Ruoyu Wang

Epigenetic modification of MiR-429 promotes liver tumour-initiating cell properties by targeting Rb binding protein 4

Objective Liver tumour-initiating cells (T-ICs) are critical for hepatocarcinogenesis. However, the underlying mechanism regulating the function of liver T-ICs remains unclear. Methods Tissue microarrays containing 242 hepatocellular carcinoma (HCC) samples were used for prognostic analysis. Magnetically activated cell sorting was used to isolate epithelial cell adhesion molecule (EPCAM)-positive cells. The gene expressions affected by miR-429 were determined by arrays. Co-immunoprecipitation was used to study interactions among retinoblastoma protein (RB1), Rb binding protein 4 (RBBP4) and E2F transcription factor 1 (E2F1). The DNA methylation status in CpG islands was detected by quantitative methylation analysis. miRNAs in microvesicles were isolated by a syringe filter system. Results The significant prognosis factor miR-429 was upregulated in HCC tissues and also in primary liver T-ICs isolated from clinical samples. The enrichment of miR-429 in EPCAM+ T-ICs contributed to hepatocyte self-renewal, malignant proliferation, chemoresistance and tumorigenicity. A novel functional axis involving miR-429, RBBP4, E2F1 and POU class 5 homeobox 1 (POU5F1 or OCT4) governing the regulation of liver EPCAM+ T-ICs was established in vitro and in vivo. The molecular mechanism regulating miR-429 expression, involving four abnormal hypomethylated sites upstream of the miR-200b/miR-200a/miR-429 cluster, was first defined in both EPCAM+ liver T-ICs and very early-stage HCC tissues. miR-429 secreted by high-expressing cells has the potential to become a proactive signalling molecule to mediate intercellular communication. Conclusions Epigenetic modification of miR-429 can manipulate liver T-ICs by targeting the RBBP4/E2F1/OCT4 axis. This miRNA might be targeted to inactivate T-ICs, thus providing a novel strategy for HCC prevention and treatment.

PTPN11/Shp2 overexpression enhances liver cancer progression and predicts poor prognosis of patients.

BACKGROUND & AIMS We have previously reported that Shp2, a tyrosine phosphatase previously known as a pro-leukemogenic molecule, suppresses the initiation of hepatocellular carcinoma (HCC). However, the role of Shp2 in HCC progression remains obscure. METHODS Shp2 expression was determined in human HCC using real-time PCR, immunoblotting and immunohistochemistry. Clinical significance of Shp2 expression was analyzed in 301 HCC tissues with clinico-pathological characteristics and follow-up information. Short hairpin RNA was utilized to investigate the function of Shp2 in hepatoma cell behavior. Role of Shp2 in HCC progression was monitored through nude mice xenograft assay. Kinase activity assay and co-immunoprecipitation were used for mechanism analysis. RESULTS Elevated expression of Shp2 was detected in 65.9% (394/598) of human HCCs, and its levels were even higher in metastasized foci. Overexpression of Shp2 correlated well with the malignant clinico-pathological characteristics of HCC and predicted the poor prognosis of patients. Interference of Shp2 expression suppressed the proliferation of hepatoma cells in vitro and inhibited the growth of HCC xenografts in vivo. Down-regulation of Shp2 attenuated the adhesion and migration of hepatoma cells and diminished metastasized HCC formation in mice. Our data demonstrated that Shp2 promotes HCC growth and metastasis by coordinately activating Ras/Raf/Erk pathway and PI3-K/Akt/mTOR cascade. Moreover, down-regulation of Shp2 enhanced the sensitivity of hepatoma cells upon sorafenib treatment, and patients with low Shp2 expression exhibited superior prognosis to sorafenib. CONCLUSIONS Shp2 promotes the progression of HCC and may serve as a prognostic biomarker for patients.

Palmitic acid induces autophagy in hepatocytes via JNK2 activation

Aim:Free fatty acid-induced lipotoxicity plays a crucial role in the progression of nonalcoholic fatty liver disease (NAFLD). In the present study we investigated the effects of a high-fat diet and free fatty acids on the autophagic process in hepatocytes in vivo and in vitro and the underlying mechanisms.Methods:LC3-II expression, a hallmark of autophagic flux, was detected in liver specimens from patients with non-alcoholic steatohepatitis (NASH) as well as in the livers of C57BL/6 mice fed a high-fat diet (HFD) up to 16 weeks. LC3-II expression was also analyzed in human SMMC-7721 and HepG2 hepatoma cells exposed to palmitic acid (PA), a saturated fatty acid. PA-induced apoptosis was detected by Annexin V staining and specific cleavage of PARP in the presence and absence of different agents.Results:LC3-II expression was markedly increased in human NASH and in liver tissues of HFD-fed mice. Treatment of SMMC-7721 cells with PA increased LC3-II expression in time- and dose-dependent manners, whereas the unsaturated fatty acid oleic acid had no effect. Inhibition of autophagy with 3MA sensitized SMMC-7721 cells to PA-induced apoptosis, whereas activation of autophagy by rapamycin attenuated PA-induced PARP cleavage. The autophagy-associated proteins Beclin1 and Atg5 were essential for PA-induced autophagy in SMMC-7721 cells. Moreover, pretreatment with SP600125, an inhibitor of JNK, effectively abrogated PA-mediated autophagy and apoptosis. Specific knockdown of JNK2, but not JNK1, in SMMC-7721 cells significantly suppressed PA-induced autophagy and enhanced its pro-apoptotic activity; whereas specific knockdown of JNK1 had the converse effect. Similar results were obtained when HepG2 cells were tested.Conclusion:JNK1 promotes PA-induced lipoapoptosis, whereas JNK2 activates pro-survival autophagy and inhibits PA lipotoxicity. Our results suggest that modulation of autophagy may have therapeutic benefits in the treatment of lipid-related metabolic diseases.

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.

The Tyrosine Kinase c-Met Contributes to the Pro-tumorigenic Function of the p38 Kinase in Human Bile Duct Cholangiocarcinoma Cells

Background: Both p38 and c-Met are implicated in the tumorigenesis of cholangiocarcinoma. Results: The inhibition of p38 inhibits human cholangiocarcinoma cell proliferation and invasion through reducing the basal activity of c-Met. Conclusion: c-Met contributes to the pro-tumorigenic action of p38 in human cholangiocarcinoma cells. Significance: We identify a novel molecular mechanism that contributes to understanding the pro-tumorigenic activity of p38 in human cholangiocarcinoma cells. Pro-tumorigenic function of the p38 kinase plays a critical role in human cholangiocarcinogenesis. However, the underlying mechanism remains incompletely understood. Here, we report that c-Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), contributes to the pro-tumorigenic ability of p38 in human cholangiocarcinoma cells. Both p38 and c-Met promote the proliferation and invasion of human cholangiocarcinoma cells. Importantly, inhibition or knockdown of p38 decreased the basal activation of c-Met. Tyrosine phosphatase inhibitor studies revealed that p38 promotes the activity of c-Met, at least in part, by inhibiting dephosphorylation of the receptor. Moreover, density enhanced phosphatase-1 (DEP-1) is involved in p38-mediated inhibiting dephosphorylation of c-Met. Furthermore, p38 inhibits the degradation of c-Met. Taken together, these data provide a potential mechanism to explain how p38 promotes human cholangiocarcinoma cell proliferation and invasion. We propose that the link between p38 and c-Met is implicated in the progression of human cholangiocarcinoma.

Serum miRNAs as predictive and preventive biomarker for pre-clinical hepatocellular carcinoma.

The extremely poor prognosis of patients with symptomatic hepatocellular carcinoma (HCC) diagnosed clinically at advanced stages suggests an urgent need for biomarkers that can be used for prospective surveillance and pre-clinical screening for early presence of pre-malignant lesions and tumors. In a retrospective longitudinal phase 3 biomarker study in seven medical centers of China, time-series and 6 months interval-serum samples were collected from chronic hepatitis B virus infected (CHB) patient cohorts at the pre-malignant or pre-clinical stages (average 6 months prior to clinical diagnosis) and CHB patients that did not develop cancer, and circulating miRNAs measured. A set of serum miRNAs including miR-193a-3p, miR-369-5p, miR-672, miR-429 and let-7i* were identified in pre-clinical HCC patients and have the potential to screen for CHB patients at high risk to develop HCC 6-12 months after miRNAs measurement. These circulating miRNAs combined with the conventional screening tools using α-fetoprotein and ultrasound, may have great promise for the prediction and prevention of HCC in high-risk populations.

Inhibition of autophagy may suppress the development of hepatoblastoma

Hepatoblastoma (HB) is a rare cancer but represents the most common liver malignancy in children under 3 years of age. Nevertheless, a clear understanding of the pathogenesis is lacking. Although the treatment of HB has been dramatically improved by combining chemotherapy regimens with surgery, its fatal outcome of fast development and recurrence makes new treatment strategies for HB, based on an improved understanding of the pathogenesis, essential. Autophagy is believed to be important in the progression of cancers. However, the role of autophagy in HB remains to be elucidated. Here, we show that autophagy is activated in HB tissues and cells under the conditions of starvation or chemotherapy, coupled with the over‐expression of autophagic‐related genes BECN1 and ATG5. Suppression of autophagy with pharmacological agents and small interfering RNAs significantly increased cell apoptosis and retarded proliferation in response to nutrition deprivation and treatment with chemotherapeutics. Our data demonstrate that the BECN1 and ATG5‐dependent phosphoinositide 3‐kinase (PI3K) signaling pathway is essential for the survival of HB cells and their tolerance to chemotherapy and starvation‐induced death, and suggests that modifying such autophagic genes may suppress the development of HB, thus offering a therapeutic potential for patients with HB.

DNA Methylation of Tumor Suppressor Genes Located on Chromosome 3p in Non-small Cell Lung Cancer

BACKGROUND AND OBJECTIVE DNA methylation is one of the mechanisms of epigenetics. Allelic loss located on chromosome 3p happen frequently and early in non-small cell lung cancer (NSCLC). The aim of this study is to detect the promoter methylation status of tumor suppressor genes (TSGs) located on chromosome 3p in NSCLC and to evaluate its correlation with clinicopathological features. METHODS A total of 78 paired NSCLC specimens and their adjacent normal tissues were collected in the study. Promoter methylation status was determined by methylation-specific polymerase chain reaction (MSP). DLEC1 gene expression was determined by RT-PCR and immunohistochemistry. RESULTS Aberrant methylation frequency of DLEC1, RASSF1A, hMLH1, RARβ and FHIT genes detected in 78 NSCLC tissues were 41.03%, 39.74%, 30.77% and 16.67%, respectively, which were all significantly higher than that in adjacent normal tissues. However, FHIT gene was not detected methylation in both cancerous and non-cancerous tissues. DLEC1 hypermethylation was associated with advanced stage (P=0.011) and lymph metastasis (P=0.019), while RASSF1A, RARβ, hMLH1 and mean methylation index (MI) were not correlated with any clinicopathological parameters. Moreover, DLEC1 gene downregulation was detected in 56.41% (44/78) NSCLC tissues and correlated with promoter hypermethylation. CONCLUSIONS Frequent hypermethylation of TSGs located on chromosome 3p was a common event contributing to NSCLC pathogenesis and DLEC1 methylation was closely correlated with loss of expression.

CYP3A5 Functions as a Tumor Suppressor in Hepatocellular Carcinoma by Regulating mTORC2/Akt Signaling

CYP3A5 is a cytochrome P450 protein that functions in the liver metabolism of many carcinogens and cancer drugs. However, it has not been thought to directly affect cancer progression. In this study, we challenge this perspective by demonstrating that CYP3A5 is downregulated in many hepatocellular carcinomas (HCC), where it has an important role as a tumor suppressor that antagonizes the malignant phenotype. CYP3A5 was downregulated in multiple cohorts of human HCC examined. Lower CYP3A5 levels were associated with more aggressive vascular invasion, poor differentiation, shorter time to disease recurrence after treatment, and worse overall patient survival. Mechanistic investigations showed that CYP3A5 overexpression limited MMP2/9 function and suppressed HCC migration and invasion in vitro and in vivo by inhibiting AKT signaling. Notably, AKT phosphorylation at Ser473 was inhibited in CYP3A5-overexpressing HCC cells, an event requiring mTORC2 but not Rictor/mTOR complex formation. CYP3A5-induced ROS accumulation was found to be a critical upstream regulator of mTORC2 activity, consistent with evidence of reduced GSH redox activity in most clinical HCC specimens with reduced metastatic capacity. Taken together, our results defined CYP3A5 as a suppressor of HCC pathogenesis and metastasis with potential utility a prognostic biomarker.

Overexpression of CHKA contributes to tumor progression and metastasis and predicts poor prognosis in colorectal carcinoma.

Aberrant expression of choline kinase alpha (CHKA) has been reported in a variety of human malignancies including colorectal carcinoma (CRC). However, the role of CHKA in the progression and prognosis of CRC remains unknown. In this study, we found that CHKA was frequently upregulated in CRC clinical samples and CRC-derived cell lines and was significantly correlated with lymph node metastasis (p = 0.028), TNM stage (p = 0.009), disease recurrence (p = 0.004) and death (p < 0.001). Survival analyses indicated that patients with higher CHKA expression had a significantly shorter disease-free survival (DFS) and disease-specific survival (DSS) than those with lower CHKA expression. Multivariate analyses confirmed that increased CHKA expression was an independent unfavorable prognostic factor for CRC patients. In addition, combination of CHKA with TNM stage was a more powerful predictor of poor prognosis than either parameter alone. Functional study demonstrated that knockdown of CHKA expression profoundly suppressed the growth and metastasis of CRC cells both in vitro and in vivo. Mechanistic investigation revealed that EGFR/PI3K/AKT pathway was essential for mediating CHKA function. In conclusion, our results provide the first evidence that CHKA contributes to tumor progression and metastasis and may serve as a novel prognostic biomarker and potential therapeutic target in CRC.