136/-1117) and 5′-TGGCTTCAGTGAGAGTTGGCTGGT-3′ (AS, +67/+44). The Ccl2 promoter

456

nucleotide sequence was confirmed by DNA sequencing and cloned into a pGL3 basic luciferase

457

vector (Promega, Madison, WI, USA) to generate a reporter plasmid. We transfected BNL-CL2 with

genomic

DNA

using

following

sense

(S)

and (S1,

27

antisense

(AS)

primers:

-2468/-2445),

458

either cytomegalovirus promoter (CMV)-T7-tagged FoxM1 plasmid (CMV-T7-FoxM1) or

459

CMV-empty expression plasmid, as well as with luciferase reporters driven by the murine Ccl2

460

promoter lesions. After seeding cells and culturing them overnight, the plasmid was transfected with

461

FuGENE HD transfection reagent (catalog E2311; Promega) according to the manufacturer’s

462

instructions. We used the 6×CDX2-Luc plasmid, a known FoxM1 reporter, as a positive control for

463

CMV-T7-FoxM1 transcriptional activity. The CMV-Renilla luciferase plasmid was used as internal

464

controls to normalize transfection efficiency. A dual luciferase assay (Promega) was performed 48 h

465

after transfection, as described previously.38

466

Chromatin Immunoprecipitation Assay

467

BNL-CL2 cells were prepared using the SimpleChIP Plus Enzymatic Chromatin IP kit

468

(catalog 9005; Cell Signaling Technology). Nuclear extracts from BNL-CL2 cells were cross-linked

469

by the addition of formaldehyde, sonicated, and used for the immunoprecipitation with FoxM1 rabbit

470

polyclonal antibodies (K-19 and C-20) (catalog sc-500, sc-502; Santa Cruz Biotechnology). Reverse

471

cross-linked chromatin immunoprecipitation DNA samples were subjected to real-time PCR using

472

the

473

5′-TCAGGTCCAGGGAAGCATTCTG-3′ (S) and 5′-TGTGTTTATTCCATGGCAAGTGGTC-3′

474

(AS).

475

Synthesis of the triantennary N-acetylgalactosamine (GalNAc)-siRNA

476

oligonucleotides

specific

to

promoter

regions

of

the

murine

Ccl2

gene:

GalNAc-siRNA conjugates were synthesized by Nihon Gene Research Laboratories Inc., 28

477

as described previously.22 The target sequences for GalNAc-siCcl2 and GalNAc-siControl (control)

478

were 5'-TGAATGAGTAGCAGCAGGTGAGTGG-3' and 5'-GGTCTGATCACTGCTCGGT-3'.40

479

The designed triantennary GalNAc-siCcl2 and control are shown in Table 1. TG mice were injected

480

subcutaneously with 3mg/kg of GalNAc-siCcl2 or control 24 hours before DOX treatment, as

481

described previously.41

482

Depletion of Macrophages

483

Clodronate liposomes were prepared as previously described.42 To deplete macrophages,

484

the mice were injected intravenously with either 50 mg/kg of body weight clodronate liposomes or

485

PBS liposomes from 3 days before the experiment. Macrophage depletion in the livers was verified

486

using F4/80 immunostaining three days after intravenous injection.

487

Induction of Chronic Liver Injury

488

To induce chronic liver injury, C57BL/6 mice were subjected to a high-fat,

489

high-cholesterol (HFHC) diet (catalog D09100308, Research Diet, New Brunswick, NJ, USA) for

490

eight weeks or an intraperitoneal administration of carbon tetrachloride (CCl4; 0.5 ml/kg body wt)

491

two times a week for four weeks. The mice were killed at 48 h after final injection.

492

Inhibition of FoxM1 Activity

493

To inhibit FoxM1 activity, we used thiostrepton (catalog T8902, Sigma-Aldrich), a FoxM1

494

inhibitor. 25, 26 In a chronic liver injury model, the mice were injected intraperitoneally with either 30

495

mg/kg bw thiostrepton dissolved in 10% dimethyl sulfoxide (DMSO/PBS) or vehicle as a control for 29

43

496

a total of 7 injections over 2 weeks, as described previously.

TG mice were injected

497

intraperitoneally with thiostrepton or vehicle for two times from 3 days before DOX treatment.

498

Clinical Samples

499

Tumor and non-tumor liver tissues were obtained from patients who underwent surgical

500

liver resection from primary liver tumors. This study was performed according to the ethical

501

guidelines of the Declaration of Helsinki. Approval to use resected samples was obtained from the

502

Institutional Review Board Committee at Osaka University Hospital (No. 13556), and written

503

informed consent was obtained from all patients.

504

Statistical Analysis

505

Statistical analysis was performed using JMP Pro 13.0 software (SAS Institute, Cary, NC,

506

USA). Individual values (symbols) and means (bar) ± standard deviations (SD; error bars) from at

507

least 3 independent experiments are shown. The Student's t test was used to analyze the significant

508

difference between the two groups and one-way analysis of variance (one-way ANOVA) test was

509

performed to analyze the difference among groups. P values less than 0.05 were considered to be

510

statistically significant.

511

30

512

REFERENCES

513

1.

El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011;365(12):1118-27.

514

2.

El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 2012;142(6):1264-73 e1.

515

516

3.

research 2011;21(1):159-68.

517

518

4.

Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer: coming of age. Nature reviews Immunology 2018;18(5):309-24.

519

520

He G, Karin M. NF-kappaB and STAT3 - key players in liver inflammation and cancer. Cell

5.

Costa RH, Kalinichenko VV, Holterman AX, Wang X. Transcription factors in liver

521

development, differentiation, and regeneration. Hepatology (Baltimore, Md)

522

2003;38(6):1331-47.

523

6.

Costa RH. FoxM1 dances with mitosis. Nature cell biology 2005;7(2):108-10.

524

7.

Kalin TV, Ustiyan V, Kalinichenko VV. Multiple faces of FoxM1 transcription factor: lessons from transgenic mouse models. Cell cycle (Georgetown, Tex) 2011;10(3):396-405.

525

526

8.

Pilarsky C, Wenzig M, Specht T, Saeger HD, Grutzmann R. Identification and validation of

527

commonly overexpressed genes in solid tumors by comparison of microarray data. Neoplasia

528

2004;6(6):744-50.

529

530

9.

Kim IM, Ackerson T, Ramakrishna S, Tretiakova M, Wang IC, Kalin TV, Major ML, Gusarova GA, Yoder HM, Costa RH, Kalinichenko VV. The Forkhead Box m1 transcription 31

531

factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer

532

research 2006;66(4):2153-61.

533

10.

Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, Lyubimov A,

534

Costa RH. Increased levels of the FoxM1 transcription factor accelerate development and

535

progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer

536

research 2006;66(3):1712-20.

537

11.

Yoshida Y, Wang IC, Yoder HM, Davidson NO, Costa RH. The forkhead box M1

538

transcription factor contributes to the development and growth of mouse colorectal cancer.

539

Gastroenterology 2007;132(4):1420-31.

540

12.

Wang Z, Park HJ, Carr JR, Chen YJ, Zheng Y, Li J, Tyner AL, Costa RH, Bagchi S,

541

Raychaudhuri P. FoxM1 in tumorigenicity of the neuroblastoma cells and renewal of the

542

neural progenitors. Cancer research 2011;71(12):4292-302.

543

13.

Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM, Dennewitz MB,

544

Shin B, Datta A, Raychaudhuri P, Costa RH. Foxm1b transcription factor is essential for

545

development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor

546

suppressor. Genes Dev 2004;18(7):830-50.

547

14.

Gusarova GA, Wang IC, Major ML, Kalinichenko VV, Ackerson T, Petrovic V, Costa RH. A

548

cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma

549

treatment. The Journal of clinical investigation 2007;117(1):99-111. 32

550

15.

Egawa M, Yoshida Y, Ogura S, Kurahashi T, Kizu T, Furuta K, Kamada Y, Chatani N,

551

Hamano M, Kiso S, Hikita H, Tatsumi T, Eguchi H, Nagano H, Doki Y, Mori M, Takehara T.

552

Increased expression of Forkhead box M1 transcription factor is associated with

553

clinicopathological features and confers a poor prognosis in human hepatocellular carcinoma.

554

Hepatology research : the official journal of the Japan Society of Hepatology

555

2017;47(11):1196-205.

556

16.

Ogura S, Yoshida Y, Kurahashi T, Egawa M, Furuta K, Kiso S, Kamada Y, Hikita H, Eguchi

557

H, Ogita H, Doki Y, Mori M, Tatsumi T, Takehara T. Targeting the mevalonate pathway is a

558

novel therapeutic approach to inhibit oncogenic FoxM1 transcription factor in human

559

hepatocellular carcinoma. Oncotarget 2018;9(30):21022-35.

560

17.

Xia L, Huang W, Tian D, Zhu H, Zhang Y, Hu H, Fan D, Nie Y, Wu K. Upregulated FoxM1

561

expression induced by hepatitis B virus X protein promotes tumor metastasis and indicates

562

poor prognosis in hepatitis B virus-related hepatocellular carcinoma. Journal of hepatology

563

2012;57(3):600-12.

564

18.

Ye H, Kelly TF, Samadani U, Lim L, Rubio S, Overdier DG, Roebuck KA, Costa RH.

565

Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and

566

mesenchymal cells of embryonic and adult tissues. Mol Cell Biol 1997;17(3):1626-41.

567

568

19.

Wang X, Kiyokawa H, Dennewitz MB, Costa RH. The Forkhead Box m1b transcription factor is essential for hepatocyte DNA replication and mitosis during mouse liver 33

569

regeneration. Proceedings of the National Academy of Sciences of the United States of

570

America 2002;99(26):16881-6.

571

20.

Wang X, Hung NJ, Costa RH. Earlier expression of the transcription factor HFH-11B

572

diminishes induction of p21(CIP1/WAF1) levels and accelerates mouse hepatocyte entry into

573

S-phase following carbon tetrachloride liver injury. Hepatology (Baltimore, Md)

574

2001;33(6):1404-14.

575

21.

Ren X, Shah TA, Ustiyan V, Zhang Y, Shinn J, Chen G, Whitsett JA, Kalin TV, Kalinichenko

576

VV. FOXM1 promotes allergen-induced goblet cell metaplasia and pulmonary inflammation.

577

Mol Cell Biol 2013;33(2):371-86.

578

22.

Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P,

579

Kel'in AV, Milstein S, Taneja N, O'Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME,

580

Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier

581

MA, Rajeev KG, Manoharan M. Multivalent N-acetylgalactosamine-conjugated siRNA

582

localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. Journal of the

583

American Chemical Society 2014;136(49):16958-61.

584

23.

Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a

585

novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology

586

2003;125(1):89-97.

587

24.

Timek DT, Shi J, Liu H, Lin F. Arginase-1, HepPar-1, and Glypican-3 are the most effective 34

588

panel of markers in distinguishing hepatocellular carcinoma from metastatic tumor on

589

fine-needle aspiration specimens. American journal of clinical pathology

590

2012;138(2):203-10.

591

25.

human cancer cells. PloS one 2009;4(5):e5592.

592

593

26.

27.

28.

29.

Raychaudhuri P, Park HJ. FoxM1: a master regulator of tumor metastasis. Cancer research 2011;71(13):4329-33.

600

601

Luedde T, Schwabe RF. NF-kappaB in the liver--linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2011;8(2):108-18.

598

599

Elsharkawy AM, Mann DA. Nuclear factor-kappaB and the hepatic inflammation-fibrosis-cancer axis. Hepatology (Baltimore, Md) 2007;46(2):590-7.

596

597

Hegde NS, Sanders DA, Rodriguez R, Balasubramanian S. The transcription factor FOXM1 is a cellular target of the natural product thiostrepton. Nature chemistry 2011;3(9):725-31.

594

595

Bhat UG, Halasi M, Gartel AL. Thiazole antibiotics target FoxM1 and induce apoptosis in

30.

Penke LR, Speth JM, Dommeti VL, White ES, Bergin IL, Peters-Golden M. FOXM1 is a

602

critical driver of lung fibroblast activation and fibrogenesis. The Journal of clinical

603

investigation 2018;128(6):2389-405.

604

31.

Feng Y, Wang L, Zeng J, Shen L, Liang X, Yu H, Liu S, Liu Z, Sun Y, Li W, Chen C, Jia J.

605

FoxM1 is overexpressed in Helicobacter pylori-induced gastric carcinogenesis and is

606

negatively regulated by miR-370. Molecular cancer research : MCR 2013;11(8):834-44. 35

607

32.

2014;147(3):577-94.e1.

608

609

Marra F, Tacke F. Roles for chemokines in liver disease. Gastroenterology

33.

Li X, Yao W, Yuan Y, Chen P, Li B, Li J, Chu R, Song H, Xie D, Jiang X, Wang H. Targeting

610

of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy

611

against hepatocellular carcinoma. Gut 2017;66(1):157-67.

612

34.

Teng KY, Han J, Zhang X, Hsu SH, He S, Wani NA, Barajas JM, Snyder LA, Frankel WL,

613

Caligiuri MA, Jacob ST, Yu J, Ghoshal K. Blocking the CCL2-CCR2 Axis Using

614

CCL2-Neutralizing Antibody Is an Effective Therapy for Hepatocellular Cancer in a Mouse

615

Model. Molecular cancer therapeutics 2017;16(2):312-22.

616

35.

Ren X, Zhang Y, Snyder J, Cross ER, Shah TA, Kalin TV, Kalinichenko VV. Forkhead box

617

M1 transcription factor is required for macrophage recruitment during liver repair. Mol Cell

618

Biol 2010;30(22):5381-93.

619

36.

Sun L, Ren X, Wang IC, Pradhan A, Zhang Y, Flood HM, Han B, Whitsett JA, Kalin TV,

620

Kalinichenko VV. The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and

621

prevents IL-13 and STAT6 signaling in allergen-exposed mice. Science signaling

622

2017;10(475).

623

37.

Wang IC, Zhang Y, Snyder J, Sutherland MJ, Burhans MS, Shannon JM, Park HJ, Whitsett

624

JA, Kalinichenko VV. Increased expression of FoxM1 transcription factor in respiratory

625

epithelium inhibits lung sacculation and causes Clara cell hyperplasia. Dev Biol 36

2010;347(2):301-14.

626

627

38.

Balli D, Ustiyan V, Zhang Y, Wang IC, Masino AJ, Ren X, Whitsett JA, Kalinichenko VV,

628

Kalin TV. Foxm1 transcription factor is required for lung fibrosis and

629

epithelial-to-mesenchymal transition. EMBO J 2013;32(2):231-44.

630

39.

Furuta K, Yoshida Y, Ogura S, Kurahashi T, Kizu T, Maeda S, Egawa M, Chatani N, Nishida

631

K, Nakaoka Y, Kiso S, Kamada Y, Takehara T. Gab1 adaptor protein acts as a gatekeeper to

632

balance hepatocyte death and proliferation during acetaminophen-induced liver injury in mice.

633

Hepatology (Baltimore, Md) 2016;63(4):1340-55.

634

40.

Taniuchi K, Cerny RL, Tanouchi A, Kohno K, Kotani N, Honke K, Saibara T, Hollingsworth

635

MA. Overexpression of GalNAc-transferase GalNAc-T3 promotes pancreatic cancer cell

636

growth. Oncogene 2011;30(49):4843-54.

637

41.

Willoughby JLS, Chan A, Sehgal A, Butler JS, Nair JK, Racie T, Shulga-Morskaya S,

638

Nguyen T, Qian K, Yucius K, Charisse K, van Berkel TJC, Manoharan M, Rajeev KG, Maier

639

MA, Jadhav V, Zimmermann TS. Evaluation of GalNAc-siRNA Conjugate Activity in

640

Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

641

Molecular therapy : the journal of the American Society of Gene Therapy 2018;26(1):105-14.

642

42.

Miura K, Yang L, van Rooijen N, Ohnishi H, Seki E. Hepatic recruitment of macrophages

643

promotes nonalcoholic steatohepatitis through CCR2. American journal of physiology

644

Gastrointestinal and liver physiology 2012;302(11):G1310-21. 37

645

43.

Weiler SME, Pinna F, Wolf T, Lutz T, Geldiyev A, Sticht C, Knaub M, Thomann S, Bissinger

646

M, Wan S, Rossler S, Becker D, Gretz N, Lang H, Bergmann F, Ustiyan V, Kalin TV, Singer

647

S, Lee JS, Marquardt JU, Schirmacher P, Kalinichenko VV, Breuhahn K. Induction of

648

Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in

649

Liver Cancer. Gastroenterology 2017;152(8):2037-51.e22.

650

651

38

652

Table 1. Designs of the GalNAc-siRNA conjugates.

653

Compound

Strand

Sequence (5' to 3')

GalNAc-siCcl2

S

UGAAUGAGUAGCAGCAGGUGAGUGG-(GalNAc)

AS

CCACUCACCUGCUGCUACUCAUUCATT

S

GGUCUGAUCACUGCUCGGU-(GalNAc)

AS

ACCGAGCAGUGAUCAGACCTT

GalNAc-siControl

654 655

S, sense strand; AS, antisense strand; GalNAc, triantennary N-acetylagalactosamine.

656

657

39

658

FIGURE LEGENDS

659

Figure 1. Spontaneous liver injury and fibrosis in hepatocyte-specific FoxM1 transgenic mice.

660

(A) Schematic illustration of strategy for generating hepatocyte-specific FoxM1 transgenic (TG)

661

mice. WT, wild-type. (B) Western blot analysis showing FoxM1 protein expression in the liver,

662

kidney, and heart of WT and TG mice after 3 days of doxycycline (DOX) treatment at 8 weeks.

663

FoxM1 protein expression was liver- and DOX-specific in the TG mice. (C) Representative images

664

of FoxM1 staining in liver sections of WT and TG mice after 3 days of DOX treatment at 8 weeks.

665

Scale bar: 100 µm (original magnification, ×200). (D) Quantification of FoxM1-positive area in liver

666

sections of WT and TG mice after 3 days of DOX treatment at 8 weeks (WT, n=8; TG, n=8). (E)

667

Serum ALT levels in WT and TG mice after 13 weeks of DOX treatment since birth (WT, n = 8; TG,

668

n = 8). (F) Representative images of H&E (top) and TUNEL (bottom) staining in liver sections of

669

WT and TG mice after 13 weeks of DOX treatment since birth. Scale bar: 200 µm (original

670

magnification, ×100). (G) Quantification of the number of TUNEL-positive hepatocytes in liver

671

sections of WT and TG mice after 13 weeks of DOX treatment since birth (WT, n = 8; TG, n = 8).

672

(H) Representative views of picrosirius red (top) and α-smooth muscle actin (bottom) staining in

673

liver sections of WT and TG mice after 13 weeks of DOX treatment since birth. Scale bar: 200 µm

674

(original magnification, ×100). (I) Quantification of picrosirius red positive area in the livers of WT

675

and TG mice after 13 weeks of DOX treatment since birth (WT, n = 8; TG, n = 8). (J) Gene

676

expression of ColIα1 and ColIα2 in the livers of WT and TG mice after 13 weeks of DOX treatment 40

677

since birth (WT, n = 8; TG, n = 8). Data are expressed as individual values and mean ± SD, **p <

678

0.01. Significance was calculated using an unpaired Student t test.

679

Figure 2. Reversible liver inflammation with macrophage recruitment induced by short-term

680

hepatocyte-specific overexpression of FoxM1. (A) Schematic illustration of short-term hepatic

681

overexpression of FoxM1. Hepatic overexpression of FoxM1 was induced by three days of

682

doxycycline (DOX) treatment (DOX on) and repressed by three subsequent days of DOX removal

683

(DOX off). Pretreatment is denoted as DOX (-). (B) Western blot analysis showing FoxM1 protein

684

expression in the livers of wild-type (WT) and transgenic (TG) mice at indicated time points. (C)

685

Serum ALT levels of WT and TG mice at indicated time points (WT, n = 8 at 8 wk; TG, n = 8 at 8

686

wk). (D) Representative images of TUNEL staining in liver sections of WT and TG mice. Top,

687

DOX(-); middle, DOX on; bottom, DOX off. Scale bar: 200 µm (original magnification, ×100). (E)

688

Quantification of the number of TUNEL-positive hepatocytes in liver sections of WT and TG mice at

689

indicated time points (WT, n = 8 at 8 wk; TG, n = 8 at 8 wk). (F) Representative images of H&E

690

(left) and F4/80 (right) staining in liver sections of WT and TG mice. Top, DOX (-); second, DOX

691

on; third, DOX on (enlarged view of boxed region of second panel); bottom, DOX off. Scale bar:

692

100 µm (original magnification, ×200). (G) Quantification of the number of CD11b+F4/80+

693

macrophages in the livers of WT and TG mice at indicated time points (WT, n = 3 at 8 wk; TG, n = 3

694

at 8 wk). (H) Quantification of hepatic gene expression of F4/80, Cd68, Nos2, Tnfα, Ccl2, Ccr2,

695

Ccl5, Arg1, and Cd206 in WT and TG mice after 3 days of DOX treatment (WT, n = 8 at 8 wk; TG, n 41

696

= 8 at 8 wk). (I) Quantification of serum CCL2 levels in WT and TG mice at indicated time points

697

(WT, n = 8 at 8 wk; TG, n = 8 at 8 wk). (J) Western blot analysis showing FoxM1 protein expression

698

in the livers of WT and TG mice after 1 day of DOX treatment. (K) Serum ALT levels of WT and TG

699

mice after 1 day of DOX treatment (WT, n = 8 at 8 wk; TG, n = 8 at 8 wk). (L) Gene expression of

700

Ccl2 (left) and serum CCL2 (right) of WT and TG mice after 1 day of DOX treatment (WT, n = 8 at

701

8 wk; TG, n = 8 at 8 wk). (M) Representative images of FoxM1 (top) and TUNEL (bottom) staining

702

in liver sections of TG mice. Left, DOX (-); middle, DOX low dose; right, DOX on. (N)

703

Quantification of FoxM1-positive area in liver sections of TG mice. DOX (-), n=8; DOX low dose,

704

n=6; DOX on, n=8 at 8 wk. (O) Quantification of the number of TUNEL-positive hepatocytes in

705

liver sections of TG mice. DOX (-), n=8; DOX low dose, n=6; DOX on, n=8 at 8 wk. (P) Serum ALT

706

levels of TG mice. DOX (-), n=8; DOX low dose, n=6; DOX on, n=8 at 8 wk. (Q) Ccl2 gene

707

expression in livers of TG mice. DOX (-), n=8; DOX low dose, n=6; DOX on, n=8 at 8 wk. Data are

708

expressed as individual values and mean ± SD, **p < 0.01. Significance was calculated using

709

one-way ANOVA test (C, E, G, I, N, O, P, Q), or Student t test (H, K, L).

710

Figure 3. Direct regulation of CCL2 by FoxM1 in hepatocytes. (A) Quantification of gene

711

expression of FoxM1 (left) and Ccl2 (right) in FoxM1 siRNA-transfected BNL-CL2 cells (n = 3 per

712

group). (B) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected BNL-CL2

713

cells at indicated time points after transfection (n = 3 per group). (C) Quantification of gene

714

expression of FoxM1 (left) and Ccl2 (right) in FoxM1 siRNA-transfected AML12 cells (n = 3 per 42

715

group). (D) Quantification of CCL2 levels in supernatant of FoxM1 siRNA-transfected AML12 cells

716

at indicated time points after transfection (n = 3 per group).

717

(Luc) reporter constructs containing −2468/+67 bp murine Ccl2 promoter and its deletion mutants

718

(−1401/+67 bp and −1136/+67 bp) and quantification of transcriptional activities induced by

719

co-transfection of T7-FoxM1 expression vector (T7-FoxM1) compared with CMV-empty vector

720

(Mock) (n=3 per group). A 6xCDX2 promoter LUC construct was used as a positive control. (F)

721

Quantification of chromatin immunoprecipitation assay to show direct binding of FoxM1 protein to

722

murine Ccl2 promoter DNA using two independent antibodies against FoxM1 (K19 and C20)

723

compared with IgG control (n = 3 per group). Data are expressed as individual values and mean ±

724

SD, **p < 0.01. Significance was calculated using an unpaired Student t test.

725

Figure 4. Hepatocytes are the major source of CCL2 production in TG mice. (A) Quantification

726

of Ccl2 gene expression in hepatocytes (left) and nonparenchymal cells (NPCs; right) from WT and

727

TG mice after 3 days of DOX treatment (n=3, each group). (B) Western blot analysis of FoxM1

728

protein expression in hepatocytes and NPCs of WT and TG mice.

729

Figure 5. FoxM1 induction has a modest effect on cell death in cultured hepatocytes in vitro.

730

(A) Western blot analysis showing FoxM1 protein expression in isolated primary hepatocytes of TG

731

mice treated with 100 ng/ml doxycycline (DOX). (B and C) Quantification of gene expression of

732

Ccl2 in primary hepatocytes from WT (B) and TG (C) mice treated with 100 ng/ml DOX (n = 4 per

43

(E) Schematic illustration of luciferase

733

group). (D and E) Quantification of CCL2 levels in supernatant of primary hepatocytes from WT (D)

734

and TG (E) mice treated with 100 ng/ml DOX (n = 4 per group). (F and G) WST assay of primary

735

hepatocytes from WT (F) and TG (G) mice treated with 100 ng/ml DOX (n = 4 per group). (H and I)

736

Caspase 3/7 activity in primary hepatocytes from WT (H) and TG (I) mice treated with 100 ng/ml

737

DOX (n = 4 per group). (J and K) Quantification of gene expression of Ccna2 in primary

738

hepatocytes from WT (J) and TG (K) mice treated with 100 ng/ml DOX (n = 4 per group). (L and M)

739

Quantification of gene expression of Ccnb1 in primary hepatocytes from WT (L) and TG (M) mice

740

treated with 100 ng/ml DOX (n = 4 per group). (N and O) Quantification of gene expression of Skp2

741

in primary hepatocytes of WT (N) and TG (O) mice treated with 100 ng/ml DOX (n = 4 per group).

742

Data are expressed as individual values and mean ± SD, * p < 0.05, **p < 0.01. Significance was

743

calculated using an unpaired Student t test.

744

Figure 6. Hepatocyte-specific CCL2 inhibition reduces liver injury in TG mice. (A)

745

Quantification of hepatic gene expression of Ccl2 (left) and serum CCL2 levels (right) in TG mice

746

treated with GalNAc-siControl (control) or GalNAc-siCcl2 (control, n = 4 at 8 wk; GalNAc-siCcl2,

747

n = 4 at 8 wk). (B) Representative images of H&E staining of TG mice treated with control or

748

GalNAc-siCcl2. Scale bar: 200 µm (original magnification, ×100). (C) Serum ALT levels of TG

749

mice treated with control or GalNAc-siCcl2 (control, n = 4 at 8 wk; GalNAc-siCcl2, n = 4 at 8 wk).

750

(D) Representative images of TUNEL staining of TG mice treated with control or GalNAc-siCcl2.

751

Scale bar: 200 µm (original magnification, ×100). (E) Quantification of the number of 44

752

TUNEL-positive hepatocytes in liver sections from control and GalNAc-siCcl2 group mice (control,

753

n = 4 at 8 wk; GalNAc-siCcl2, n = 4 at 8 wk). (F) Representative images of F4/80 staining of TG

754

mice treated with control or GalNAc-siCcl2 (bottom, enlarged views of the boxed regions in the top

755

image). Scale bar: 100 µm (original magnification, ×200). (G) Representative images of picrosirius

756

red staining in liver sections from TG mice treated with control or GalNAc-siCcl2. Scale bar: 200

757

µm (original magnification, ×100). (H) Quantification of picrosirius red positive area (%) in the

758

livers of control and GalNAc-siCcl2 group mice (control, n = 4 at 8 wk; GalNAc-siCcl2, n = 4 at 8

759

wk). (I) Gene expression of ColIα1 (left) and ColIα2 (right) in the livers of control and

760

GalNAc-siCcl2 group mice (control, n = 4 at 8 wk; GalNAc-siCcl2, n = 4 at 8 wk). Data are

761

expressed as individual values and mean ± SD, **p < 0.01. Significance was calculated using an

762

unpaired Student t test.

763

Figure 7. Macrophage recruitment is required for spontaneous liver inflammation in

764

transgenic mice. (A) Representative images of F4/80 staining in liver sections of 3-day DOX-treated

765

wild-type (WT) and transgenic (TG) mice injected with PBS or clodronate liposomes. Top, WT

766

mice; middle, TG mice; bottom, enlarged views of boxed region of middle panels. Scale bar: 200 µm

767

(original magnification, ×100). (B) Quantification of hepatic gene expression of F4/80 (left) and

768

Cd68 (right) in 3-day DOX-treated WT and TG mice injected with PBS or clodronate liposomes.

769

(PBS-treated WT mice, n = 4 at 8 wk; PBS-treated TG mice, n = 4 at 8 wk; clodronate

770

liposome–treated WT mice, n = 4 at 8 wk; clodronate liposome–treated WT mice, n = 4 at 8 wk). (C) 45

771

Quantification of serum ALT levels in 3 days DOX-treated WT and TG mice injected with PBS or

772

clodronate liposomes (PBS-treated WT mice, n = 4 at 8 wk; PBS-treated TG mice, n = 4 at 8 wk;

773

clodronate liposome–treated WT mice, n = 4 at 8 wk; clodronate liposome–treated WT mice, n = 4 at

774

8 wk). (D) Representative images of TUNEL staining in liver sections of 3-day DOX-treated WT and

775

TG mice injected with PBS or clodronate liposomes. Scale bar: 200 µm (original magnification,

776

×100). (E) Quantification of the number of TUNEL-positive hepatocytes in liver sections of 3-day

777

DOX-treated WT and TG mice injected with PBS or clodronate liposomes (PBS-treated WT mice, n

778

= 4 at 8 wk; PBS-treated TG mice, n = 4 at 8 wk; clodronate liposome–treated WT mice, n = 4 at 8

779

wk; clodronate liposome–treated WT mice, n = 4 at 8 wk). Data are expressed as individual values

780

and mean ± SD, ** p < 0.01. Significance was calculated using one-way ANOVA test.

781

Figure 8. Long-term hepatocyte-specific overexpression of FoxM1 causes hepatocarcinogenesis

782

with significant fibrosis in the background liver. (A) Gross appearance of livers in wild-type (WT)

783

and transgenic (TG) mice after 12 months of doxycycline (DOX) treatment since birth. Arrowheads

784

indicate liver tumors in TG mice. (B) Representative images of H&E (top), Arginase-1 (middle) and

785

Glypican-3 (bottom) staining of WT and TG mice after 12 months of DOX treatment since birth.

786

Non-tumor (NT) lesions in WT mice (left), non-tumor lesions in TG mice, denoted as TG(NT)

787

(middle), and tumor lesions in TG mice, which are denoted as TG(T) (right). Scale bar: 100 µm

788

(original magnification, ×400). (C) Quantification of tumor numbers (left), maximal size (mm,

789

middle), tumor incidence (%, right) in WT and TG mice after 12 months of DOX treatment since 46

790

birth (WT mice, n = 10; TG mice, n = 10). (D) Representative images of Ki-67 staining of WT and

791

TG mice after 12 months of DOX treatment since birth. Scale bar: 200 µm (original magnification,

792

×100). (E) Quantification of the number of Ki-67 positive hepatocytes in liver sections of NT

793

lesions in WT mice, NT lesions in TG mice, denoted as TG(NT), and tumor lesions in TG mice,

794

which are denoted as TG(T). (WT mice, n = 10; TG mice, n = 10). (F) Representative images of

795

picrosirius red staining of WT and TG mice after 12 months of DOX treatment since birth. NT,

796

non-tumor, T, tumor. Scale bar: 200 µm (original magnification, ×100). (G) Quantification of

797

picrosirius red positive area (%) in liver sections of WT and TG mice (WT mice, n = 10; TG mice, n

798

= 10). (H) Quantification of ColIα1 and ColIα2 gene expression in the livers of WT and TG mice

799

(WT mice, n = 10; TG mice, n = 10). Data are expressed as individual values and mean ± SD, NT,

800

non-tumor; T, tumor. * p < 0.05, ** p < 0.01. Significance was calculated using one-way ANOVA

801

test (E), or Student t test (C, G, H).

802

Figure 9. Increased expression of FoxM1 in liver sections of patients with chronic liver diseases

803

and murine model of liver injury. (A) Representative images of FoxM1 staining in liver sections of

804

patients with chronic liver diseases. Scale bar: 100 µm (original magnification, ×400).

805

Quantification of the FoxM1-positive area was indicated on the lower left. NL, normal liver; CHB,

806

chronic hepatitis B; CHC, chronic hepatitis C; NASH, nonalcoholic steatohepatitis; LC, liver

807

cirrhosis; HCC, hepatocellular carcinoma. (B) Quantification of hepatic FoxM1 expression (left) and

808

serum ALT levels (right) in C57BL/6 mice fed a high-fat and high-cholesterol (HFHC) diet for 8 47

809

weeks or treated with carbon tetrachloride (CCl4) for 4 weeks. Mice fed a normal diet (ND) were

810

used as the control (ND, n = 6 at 14 wk; HFHC, n = 6 at 14 wk; CCl4, n = 4 at 14 wk). (C)

811

Representative images of FoxM1 staining (left) and quantification of FoxM1-positive area (right) in

812

liver sections of C57BL/6 mice fed a ND or a HFHC diet or treated with CCl4. ND-fed mice were

813

used as the control. Scale bar: 100 µm (original magnification, ×200). Quantification positive rate

814

was indicated on the lower left. Data are expressed as individual values and mean ± SD, **p < 0.01.

815

Significance was calculated using an unpaired Student t test.

816

Figure 10. FoxM1 inhibitor reduces liver inflammation in mice fed a high-fat, high-cholesterol

817

diet. (A) Serum ALT levels of C57BL/6 mice fed a high-fat, high-cholesterol diet for 8 weeks and

818

treated with vehicle (control) or thiostrepton for 2 weeks (control, n = 8 at 14 wk; thiostrepton, n = 8

819

at 14 wk). (B) Representative images of H&E staining of control and thiostrepton groups. Scale bar:

820

200 µm (original magnification, ×100). (C) Representative images of TUNEL staining of control

821

and thiostrepton groups. Scale bar: 200 µm (original magnification, ×100). (D) Quantification of the

822

number of TUNEL-positive hepatocytes in liver sections of control and thiostrepton group (control, n

823

= 8; thiostrepton, n = 8). (E) Representative images of F4/80 staining of C57BL/6 mice treated with

824

vehicle or thiostrepton (bottom, enlarged views of top boxed regions). Scale bar: 100 µm (original

825

magnification, ×200). (F) Quantification of F4/80 and Ccl2 gene expression in the livers of control

826

and thiostrepton group (control, n = 8; thiostrepton, n = 8). (G) Serum CCL2 levels of control and

827

thiostrepton group (control, n = 8; thiostrepton, n = 8). (H) Representative images of Ki-67 staining 48

828

in control and thiostrepton group (control, n = 8; thiostrepton, n = 8). Scale bar: 100 µm (original

829

magnification, ×200). (I) Quantification of the number of Ki-67 positive hepatocytes in liver sections

830

of control and thiostrepton group (control, n = 8; thiostrepton, n = 8). Data are expressed as

831

individual values and mean ± SD, ** p < 0.01. Significance was calculated using an unpaired

832

Student t test.

833

Figure 11. FoxM1 inhibitor reduces liver inflammation in transgenic mice. (A) Serum ALT

834

levels of TG mice treated with vehicle (control) or thiostrepton every 3 days from 3 days before the

835

experiment (control, n = 8 at 8 wk; thiostrepton, n = 8 at 8 wk). Hepatic overexpression of FoxM1

836

was induced by 3 days of doxycycline treatment. (B) Representative images of H&E staining of TG

837

mice treated with vehicle or thiostrepton. Scale bar: 100 µm (original magnification, ×200). (C)

838

Representative images of TUNEL staining of TG mice treated with vehicle or thiostrepton. Scale bar:

839

200 µm (original magnification, ×100). (D) Quantification of the number of TUNEL-positive

840

hepatocytes in liver sections of control and thiostrepton group (control, n = 8 at 8 wk; thiostrepton, n

841

= 8 at 8 wk). (E) Representative images of F4/80 staining of TG mice treated with vehicle or

842

thiostrepton (bottom, enlarged views of top boxed regions). Scale bar: 100 µm (original

843

magnification, ×200). (F) Quantification of hepatic gene expression of F4/80 and Ccl2 in TG mice

844

treated with vehicle or thiostrepton (control, n = 8 at 8 wk; thiostrepton, n = 8 at 8 wk). (G) Serum

845

CCL2 levels in TG mice treated with vehicle or thiostrepton (control, n = 8 at 8 wk; thiostrepton, n =

846

8 at 8 wk). (H) Representative images of Ki-67 staining in control and thiostrepton group (control, n 49

847

= 8; thiostrepton, n = 8). Scale bar: 100 µm (original magnification, ×200). (I) Quantification of the

848

number of Ki-67 positive hepatocytes in liver sections of control and thiostrepton group (control, n =

849

8; thiostrepton, n = 8). Data are expressed as individual values and mean ± SD, * p < 0.05, ** p <

850

0.01. Significance was calculated using an unpaired Student t test.

50

TetO7-FoxM1 Rosa26-LSL-rtTA

FoxM1 DOX (-)

TG mice TetO7-FoxM1 Rosa26-LSL-rtTA Alb-Cre

D

TG

DOX (+)

DOX (-)

WT

200

100

WT

40 20 0

WT

TG

G

TG

** 4 WT TG 2

0 WT

TG

WT WT TG

I WT

TG

Picrosirius red positive area (%)

H αSMA Picrosirius red

WT TG

60

** 8

4

0

WT

TG

TG

J Relative expression of mRNA /Gapdh (fold of control)

0

F

80

TUNEL-positive hepatocytes (/field)

WT TG

GAPDH **

H&E

**

GAPDH FoxM1

DOX (+)

TUNEL

Serum ALT (U/L)

E

Liver Kidney Heart WT TG WTTG WTTG

Alb-Cre

WT mice TetO7-FoxM1 Rosa26-LSL-rtTA

C

B

Rosa26-LSL-rtTA

FoxM1-positive area (%)

Figure 1 A TetO7-FoxM1

**

WT TG

40

** 20

0

Col1α1

Col1α2

Figure 2 A DOX (-) 8 weeks♂ WT or TG

DOX on

DOX(+) 3 days

DOX (-)

B

DOX off

DOX on

DOX off

FoxM1 WT

DOX(-) 3 days

GAPDH FoxM1

C

**

**

TG GAPDH

**

Serum ALT (U/L)

600 WT TG

400 200

0 DOX (-) TUNEL WT

TG

DOX off

E

DOX (-) DOX on DOX off

DOX (-)

DOX on

DOX off

**

40

** WT TG

20

0 DOX (-) H&E WT

F4/80 TG

WT

TG

DOX on

DOX off

G ** Macrophages (x106 cells / g liver)

F

** TUNEL-positive hepatocytes (/field)

D

DOX on

WT TG **

**

0.6

0.4

0.2

0

DOX (-)

DOX on DOX off

H

Relative expression of mRNA / Gapdh (fold of control)

Figure 2 **

600 400

WT TG

** 200 **

**

**

**

**

0 F4/80

Nos2

Tnfα

Ccl2

Ccr2

CcI5

Arg1

Cd206

**

** Serum CCL2 (pg/mL)

I

Cd68 **

600

WT TG

400 200

0

DOX (-)

DOX on

J

DOX off

DOX 1day WT

TG

FoxM1 GAPDH

30 20 10 0

L

DOX 1day

10

**

8 6 4 2 0

DOX 1day

200 Serum CCL2 (pg/mL)

WT TG

Ccl2 mRNA (fold of control)

Serum ALT (U/L)

K

WT TG

150 100 50 0

DOX 1day

Figure 2

DOX (-)

DOX low dose

DOX on

M FoxM1

80

**

**

60 40 20 0 DOX DOX DOX (-) low dose on

Serum ALT (U/L)

P 600

**

**

400 200

0

DOX DOX DOX (-) low dose on

Q

Ccl2 mRNA (fold of control)

O FoxM1-positive area (%)

N

TUNEL-positive hepatocytes (/field)

TUNEL

40

**

**

30 20 10 0 DOX DOX DOX (-) low dose on **

**

400

200

0 DOX DOX DOX (-) low dose on

BNL-CL2 **

BNL-CL2 **

1

** 6000 4000 2000 0 0 24 48 72 Time post transfection (h)

0 AML12 **

D

AML-12 **

AML-12

1

CCL2 (pg/mL)

Ccl2 mRNA (fold of control)

Foxm1 mRNA (fold of control)

** 1

Control siFoxm1

**

Ccl2 mRNA (fold of control)

Foxm1 mRNA (fold of control)

1

0

C

BNL-CL2

B CCL2 (pg/mL)

Figure 3 A

30000

Control siFoxm1

**

20000 10000 0

0

0

0 24 48 72 Time post transfection (h) FoxM1 (K19) FoxM1 (C20) IgG

F Mock T7-FoxM1

FoxM1 binding site

+67

-1401

**

Luc +67 Luc -1136

+67 **

6xCDX2 promoter 0

2

4

6

8

Luciferase fold induction

Relative ratio to IgG Control

Luc -2468

** **

**

Mouse Ccl2 promoter construct

E

2

1

0 Ccl2 Control promoter primer

Ccl2 mRNA (fold of control)

Figure 4 12 A

B

** WT TG

8

Hepatocytes WT FoxM1 GAPDH

4

0

Hepatocytes

NPCs

TG

NPCs WT

TG

Figure 5 A

WT

DOX (-)

DOX (+)

TG DOX DOX (-) (+)

FoxM1 GAPDH

B

C

WT

TG **

Ccl2 mRNA (fold of control)

Ccl2 mRNA (fold of control)

DOX(-) DOX(+)

1.2 0.8 0.4

3 2 1 0

0 WT

D

DOX(-) DOX(+)

TG **

E DOX(-) DOX(+)

4000

2000

CCL2 (pg/mL)

CCL2 (pg/mL)

12000

4000

0

0 WT *

F

G

TG * 1

DOX(-) DOX(+)

WST assay (fold of control)

WST assay (fold of control)

1

0

DOX(-) DOX(+)

8000

DOX(-) DOX(+)

0

Skp2 mRNA (fold of control)

L 0 WT

1

N

6

4

2

0 DOX(-) DOX(+)

0 WT

2

WT

DOX(-) DOX(+) Ccna2 mRNA (fold of control)

Caspase 3/7 activity (fold of control) 1.2

DOX(-) DOX(+)

1 Ccnb1 mRNA (fold of control)

Ccna2 mRNA (fold of control)

J 1.6

DOX(-) DOX(+)

0.8

0.4 Caspase 3/7 activity (fold of control)

WT

Skp2 mRNA (fold of control)

Ccnb1 mRNA (fold of control)

Figure 5 H I

K

M

0

8 TG **

2

O

12

8

4

0

DOX(-) DOX(+)

1

0 TG

1 DOX(-) DOX(+)

0 TG **

** 12

8 DOX(-) DOX(+)

4

0

TG

*

DOX(-) DOX(+)

** 1.5 1 0.5

0

400

control GalNAc-siCcl2

200

0 GalNAc-siCcl2

C

TUNEL

GalNAc-siCcl2

F4/80

0 ** 16 12 8 4 0

control

GalNAc-siCcl2

control GalNAc-siCcl2

500

E control

**

1000

TUNEL-positive hepatocytes (/field)

D

F

1500 Serum ALT (U/L)

control

H&E

B

**

CCL2 (pg/L)

Ccl2 mRNA (fold of control)

Figure 6 A

control GalNAc-siCcl2

Picrosirius red positive area (%)

control

H

control GalNAc-siCcl2

0.6

0.4

0.2

0 2

1

0 Col1α1 Relative expression of mRNA /Gapdh (fold of control)

G

Relative expression of mRNA /Gapdh (fold of control)

Picrosirius red

Figure 6

GalNAc-siCcl2

I *

1.2

0.8

0.4

0 Col1α2

control GalNAc-siCcl2

Figure 7 A

PBS

B

Clodronate

WT TG

WT

TG

**

4

2

20

10

0

0

PBS

PBS Clodronate

**

Serum ALT (U/L)

600

D

** **

WT TG WT 400

200 TG 0 PBS Clodronate

Clodronate

E PBS

Clodronate

TUNEL-positive hepatocytes (/field)

C

**

** Cd68 mRNA (fold of control)

F4/80 mRNA (fold of control)

**

**

**

WT TG

** **

20

10

0

**

PBS Clodronate

Figure 8 A WT

TG

WT

Glypican-3

Arginase-1

H&E

B

TG(NT)

TG(T)

2

0

D Ki-67

TG T

F

WT

TG

T

NT 12

8

4

Tumor incidence (%)

**

0

WT

NT

Picrosirius red

12

10

8

6

4

2

0 100

G

** **

50

**

12

8

4

0

Col1α2 mRNA (fold of control)

4 **

Col1α1 mRNA (fold of control)

6

Maximal size (mm)

**

Ki-67 positive hepatocytes (/field)

8

Picrosirius red positive area (%)

Tumor number

Figure 8

C WT TG

100

0

E ** *

200

150 WT

TG (NT)

TG (T)

0

H

WT TG WT TG

8

6

4

2

0 **

Figure 9 **

B

CHB

0.4%

** Serum ALT (U/L)

NL

FoxM1

Foxm1 mRNA (fold of control)

A

** 400

60

40 20

300 200 100

0

0 ND HFHC CCl4

ND HFHC CCl4

24.0%

**

CHC

C

0.7% 33.6%

31.0% Normal HFHC CCl4

LC

42.0%

HCC

36.9%

42.2%

FoxM1-positive area (%)

** 20.0%

NASH

**

60 40 20 0 ND HFHC CCl4

Figure 10 A

B

**

control

control thiostrepton

80 60

thiostrepton

H&E

Serum ALT (U/L)

100

40 20 0

D thiostrepton

TUNEL

control

TUNEL-positive hepatocytes (/field)

C

E

control

thiostrepton

**

4

control thiostrepton

3 2 1 0

F

control thiostrepton ** 3 Ccl2 mRNA (fold of control)

F4/80

F4/80 mRNA (fold of control)

1.2

0.8

0.4

0

I control

150 100 50 0

Ki-67

Serum CCL2 (pg/mL)

H thiostrepton

1

0 30 Ki-67 positive hepatocytes (/field)

**

G

2

20

10

0

control thiostrepton

Figure 11 **

A

B

1500 1000 500 0

D thiostrepton

TUNEL

control

E

control

thiostrepton

control thiostrepton

** TUNEL-positive hepatocytes (/field)

C

thiostrepton

H&E

Serum ALT (U/L)

control control thiostrepton

2000

60 40

20

0

F

control thiostrepton

1.2 0.8 0.4

* Ccl2 mRNA (fold of control)

F4/80

F4/80 mRNA (fold of control)

*

0

2500

1500 1000 500 0

1 0

control thiostrepton

I control

2000

2

thiostrepton

Ki-67 positive hepatocytes (/field)

**

H

Ki-67

Serum CCL2 (pg/mL)

G

3

2

1

0