Weisi Liu

EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma

The CXC chemokine receptor 4 (CXCR4) exerts a variety of functions at different steps of hepatocellular carcinoma (HCC) progression. The molecular mechanisms and therapeutic value of CXCR4 in the development of HCC remain undefined. Here we show that aberrant CXCR4 overexpression is associated with poor prognosis and aggressive characteristics of HCC. Suppression of CXCR4 activity via CXCR4 knockdown, AMD3100 or neutralizing antibody administration inhibits hepatoma cell tumorigenesis in vitro and in vivo. CXCR4 overexpression displays the opposite effects. Using Mir library screening we identify miR-622 as a regulator of CXCR4. Further studies show that miR-622 directly target the 3′ untranslated region of CXCR4 and is transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. EZH2/miR-622 promotes tumorigenesis through CXCR4. EZH2-mediated loss of miR-622 is found to correlate with CXCR4 overexpression and unfavourable prognosis in HCC patients. This study establishes EZH2/miR-622/CXCR4 as a potential adverse prognostic factor and therapeutic target for HCC patients.

Decreased Expression of Hepatocyte Nuclear Factor 4α (Hnf4α)/MicroRNA-122 (miR-122) Axis in Hepatitis B Virus-associated Hepatocellular Carcinoma Enhances Potential Oncogenic GALNT10 Protein Activity

Background: GALNT, the initial enzyme in mucin-type O-glycosylation, plays critical roles in cancer etiology. Results: GALNT10-induced cellular proliferation was associated with EGFR activation mediated by down-regulation of miR-122 in HBV-associated HCC. Conclusion: A regulatory pathway of Hnf4α/miR-122/GALNT10/EGFR may represent a possible mechanism underlying HBV-associated hepatocarcinogenesis. Significance: This finding provides a novel role for O-glycosylation in HCC pathogenesis. MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed that GALNT10 indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis.

Klotho Endows Hepatoma Cells with Resistance to Anoikis via VEGFR2/PAK1 Activation in Hepatocellular Carcinoma

Klotho was originally characterized as an aging suppressor gene that predisposed Klotho-deficient mice to premature aging-like syndrome. Although Klotho was recently reported to exhibit tumor suppressive properties during various malignant transformations, the functional role and molecular mechanism of Klotho in hepatocarcinogenesis remains poorly understood. In our present study, immunohistochemical Klotho staining levels in a clinical follow-up of 52 hepatoma patients were significantly associated with liver cirrhosis, tumor multiplicity and venous invasion. The overall survival rate of hepatoma patients with high Klotho expression was significantly lower than those patients with low Klotho expression. Moreover, Klotho overexpression increased cellular migration, anchorage-independent growth, and anoikis resistance in hepatoma cells. Klotho overexpression elevated p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase activity inhibition with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor IPA3 treatment reversed anoikis resistance in Klotho-overexpressed hepatoma cells. More importantly, the pivotal significance of upregulated VEGFR2 protein levels mediated by Klotho expression was confirmed by VEGFR2 inhibitor Axitinib and blocking antibody treatment in hepatoma cells. Axitinib treatment sensitized anoikis was reversed by constitutive active mutant PAK1 T423E coexpression in Klotho-overexpressed hepatoma cells. Conversely, knockdown of Klotho reduced VEGFR2/PAK1 dependent anoikis resistance, which could be reversed by PAK1 T423E. These results revealed a novel oncogenic function of Klotho in promoting anoikis resistance via activating VEGFR2/PAK1 signaling, thus facilitating tumor migration and invasion during hepatoma progression, which could provide a putative molecular mechanism for tumor metastasis.

N-acetylglucosaminyltransferase V confers hepatoma cells with resistance to anoikis through EGFR/PAK1 activation

Elevated expression and activity of N-acetylglucosaminyltransferase V (Mgat5) in hepatocellular carcinoma (HCC) is a common early event involved in tumor invasion during hepatocarcinogenesis. A better understanding of the functional role and the molecular mechanism for Mgat5-targeted protein and downstream signaling pathway behind hepatoma invasion and metastasis is urgently needed. Here, we show that Mgat5 overexpression promoted anchorage-independent growth and inhibited anoikis in hepatoma cells. This effect was reversed by glycosyltransferase inactive mutant Mgat5 L188R transfection, α-mannosidase II inhibitor swainsonine treatment and N-acetyl glucosamine (GlcNAc) phosphotransferase (GPT) inhibitor tunicamycin administration. Mgat5 overexpression increased p21-activated kinase 1 (PAK1) expression and shRNA-mediated PAK1 knockdown and kinase inactivation with kinase dead mutant PAK1 K299R coexpression or allosteric inhibitor P21-activated kinase inhibitor III (IPA3) treatment reversed anoikis resistance in Mgat5-overexpressed hepatoma cells. Furthermore, Mgat5 overexpression upregulated β-1-6-GlcNAc branched N-glycosylation and following phosphorylation of epidermal growth factor receptor (EGFR) in hepatoma cells. EGFR tyrosine kinase inhibitors AG1478 and Iressa treatment declined anchorage-independent growth and anoikis resistance, which could be rescued by constitutive active mutant PAK1 T423E coexpression in Mgat5-overexpressed hepatoma cells. Conversely, knockdown of Mgat5 reduced EGFR/PAK1-dependent anoikis resistance, which could be reversed by PAK1 T423E. These results identified Mgat5-mediated β-1-6-GlcNAc branched N-glycosylation and following activation of EGFR as a potential novel upstream molecular event for PAK1-induced anoikis resistance in hepatoma cells, implicating that molecular targeted therapeutics against Mgat5/EGFR/PAK1 might open a new avenue for personalized medicine in advanced-stage HCC patients.

Polycomb-mediated loss of microRNA let-7c determines inflammatory macrophage polarization via PAK1-dependent NF-κB pathway.

Serine/threonine kinase family members p21-activated kinases (PAKs) are important regulators of cytoskeletal remodeling and cell motility in mononuclear phagocytic system, but their role in macrophage differentiation and polarization remains obscure. We have shown here that inflammatory stimuli induced PAK1 overexpression in human and murine macrophages. Elevated expression of PAK1 contributed to macrophage M1 polarization and lipopolysaccharide (LPS)-induced endotoxin shock. We further observed that epigenetic loss of microRNA let-7c due to enhancer of zeste homolog 2 (EZH2) upregulation determined PAK1 elevation and inflammatory phenotype in M1 macrophages. EZH2/let-7c/PAK1 axis promotes macrophage M1 polarization via NIK-IKK-NF-κB signaling. Moreover, pharmacological and genetic ablation with EZH2/let-7c/PAK1 axis blunted inflammatory phenotype in M1 macrophages. Critically, either myeloid-restricted PAK1 deletion (PAK1Lyz2cre) or pharmacological and genetic ablation with EZH2/let-7c/PAK1 signal resulted in resistance to LPS-induced endotoxin shock via blunting macrophage M1 polarization. PAK1, therefore, is an essential controller of inflammatory macrophage polarization, regulating immune responses against pathogenic stimuli.

Sorafenib suppresses growth and survival of hepatoma cells by accelerating degradation of enhancer of zeste homolog 2

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes that regulate cancer cell growth and survival. It is overexpressed in hepatocellular carcinoma (HCC) with a clinical significance that remains obscure. Sorafenib, a multikinase inhibitor, has been used as a first‐line therapeutic drug and shown clinical efficiency for advanced‐stage HCC patients. In the present study, we found that sorafenib lowered the protein level of EZH2 through accelerating proteasome‐mediated EZH2 degradation in hepatoma cells. Overexpression of EZH2 reversed sorafenib‐induced cell growth arrest, cell cycle arrest, and cell apoptosis dependent on histone methyltransferase activity in hepatoma cells. More importantly, shRNA‐mediated EZH2 knockdown or EZH2 inhibition with 3‐deazaneplanocin A treatment promoted sorafenib‐induced hepatoma cell growth arrest and apoptosis. Sorafenib altered the hepatoma epigenome by reducing EZH2 and H3K27 trimethylation. These results revealed a novel therapeutic mechanism underlying sorafenib treatment in suppressing hepatoma growth and survival by accelerating EZH2 degradation. Genetic deletion or pharmacological ablation of EZH2 made hepatoma cells more sensitive to sorafenib, which helps provide a strong framework for exploring innovative combined therapies for advanced‐stage HCC patients.

Galectin-9 predicts postoperative recurrence and survival of patients with clear-cell renal cell carcinoma

Galectin-9 (Gal-9), a member of animal lectin family with evolutionary conserved carbohydrate recognition domains, has been reported to exert a large variety of functional roles in tumorigenesis due to its β-galactoside-binding affinity. The aim of this study is to evaluate the expression and prognostic significance of Gal-9 in patients with clear-cell renal cell carcinoma (ccRCC). The expression of Gal-9 was assessed by immunohistochemistry in 196 patients with ccRCC who underwent nephrectomy. In the cohort, 48 patients died and 61 patients suffered recurrence. Kaplan–Meier method with log–rank test was applied to compare survival curves. The authors employed univariate and multivariate Cox regression models to evaluate the prognostic value of Gal-9 expression in overall survival (OS) and recurrence-free survival (RFS). In patients with ccRCC, Gal-9 expression, which was positively associated with tumor size (P = 0.014), Fuhrman grade (P = 0.010), and necrosis (P = 0.025), was determined to be an independent prognostic indicator for OS (hazard ratio [HR] 2.394; P = 0.005) and RFS (HR 2.096; P = 0.006). High expression of Gal-9 was associated with poor survival (P = 0.001) and early recurrence (P = 0.006). Furthermore, Gal-9 expression could significantly stratify the patients in early (grades I + II) tumor, node, and metastasis (TNM) stage (OS: P = 0.005; RFS: P = 0.041) and low (grades 1 + 2) Fuhrman grade (OS: P = 0.004; RFS: P = 0.006). The prognostic accuracy of TNM, SSIGN, and UISS prognostic models was improved when Gal-9 expression was added. Gal-9 expression is a potential independent prognostic factor for OS and RFS in patients with ccRCC.

p21-Activated kinase 4 predicts early recurrence and poor survival in patients with nonmetastatic clear cell renal cell carcinoma.

BACKGROUND p21-Activated kinase 4 (PAK4), a serine/threonine kinase implicated in the cytoskeleton organization to orchestrate cell morphology, adhesion, and motility, is associated with angiogenesis and vessel branching, which are important events in the progression of clear cell renal cell carcinoma (ccRCC). We investigated the effect of PAK4 expression on recurrence and survival among patients with nonmetastatic ccRCC following surgery. METHODS PAK4 expression was assessed, using immunohistochemistry, in 376 patients with nonmetastatic ccRCC after nephrectomy, where data of 187 patients were obtained from 2013 to 2014 and of 189 patients were obtained from 2008. Kaplan-Meier and Cox regression analyses were used to associate PAK4 expression with overall survival and recurrence-free survival. RESULTS Overall, 41.2% and 36.5% of specimens exhibited high PAK4 expression in 2 cohorts. Patients with high PAK4 expression were prone to possess high Fuhrman grade and tumor necrosis. Moreover, high PAK4 intensity was significantly associated with poor overall survival and recurrence-free survival. PAK4 expression remained an independent adverse prognosticator after adjusting for other well-established factors. Furthermore, in subgroups stratified by Fuhrman grade or T category, patients with high PAK4 intensity had an increased risk of recurrence and death. After adjusting for age, high PAK4 expression was an adverse prognostic marker in subgroup of low Fuhrman grade and in subgroup of early T category. CONCLUSION PAK4 expression is an independent adverse prognostic biomarker for recurrence and survival among patients with low-risk ccRCC after nephrectomy.

High expression of interleukin-11 is an independent indicator of poor prognosis in clear-cell renal cell carcinoma

Interleukin‐11 (IL‐11), a member of the IL‐6 family of cytokines, exerts pleiotropic oncogenic activities by stimulating angiogenesis and metastasis in many cancer types. The present study aims to evaluate the impact of IL‐11 expression on recurrence and mortality of patients with clear‐cell renal cell carcinoma (ccRCC). We retrospectively enrolled 193 ccRCC patients undergoing nephrectomy at a single center. Clinicopathologic features, recurrence‐free survival (RFS) and overall survival (OS) were recorded. IL‐11 intensity was assessed by immunohistochemistry in tumor specimens. The Kaplan–Meier method was applied to compare survival curves. Cox regression models were used to analyze the impact of prognostic factors on RFS and OS. The concordance index (C‐index) was calculated to assess predictive accuracy. High IL‐11 expression is associated with increased risk of recurrence and poor survival for ccRCC patients (P < 0.001 and P < 0.001, respectively), especially those with early‐stage disease (TNM stage I + II). Multivariate analyses confirmed that IL‐11 expression was an independent prognostic factor for RFS and OS (P = 0.006 and P = 0.008, respectively). The predictive accuracy of well‐established prognostic models was improved when IL‐11 expression was integrated. In conclusion, high IL‐11 expression is an independent predictor of poor prognosis in ccRCC patients. It may help identify patients who could benefit from additional treatments and closer follow up.

Decreased Expression of SETD2 Predicts Unfavorable Prognosis in Patients With Nonmetastatic Clear-Cell Renal Cell Carcinoma

AbstractDNA sequencing revealed that mutations in SETD2 occur in 3% to 12% of clear-cell renal cell carcinoma (ccRCC) cases and are associated with poor clinical outcome. In this study, we used an immunohistochemistry (IHC) assay to evaluate the impact of SETD2 loss, with expression of H3K36me3, a nonredundantly histone modification by SETD2, on recurrence and survival of nonmetastatic ccRCC patients after nephrectomy.SETD2 and H3K36me3 were assessed in 192 nonmetastatic ccRCC patients enrolled retrospectively from a single institution. Kaplan–Meier and Cox regression analysis were used to associate prespecified SETD2/H3K36me3 score with overall survival (OS) and recurrence-free survival (RFS). And a nomogram was constructed to predict OS at 10 years.Patients with low expression of SETD2 were prone to possess large tumor size and advanced pT stage. And low H3K36me3 expression was associated with larger tumor size. A prespecified combined score based on SETD2 and H3K36me3 expression remained an independent prognosticator for OS and RFS, which was associated with tumor size, pT stage, and sarcomatoid. Furthermore, using prespecified SETD2/H3K36me3 score could stratify nonmetastatic ccRCC patients into different risk subgroups, especially in patients dichotomized by pT stage and Fuhrman grade, respectively. Finally, the C-index for predicting OS increased from 0.727 to 0.747, after adding SETD2/H3K36me3 score to pT stage and Fuhrman grade.The combined score based on expression of SETD2 and H3K36me3 using IHC could predict poor clinical outcomes in nonmetastatic ccRCC patients, and it may benefit preoperative risk stratification and guide treatment planning in the future.