TRIB3-TRIM8 complex drives NAFLD progression by regulating HNF4α stability.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress of hepatocytes plays a causative role in non-alcoholic fatty liver disease (NAFLD). Reduced expression of hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. Whether ER stress regulates HNF4α expression remains unknown. The aim of this study was to delineate the machinery of HNF4α protein degradation and explore a therapeutic strategy based on protecting HNF4α stability during NAFLD progression. METHODS: Correlation of HNF4α and tribbles homologue 3 (TRIB3), an ER stress sensor, was evaluated in human and mouse NAFLD tissues. RNA-sequencing, mass spectrometry analysis, co-immunoprecipitation, in vivo and in vitro ubiquitination assays were used to elucidate the mechanisms of TRIB3-mediated HNF4α degradation. Molecular docking and co-immunoprecipitation analyses were performed to identify a cell-penetrating peptide that ablates the TRIB3-HNF4α interaction. RESULTS: TRIB3 directly interacts with HNF4α and mediates ER stress-induced HNF4α degradation. TRIB3 recruits tripartite motif containing 8 (TRIM8) to form an E3 ligase complex that catalyzes K48-linked polyubiquitination of HNF4α on lysine 470. Abrogating the degradation of HNF4α attenuated the effect of TRIB3 on a diet-induced NAFLD model. Moreover, the TRIB3 gain-of-function variant p.Q84R is associated with NAFLD progression in patients, and induces lower HNF4α levels and more severe hepatic steatosis in mice. Importantly, disrupting the TRIB3-HNF4α interaction using a cell-penetrating peptide restores HNF4α levels and ameliorates NAFLD progression in mice. CONCLUSIONS: Our findings unravel the machinery of HNF4α protein degradation and indicate that targeting TRIB3-TRIM8 E3 complex-mediated HNF4α polyubiquitination may be an ideal strategy for NAFLD therapy. IMPACT AND IMPLICATIONS: Reduced expression of hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. However, the mechanism of HNF4α protein degradation remains unknown. Herein, we reveal that TRIB3-TRIM8 E3 ligase complex is responsible for HNF4α degradation during NAFLD. Inhibiting the TRIB3-HNF4α interaction effectively stabilized HNF4α protein levels and transcription factor activity in the liver and ameliorated TRIB3-mediated NAFLD progression. Our findings demonstrate that disturbing the TRIM8-TRIB3-HNF4α interaction may provide a novel approach to treat NAFLD and even other liver diseases by stabilizing the HNF4α protein.

miR-541 is associated with the prognosis of liver cirrhosis and directly targets JAG2 to inhibit the activation of hepatic stellate cells.

BACKGROUND: The activation of hepatic stellate cells (HSCs) has been emphasized as a leading event of the pathogenesis of liver cirrhosis, while the exact mechanism of its activation is largely unknown. Furthermore, the novel non-invasive predictors of prognosis in cirrhotic patients warrant more exploration. miR-541 has been identified as a tumor suppressor in hepatocellular carcinoma and a regulator of fibrotic disease, such as lung fibrosis and renal fibrosis. However, its role in liver cirrhosis has not been reported. METHODS: Real-time PCR was used to detect miR-541 expression in the liver tissues and sera of liver cirrhosis patients and in the human LX-2. Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the activation of LX-2. Bioinformatics analysis and a luciferase reporter assay were conducted to investigate the target gene of miR-541. RESULTS: miR-541 was downregulated in the tissues and sera of patients with liver cirrhosis, which was exacerbated by deteriorating disease severity. Importantly, the lower expression of miR-541 was associated with more episodes of complications including ascites and hepatic encephalopathy, a shorter overall lifespan, and decompensation-free survival. Moreover, multivariate Cox's regression analysis verified lower serum miR-541 as an independent risk factor for liver-related death in cirrhotic patients (HR = 0.394; 95% CI: 0.164-0.947; P = 0.037). miR-541 was also decreased in LX-2 cells activated by TGF-ß and the overexpression of miR-541 inhibited the proliferation, activation and hydroxyproline secretion of LX-2 cells. JAG2 is an important ligand of Notch signaling and was identified as a direct target gene of miR-541. The expression of JAG2 was upregulated in the liver tissues of cirrhotic patients and was inversely correlated with miR-541 levels. A rescue assay further confirmed that JAG2 was involved in the function of miR-541 when regulating LX-2 activation and Notch signaling. CONCLUSIONS: Dysregulation of miR-541/JAG2 axis might be a as a new mechanism of liver fibrosis, and miR-541 could serve as a novel non-invasive biomarker and therapeutic targets for liver cirrhosis.

Inhibition of protein arginine methyltransferase 1 alleviates liver fibrosis by attenuating the activation of hepatic stellate cells in mice.

Protein arginine methyltransferase 1 (PRMT1) has been reported to be involved in various diseases. The expression of PRMT1 was increased in cirrhotic livers from human patients. However, the role of PRMT1 in hepatic fibrogenesis remains largely unexplored. In this study, we investigated the effect of PRMT1 on hepatic fibrogenesis and its underlying mechanism. We found that PRMT1 expression was significantly higher in fibrotic livers of the mice treated with thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Immunofluorescence staining revealed that PRMT1 expression was augmented in both hepatocytes and hepatic stellate cells (HSCs) in the fibrotic livers. Applying a selective inhibitor of PRMT1, PT1001B, significantly suppressed PRMT1 activity and mitigated liver fibrosis in mice. Hepatocyte-specific Prmt1 knockout did not affect liver fibrosis in mice. PRMT1 overexpression promoted the expression of fibrotic genes in the LX-2 cells, whereas knockdown of PRMT1 or treatment with PT1001B exhibited reversal effects, suggesting that PRMT1 plays an important role in HSC activation. Additionally, HSC-specific Prmt1 knockout attenuated HSC activation and liver fibrosis in TAA-induced fibrotic model. RNA-seq analysis revealed that Prmt1 knockout in HSCs significantly suppressed pro-inflammatory NF-κB and pro-fibrotic TGF-ß signals, and also downregulated the expression of pro-fibrotic mediators in mouse livers. Moreover, treatment with PT1001B consistently inhibited hepatic inflammatory response in fibrotic model. In conclusion, PRMT1 plays a vital role in HSC activation. Inhibition of PRMT1 mitigates hepatic fibrosis by attenuating HSC activation in mice. Therefore, targeting PRMT1 could be a feasible therapeutic strategy for liver fibrosis.

Deletion of Sox9 in the liver leads to hepatic cystogenesis in mice by transcriptionally downregulating Sec63.

Hepatic cysts are found in heterogeneous disorders with different pathogeneses, of which simple hepatic cysts and polycystic liver diseases are two major types. The process of hepatic cytogenesis for these two diseases is caused by defects in remodelling of the ductal plate during biliary tract development, which is called ductal plate malformation. SOX9 is a transcription factor participating in the process of bile duct development, and thus, its dysregulation may play important roles in hepatic cystogenesis. SEC63 encodes an endoplasmic reticulum membrane protein that is mutated in human autosomal dominant polycystic liver disease. However, the transcriptional regulation of SEC63 is largely unknown. In the present study, a liver-specific Sox9 knockout (Sox9LKO ) mouse was generated to investigate the roles and underlying mechanism of SOX9 in hepatic cystogenesis. We found that hepatic cysts began to be observed in Sox9LKO mice at 6 months of age. The number and size of cysts increased with age in Sox9LKO mice. In addition, the characteristics of hepatic cytogenesis, including the activation of proliferation, absence of primary cilium, and disorder of polarity in biliary epithelial cells, were detected in the livers of Sox9LKO mice. RNAi silencing of SOX9 in human intrahepatic biliary epithelial cells (HIBEpic) resulted in increased proliferation and reduced formation of the primary cilium. Moreover, Sec63 was downregulated in primary biliary epithelial cells from Sox9LKO mice and SEC63 in HIBEpic transfected with siSOX9. Chromatin immunoprecipitation assays and luciferase reporter assays further demonstrated that SOX9 transcriptionally regulated the expression of SEC63 in biliary epithelial cells. Importantly, the overexpression of SEC63 in HIBEpic partially reversed the effects of SOX9 depletion on the formation of primary cilia and cell proliferation. These findings highlight the biological significance of SOX9 in hepatic cytogenesis and elucidate a novel molecular mechanism underlying hepatic cytogenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

miR-541 potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy.

OBJECTIVE: Autophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated microRNA-autophagy axis. DESIGN: Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality. RESULTS: The expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment. CONCLUSIONS: Dysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients' responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.

The HNF1α-regulated lncRNA HNF1A-AS1 reverses the malignancy of hepatocellular carcinoma by enhancing the phosphatase activity of SHP-1.

BACKGROUND: Our previous study has demonstrated that hepatocyte nuclear factor 1α (HNF1α) exerts potent therapeutic effects on hepatocellular carcinoma (HCC). However, the molecular mechanisms by which HNF1α reverses HCC malignancy need to be further elucidated. METHODS: lncRNA microarray was performed to identify the long noncoding RNAs (lncRNAs) regulated by HNF1α. Chromatin immunoprecipitation and luciferase reporter assays were applied to clarify the mechanism of the transcriptional regulation of HNF1α to HNF1A antisense RNA 1 (HNF1A-AS1). The effect of HNF1A-AS1 on HCC malignancy was evaluated in vitro and in vivo. RNA pulldown, RNA-binding protein immunoprecipitation and the Bio-Layer Interferometry assay were used to validate the interaction of HNF1A-AS1 and Src homology region 2 domain-containing phosphatase 1 (SHP-1). RESULTS: HNF1α regulated the expression of a subset of lncRNAs in HCC cells. Among these lncRNAs, the expression levels of HNF1A-AS1 were notably correlated with HNF1α levels in HCC cells and human HCC tissues. HNF1α activated the transcription of HNF1A-AS1 by directly binding to its promoter region. HNF1A-AS1 inhibited the growth and the metastasis of HCC cells in vitro and in vivo. Moreover, knockdown of HNF1A-AS1 reversed the suppressive effects of HNF1α on the migration and invasion of HCC cells. Importantly, HNF1A-AS1 directly bound to the C-terminal of SHP-1 with a high binding affinity (KD = 59.57 ± 14.29 nM) and increased the phosphatase activity of SHP-1. Inhibition of SHP-1 enzymatic activity substantially reversed the HNF1α- or HNF1A-AS1-induced reduction on the metastatic property of HCC cells. CONCLUSIONS: Our data revealed that HNF1A-AS1 is a direct transactivation target of HNF1α in HCC cells and involved in the anti-HCC effect of HNF1α. HNF1A-AS1 functions as phosphatase activator through the direct interaction with SHP-1. These findings suggest that regulation of the HNF1α/HNF1A-AS1/SHP-1 axis may have beneficial effects in the treatment of HCC.

p.Q511L mutation of HNF1α in hepatocellular carcinoma suppresses the transcriptional activity and the anti-tumor effect of HNF1α.

Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that regulates many aspects of hepatocyte functions. Our previous studies have demonstrated that HNF1α has potent therapeutic effects on hepatocellular carcinoma (HCC). Mutations in HNF1α gene are frequently associated with maturity-onset diabetes of the young type 3 (MODY3) and hepatocellular adenomas. However, the association of HNF1α mutation and HCC remains elusive. In this study, the point mutation of HNF1α gene with c.A1532 > T/p.Q511L was identified in an HCC patient by exon-capture high-throughput sequencing. Mutation of c.A1532 > T/p.Q511L in HNF1α gene was only detected in the tumor tissue but not in the adjacent non-tumorous liver tissue of the patient. Luciferase reporter assay and real-time PCR revealed that mutation of p.Q511L reduced the transcriptional activity of HNF1α. Immunofluorescence staining and subcellular fraction analysis revealed that mutation of p.Q511L disturbed the intracellular localization of HNF1α in HCC cells. Moreover, the inhibitory effect of HNF1α on the proliferation, migration and invasion in HCC cells was also partially abolished by the mutation of p.Q511L. Our data suggested that the missense mutation of HNF1α (p.Q511L) may associate with the progression of HCC.

Abrogating K458 acetylation enhances hepatocyte nuclear factor 4α (HNF4α)-induced differentiation therapy for hepatocellular carcinoma.

OBJECTIVES: In this study we aimed to assess the impact of acetylation of hepatocyte nuclear factor 4α (HNF4α) on lysine 458 on the differentiation therapy of hepatocellular carcinoma (HCC). METHODS: Periodic acid-Schiff (PAS) staining, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and senescence-associated ß-galactosidase (SA-ß-gal) activity analysis were performed to assess the differentiation of HCC cells. HNF4α protein was detected by western blot and immunohistochemistry (IHC). The effects of HNF4α-K458 acetylation on HCC malignancy were evaluated in HCC cell lines, a Huh-7 xenograft mouse model, and an orthotopic model. The differential expression genes in Huh-7 xenograft tumors were screened by RNA-sequencing analysis. RESULTS: K458R significantly enhanced the inhibitory effect of HNF4α on the malignancy of HCC cells, whereas K458Q reduced the inhibitory effects of HNF4α. Moreover, K458R promoted, while K458Q decreased, HNF4α-induced HCC cell differentiation. K458R stabilized HNF4α, while K458Q accelerated the degradation of HNF4α via the ubiquitin proteasome system. K458R also enhanced the ability of HNF4α to inhibit cell growth of HCC in the Huh-7 xenograft mouse model and the orthotopic model. RNA-sequencing analysis revealed that inhibiting K458 acetylation enhanced the transcriptional activity of HNF4α without altering the transcriptome induced by HNF4α in HCC. CONCLUSION: Our data revealed that inhibiting K458 acetylation of HNF4α might provide a more promising candidate for differential therapy of HCC.

Tripartite motif 8 promotes the progression of hepatocellular carcinoma via mediating ubiquitination of HNF1α.

Tripartite motif 8 (TRIM8) is an E3 ligase that plays dual roles in various tumor types. The biological effects and underlying mechanism of TRIM8 in hepatocellular carcinoma (HCC) remain unknown. Hepatocyte nuclear factor 1α (HNF1α) is a key transcriptional factor that plays a significant role in regulating hepatocyte differentiation and liver function. The reduced expression of HNF1α is a critical event in the development of HCC, but the underlying mechanism for its degradation remains elusive. In this study, we discovered that the expression of TRIM8 was upregulated in HCC tissues, and was positively correlated with aggressive tumor behavior of HCC and shorter survival of HCC patients. Overexpression of TRIM8 promoted the proliferation, colony formation, invasion, and migration of HCC cells, while TRIM8 knockdown or knockout exerted the opposite effects. RNA sequencing revealed that TRIM8 knockout suppresses several cancer-related pathways, including Wnt/ß-catenin and TGF-ß signaling in HepG2 cells. TRIM8 directly interacts with HNF1α, promoting its degradation by catalyzing polyubiquitination on lysine 197 in HCC cells. Moreover, the cancer-promoting effects of TRIM8 in HCC were abolished by the HNF1α-K197R mutant in vitro and in vivo. These data demonstrated that TRIM8 plays an oncogenic role in HCC progression through mediating the ubiquitination of HNF1α and promoting its protein degradation, and suggests targeting TRIM8-HNF1α may provide a promising therapeutic strategy of HCC.

SRY-Box transcription factor 9 triggers YAP nuclear entry via direct interaction in tumors.

The translocation of YAP from the cytoplasm to the nucleus is critical for its activation and plays a key role in tumor progression. However, the precise molecular mechanisms governing the nuclear import of YAP are not fully understood. In this study, we have uncovered a crucial role of SOX9 in the activation of YAP. SOX9 promotes the nuclear translocation of YAP by direct interaction. Importantly, we have identified that the binding between Asp-125 of SOX9 and Arg-124 of YAP is essential for SOX9-YAP interaction and subsequent nuclear entry of YAP. Additionally, we have discovered a novel asymmetrical dimethylation of YAP at Arg-124 (YAP-R124me2a) catalyzed by PRMT1. YAP-R124me2a enhances the interaction between YAP and SOX9 and is associated with poor prognosis in multiple cancers. Furthermore, we disrupted the interaction between SOX9 and YAP using a competitive peptide, S-A1, which mimics an α-helix of SOX9 containing Asp-125. S-A1 significantly inhibits YAP nuclear translocation and effectively suppresses tumor growth. This study provides the first evidence of SOX9 as a pivotal regulator driving YAP nuclear translocation and presents a potential therapeutic strategy for YAP-driven human cancers by targeting SOX9-YAP interaction.

Hsa_Circ_0098181 Suppresses Hepatocellular Carcinoma by Sponging miR-18a-3p and Targeting PPARA.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, and its incidence is still high in China. This study aimed to investigate the circular RNAs (circRNAs) involved in the development of HCC and elucidate the mechanism. RNA sequencing found 72 downregulated circRNAs and 88 upregulated circRNAs in human HCC tissues, including hsa_circ_0098181, hsa_circ_0072309, hsa_circ_0000831, and hsa_circ_0000231. The reduction of hsa_circ_0098181 was confirmed in eight paired human HCC tissues, hepatoma cell lines, and CCL4/DEN-induced mouse HCC models by RT-qPCR. The FISH assay revealed that hsa_circ_0098181 is mainly located in the cytoplasm of hepatocytes in the paratumor tissues. Further log-rank analysis performed in 91 HCC patients demonstrated that low expression of hsa_circ_0098181 was related to poor prognosis. The plasmid and lentivirus overexpressing hsa_circ_0098181 were delivered into HCC cell lines. After hsa_circ_0098181 was upregulated, the proliferation, invasion, migration, and colony formation of HCC cell lines were inhibited, and the apoptosis was promoted. Moreover, exogenous hsa_circ_0098181 delivery mitigated the tumor formation ability of Huh7 in Balb/C nude mice. The dual-luciferase reporter assay and the RIP assay verified that hsa_circ_0098181 sponged miR-18a-3p to regulate PPARA. In addition, a rescue experiment found miR-18a-3p mimic partly reversed the suppression of hsa_circ_0098181 on proliferation, invasion, and migration of HCC cell lines. In conclusion, hsa_circ_0098181 can repress the development of HCC through sponging miR-18a-3p and promoting the expression of PPARA in vitro and in vivo, and hsa_circ_0098181 might be a therapeutic target for HCC.

Targeting p300/CBP Attenuates Hepatocellular Carcinoma Progression through Epigenetic Regulation of Metabolism.

Targeting epigenetics in cancer has emerged as a promising anticancer strategy. p300/CBP is a central regulator of epigenetics and plays an important role in hepatocellular carcinoma (HCC) progression. Tumor-associated metabolic alterations contribute to the establishment and maintenance of the tumorigenic state. In this study, we used a novel p300 inhibitor, B029-2, to investigate the effect of targeting p300/CBP in HCC and tumor metabolism. p300/CBP-mediated acetylation of H3K18 and H3K27 increased in HCC tissues compared with surrounding noncancerous tissues. Conversely, treatment with B029-2 specifically decreased H3K18Ac and H3K27Ac and displayed significant antitumor effects in HCC cells in vitro and in vivo. Importantly, ATAC-seq and RNA-seq integrated analysis revealed that B029-2 disturbed metabolic reprogramming in HCC cells. Moreover, B029-2 decreased glycolytic function and nucleotide synthesis in Huh7 cells by reducing H3K18Ac and H3K27Ac levels at the promoter regions of amino acid metabolism and nucleotide synthesis enzyme genes, including PSPH, PSAT1, ALDH18A1, TALDO1, ATIC, and DTYMK. Overexpression of PSPH and DTYMK partially reversed the inhibitory effect of B029-2 on HCC cells. These findings suggested that p300/CBP epigenetically regulates the expression of glycolysis-related metabolic enzymes through modulation of histone acetylation in HCC and highlights the value of targeting the histone acetyltransferase activity of p300/CBP for HCC therapy. SIGNIFICANCE: This study demonstrates p300/CBP as a critical epigenetic regulator of glycolysis-related metabolic enzymes in HCC and identifies the p300/CBP inhibitor B029-2 as a potential therapeutic strategy in this disease.

Targeting PRMT5 Activity Inhibits the Malignancy of Hepatocellular Carcinoma by Promoting the Transcription of HNF4α.

Background: Liver cancer stem cells (LCSCs) are responsible for the initiation, progression and chemoresistance of liver cancer. However, no agent targeting LCSC is available in the clinic to date. Here, we investigated the effects of targeting protein arginine methyltransferase 5 (PRMT5), an epigenetic regulator, on LCSCs and HCC using a novel PRMT5 inhibitor DW14800. Methods: Tumor spheroid formation culture was used to enrich LCSCs and assess their self-renewal capability. Human alpha-1-antitrypsin (A1AT) ELISA, acetylated low-density lipoprotein (ac-LDL) uptake, periodic acid-Schiff (PAS) reactions and senescence associated ß-galactosidase (SA-ß-gal) activity assays were performed to examine the differentiation status of HCC cells. The effects of DW14800 on HCC malignancy were assessed in HCC cell lines and on an HCC xenograft model in mice. Chromatin immunoprecipitation was applied to clarify the transcriptional regulation of HNF4α by PRMT5-mediated Histone H4 arginine-3 symmetrical dimethylation (H4R3me2s). Results: Quantitative real-time PCR revealed that the expression of PRMT5 was upregulated in LCSCs. DW14800 specifically decreased the symmetrical dimethylation of arginine residues in HCC cells. Treatment of DW14800 suppressed the self-renewal capacity of LCSCs while re-establishing hepatocyte-specific characteristics in HCC cells. DW14800 displayed antitumor effects in HCC cells in vitro and in xenograft HCC in vivo. Importantly, ChIP assay showed that PRMT5 and H4R3me2s bound to the promoter region of HNF4α gene, and DW14800 increased the expression of HNF4α via reducing the H4R3me2s levels and enhancing the transcription of HNF4α. Conclusions: Our data revealed the significance of targeting PRMT5 activity in LCSC elimination and HCC differentiation, and proposed that DW14800 may represent a promising therapeutic agent for HCC in the clinic.

SHP-1 Acts as a Tumor Suppressor in Hepatocarcinogenesis and HCC Progression.

Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1, also known as PTPN6) is a nonreceptor protein tyrosine phosphatase that acts as a negative regulator of inflammation. Emerging evidence indicates that SHP-1 plays a role in inhibiting the progression of hepatocellular carcinoma (HCC). However, the role of SHP-1 in hepatocarcinogenesis remains unknown. Here, we find that levels of SHP-1 are significantly downregulated in human HCC tissues compared with those in noncancerous tissues (P < 0.001) and inversely correlate with tumor diameters (r = -0.4130, P = 0.0002) and serum α-fetoprotein levels (P = 0.047). Reduced SHP-1 expression was associated with shorter overall survival of patients with HCC with HBV infection. Overexpression of SHP-1 suppressed proliferation, migration, invasion, and tumorigenicity of HCC cells, whereas knockdown of SHP-1 enhanced the malignant phenotype. Moreover, knockout of Ptpn6 in hepatocytes (Ptpn6HKO ) enhanced hepatocarcinogenesis induced by diethylnitrosamine (DEN) as well as metastasis of primary liver cancer in mice. Furthermore, systemic delivery of SHP-1 by an adenovirus expression vector exerted a therapeutic effect in an orthotopic model of HCC in NOD/SCID mice and DEN-induced primary liver cancers in Ptpn6HKO mice. In addition, SHP-1 inhibited the activation of JAK/STAT, NF-κB, and AKT signaling pathways, but not the MAPK pathway in primary hepatocytes from DEN-treated mice and human HCC cells. Together, our data implicate SHP-1 as a tumor suppressor of hepatocarcinogenesis and HCC progression and propose it as a novel prognostic biomarker and therapeutic target of HCC.Significance: The nonreceptor protein tyrosine phosphatase SHP-1 acts as a tumor suppressor in hepatocellular carcinoma. Cancer Res; 78(16); 4680-91. ©2018 AACR.

An HNF1α-regulated feedback circuit modulates hepatic fibrogenesis via the crosstalk between hepatocytes and hepatic stellate cells.

Hepatocytes are critical for the maintenance of liver homeostasis, but its involvement in hepatic fibrogenesis remains elusive. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that plays a key role in hepatocyte function. Our previous study revealed a significant inhibitory effect of HNF1α on hepatocellular carcinoma. In this study, we report that the expression of HNF1α is significantly repressed in both human and rat fibrotic liver. Knockdown of HNF1α in the liver significantly aggravates hepatic fibrogenesis in either dimethylnitrosamine (DMN) or bile duct ligation (BDL) model in rats. In contrast, forced expression of HNF1α markedly alleviates hepatic fibrosis. HNF1α regulates the transcriptional expression of SH2 domain-containing phosphatase-1 (SHP-1) via directly binding to SHP-1 promoter in hepatocytes. Inhibition of SHP-1 expression abrogates the anti-fibrotic effect of HNF1α in DMN-treated rats. Moreover, HNF1α repression in primary hepatocytes leads to the activation of NF-κB and JAK/STAT pathways and initiates an inflammatory feedback circuit consisting of HNF1α, SHP-1, STAT3, p65, miR-21 and miR-146a, which sustains the deregulation of HNF1α in hepatocytes. More interestingly, a coordinated crosstalk between hepatocytes and hepatic stellate cells (HSCs) participates in this positive feedback circuit and facilitates the progression of hepatocellular damage. Our findings demonstrate that impaired hepatocytes play an active role in hepatic fibrogenesis. Early intervention of HNF1α-regulated inflammatory feedback loop in hepatocytes may have beneficial effects in the treatment of chronic liver diseases.