Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 14 (1), 91-103

Heat Shock Transcription Factor 1 Is a Key Determinant of HCC Development by Regulating Hepatic Steatosis and Metabolic Syndrome

Affiliations

Heat Shock Transcription Factor 1 Is a Key Determinant of HCC Development by Regulating Hepatic Steatosis and Metabolic Syndrome

Xiongjie Jin et al. Cell Metab.

Abstract

Hepatocellular carcinoma (HCC) occurrence and progression are linked tightly to progressive hepatic metabolic syndrome associated with insulin resistance, hepatic steatosis, and chronic inflammation. Heat shock transcription factor 1 (HSF1), a major transactivator of stress proteins, increases survival by protecting cells against environmental stressors. It has been implicated in the pathogenesis of cancer, but specific mechanisms by which HSF1 supports cancer development remain elusive. We propose a pathogenic mechanism whereby HSF1 activation promotes growth of premalignant cells and HCC development by stimulating lipid biosynthesis and perpetuating chronic hepatic metabolic disease induced by carcinogens. Our work shows that inactivation of HSF1 impairs cancer progression, mitigating adverse effects of carcinogens on hepatic metabolism by enhancing insulin sensitivity and sensitizing activation of AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis and inhibitor of lipid synthesis. HSF1 is a potential target for the control of hepatic steatosis, hepatic insulin resistance, and HCC development.

Figures

Figure 1
Figure 1. Deletion of hsf1 inhibits DEN-induced HCC formation
(A) Representative macroscopic pictures of livers from wild-type (WT) and hsf1-/- mice 7 and 10 months after DEN injection. Arrowhead indicates tumor nodules. m, month. (B) Quantification of liver tumors in WT (n=15), hsf1+/- (n=7), and hsf1-/- (n=16) mice 7 months after DEN injection. Bars represent mean ± SD. Statistical significance is indicated (* p<0.05, ** p<0.01, *** p< 0.001). (C) Histological analysis (H&E) of livers from WT, hsf1+/- and hsf1-/- mice 7 months after DEN injection. Representative sections show no steatosis or HCC in hsf1-/- mice. WT and hsf1+/- mice display hepatic steatosis and HCC. Arrowheads depict HCC border (scale bar 100 μm). (D) Immunohistochemical analysis of liver sections for alpha-fetoprotein (α-FP) expression from WT and hsf1-/- mice at the indicated time points after DEN-treatment at 14 days of age (0). Positive staining is present in livers of both genotypes during early postnatal period that decreases and disappeared around 1 month of age. α-FP was prominently re-expressed within HCCs developed in DEN-treated WT mice (arrows). Western blot analysis of α-FP expression in liver extracts from WT or hsf1-/- mice at indicated time after DEN-treatment (right panel).
Figure 2
Figure 2. Comparable levels of liver damage and compensatory proliferation during early stage of DEN-induced HCC between WT and hsf1-/- mice
(A, B) Ki67 and TUNEL staining of liver sections from WT or hsf1-/- mice at the indicated time points after DEN injection (left panels). Statistical analysis of Ki67+ proliferating cells or TUNEL+ apoptotic hepatocytes per high-power-field (HPF) is presented (right panels). Scale bar, 20μm (Ki67) and 10μm (TUNEL). Bars are mean ± SD; (n=5 mice per group). (C) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Bars are mean ± SD; (n=5 mice per group). (D) Expression of cytokines in livers of DEN-treated mice by real-time RT-PCR (upper panels). Serum cytokine levels of untreated or DEN-injected mice measured by ELISA (lower panels). Bars are mean ± SD; (n=5 mice per group). For all panels statistical significance is indicated (* p<0.05, ** p<0.01, *** p<0.001). d, day and m, month. 0, WT or hsf1-/- mice at 14 days of age without DEN treatment.
Figure 3
Figure 3. Hsf1 deletion prevents chronic hepatic steatosis induced by DEN treatment
(A) Representative liver sections stained with Oil red-O at indicated times after DEN injection. Counterstained with hematoxylin. 0, indicates WT or hsf1-/- mice at 14 days of age without DEN treatment. Scale bar, 10μm. (B) Levels of triglycerides and non-esterified fatty acids (NEFA) in circulation and livers of untreated (Control) or DEN-treated mice at 7 months of age. Bars are mean ± SD; (n=5 mice per group). Statistical significance is indicated * p<0.05, ** p<0.01. (C) Levels of cholesterol in HDL or LDL/VLDL and total cholesterol in the sera of untreated (control) or DEN-treated mice at 7 months of age. Bars are mean ± SD; (n=5 mice per group). Statistical significance is indicated * p<0.05, ** p<0.01.
Figure 4
Figure 4. Loss of HSF1 protects from DEN-induced hepatic steatosis through inhibition of lipid synthesis
(A) Growth curves of untreated or DEN-treated WT and hsf1-/- mice on standard chow diet (CD). Bars are mean ± SD; (n=10 mice per group). * p<0.05 for WT-DEN-treated compare to WT-untreated and ** p<0.01 for WT-untreated compared to hsf1-/- untreated mice. (B) Weight of epididymal adipose tissue (WAT) of 6- or 10-month-old WT and hsf1-/- mice, untreated (Control) or DEN treated mice. Bars are mean ± SD; (n=10 mice per group). (C) Real-time RT-PCR analysis of indicated genes in the liver of untreated or DEN-treated 7-month-old WT or hsf1-/- mice. Data are presented as relative levels of mRNA expression. Bars are mean ± SD; (n=5 mice per group). Sequences of the primers used are indicated in Table S1. (D) Western blot analysis of liver extracts from 7-month-old untreated (-DEN, control) or DEN-treated (+DEN) WT and hsf1-/- mice using antibody against PPARγ. Quantification of protein expression level normalized to β-actin loading control, Bars are mean ± SD; (n=8 mice per group) (right panels). For panels B and C, insert in the upper corner. For all panels statistical significance is indicated (* p<0.05, ** p<0.01, *** p<0.001).
Figure 5
Figure 5. Insulin sensitivity and glucose tolerance is enhanced in hsf1 deficient mice
(A) Glucose tolerance test (GTT) in 16-hr-fasted untreated (top panels), or DEN-treated (lower panels), wild-type (WT) (●) or hsf1-/- (○) mice. Bars are mean ± SD: (n=8 mice per group). (B) Insulin tolerance test (ITT) in 16-hr-fasted untreated or DEN-treated WT (●) or hsf1-/- (○) mice, at 7 months of age. Bars are mean ± SD; (n= 8 mice per group). (C) Blood glucose in fed (Fed) or 16-hr-fasted (Fast) untreated or DEN-treated mice at 1 or 7 months of age. Bars are mean ± SD; (n= 5 mice per group). (D) Plasma insulin levels determined by ELISA in fed (Fed) or 16-hr-fasted (Fast) WT or hsf1-/- mice, untreated or DEN-treated, at the indicated ages. Bars are mean ± SD; (n= 5 mice per group). (E) mRNA levels of G6Pase and PEPCK in the liver of CD (Fed) or fasted (Fast) mice, untreated or DEN treated, at 7 months of age, Bars are mean ± SD; (n= 4 mice per group). Sequences of the primers used are indicated in Table S1. For all panels statistical significance is indicated (* p<0.05, ** p<0.01, *** p<0.001).
Figure 6
Figure 6. Enhanced insulin receptor (IR) signaling in the absence of HSF1
(A) Immunoblot analyses of basal and insulin-stimulated total and p-Y-IR, total and p-Ser IRS1, and total IRS2 levels in livers of 7-months old WT and hsf1-/- mice. Mice were fasted for 16 hrs, and injected with saline or insulin 0.75 mU/g i.p. Liver extracts prepared from individual mice 10 min after insulin injection and blots were quantified by densitometry. Level of phosphorylated protein normalized to total level was expressed as relative fold-increase to control (WT without insulin) that was arbitrarily set at 1 (100%). Bars are mean ± SD; (n= 5 mice per group). (B) Liver extracts prepared 10 min after insulin or saline injection from individual WT or hsf1-/- mice were immunoprecipitated using IRS1 antibody and processed for immunoblot analysis with antibodies to p-Tyr, IRS-1, or PI3K p85. ns, non-specific antibody (negative control) (left panel). Ratio of p-Tyr (P-Y) IRS1 to total IRS1 signal intensity is indicated. Whole liver extracts of mice in the left panel were probed with antibody to p-Tyr, IRS1, PI3K p85 or β-actin. Relative molecular sizes corresponding to p-Tyr IRS1 and p-Tyr IR are indicated (right panel). (C) Activation of AKT and mTOR signaling in the liver extracts from panel A was examined by immunoblot analysis using indicated antibodies. Quantification of blots is presented (right panels). Bars are mean ± SD; (n= 5 mice per group). For all panels: Statistical significance is indicated (* p<0.05, ** p<0.01, *** p<0.001). Bars indicate WT (filled) or hsf1-/- (open) mice.
Figure 7
Figure 7. HSF1 promotes hepatic metabolic adaptation by modulating proteostasis and influences energy metabolic pathways in response to nutrient availability and insulin stimulation
(A) Increased insulin sensitivity in hsf1-/- mice results in enhanced AMPK activation: Immunoblot analyses of basal, and insulin stimulated p-AMPK levels in livers of WT and hsf1-/- mice. 7 months-old mice were fasted for 16 hrs and injected with saline or insulin (0.75 mU/g i.p.). Liver extracts prepared 10 min after insulin or saline injection were examined and blots were quantified (right panels). Level of p-AMPK or p-ACC normalized to total protein level was expressed as relative fold-increase to control (WT without insulin). Bars are mean ± SD; (n=5 mice per group). (B) Expression of HSF1, AMPK, and target genes: under nutrient available conditions: Representative Western blot of liver extracts from 7 months-old mice, fasted for 24 hrs, or refed ad libitum for 3 hours, using the indicated antibodies. The ratio of p-AMPK or p-ACC normalized to total protein level is given. Real-time RT-PCR analysis of genes that encode for HSPs in liver of hsf1-/- or WT mice (lower panels). Data are presented as relative mRNA expression. Bars are mean ± SD; (n=3-5 mice per group). In all panels, bars indicate WT (filled) or hsf1-/- (open) mice. Statistical significance is indicated (* p<0.05, ** p<0.01, *** p<0.001).

Similar articles

See all similar articles

Cited by 57 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback