Qingyu Shen

Sirtuin7 oncogenic potential in human hepatocellular carcinoma and its regulation by the tumor suppressors MiR-125a-5p and MiR-125b.

Sirtuins are nicotinamide adenine dinucleotide oxidized form (NAD+)‐dependent deacetylases and function in cellular metabolism, stress resistance, and aging. For sirtuin7 (SIRT7), a role in ribosomal gene transcription is proposed, but its function in cancer has been unclear. In this study we show that SIRT7 expression was up‐regulated in a large cohort of human hepatocellular carcinoma (HCC) patients. SIRT7 knockdown influenced the cell cycle and caused a significant increase of liver cancer cells to remain in the G1/S phase and to suppress growth. This treatment restored p21WAF1/Cip1, induced Beclin‐1, and repressed cyclin D1. In addition, sustained suppression of SIRT7 reduced the in vivo tumor growth rate in a mouse xenograft model. To explore mechanisms in SIRT7 regulation, microRNA (miRNA) profiling was carried out. This identified five significantly down‐regulated miRNAs in HCC. Bioinformatics analysis of target sites and ectopic expression in HCC cells showed that miR‐125a‐5p and miR‐125b suppressed SIRT7 and cyclin D1 expression and induced p21WAF1/Cip1‐dependent G1 cell cycle arrest. Furthermore, treatment of HCC cells with 5‐aza‐2′‐deoxycytidine or ectopic expression of wildtype but not mutated p53 restored miR‐125a‐5p and miR‐125b expression and inhibited tumor cell growth, suggesting their regulation by promoter methylation and p53 activity. To show the clinical significance of these findings, mutations in the DNA binding domain of p53 and promoter methylation of miR‐125b were investigated. Four out of nine patients with induced SIRT7 carried mutations in the p53 gene and one patient showed hypermethylation of the miR‐125b promoter region. Conclusion: Our findings suggest the oncogenic potential of SIRT7 in hepatocarcinogenesis. A regulatory loop is proposed whereby SIRT7 inhibits transcriptional activation of p21WAF1/Cip1 by way of repression of miR‐125a‐5p and miR‐125b. This makes SIRT7 a promising target in cancer therapy. (HEPATOLOGY 2013)

MiR-145 functions as a tumor suppressor by directly targeting histone deacetylase 2 in liver cancer

Aberrant regulation of histone deacetylase 2 (HDAC2) plays a pivotal role in the development of hepatocellular carcinoma (HCC), but, the underlying mechanism leading to HDAC2 overexpression is not well understood. We performed microRNA (miRNA) profiling analysis in a subset of HCCs, and identified four down-regulated miRNAs that may target HDAC2 in HCC. Ectopic expression of miRNA mimics evidenced that miR-145 suppresses HDAC2 expression in HCC cells. This treatment repressed cancer cell growth and recapitulated HDAC2 knockdown effects on HCC cells. In conclusion, we suggest that loss or suppression of miR-145 may cause aberrant overexpression of HDAC2 and promote HCC tumorigenesis.

MicroRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver cancer

BACKGROUND & AIMS Most common reason behind changes in histone deacetylase (HDAC) function is its overexpression in cancer. However, among HDACs in liver cancer, HDAC6 is uniquely endowed with a tumor suppressor, but the mechanism underlying HDAC6 inactivation has yet to be uncovered. METHODS Microarray profiling and target prediction programs were used to identify miRNAs targeting HDAC6. A series of inhibitors, activators and siRNAs was introduced to validate regulatory mechanisms for microRNA-221-3p (miR-221) governing HDAC6 in hepatocarcinogenesis. RESULTS Comprehensive miRNA profiling analysis identified seven putative endogenous miRNAs that are significantly upregulated in hepatocellular carcinoma (HCC). While miR-221 was identified as a suppressor of HDAC6 by ectopic expression of miRNA mimics in Dicer knockdown cells, targeted-disruption of miR-221 repressed cancer cell growth through derepressing HDAC6 expression. Suppression of HDAC6 via miR-221 was induced by JNK/c-Jun signaling in liver cancer cells but not in normal hepatic cells. Additionally, cytokine-induced NF-κBp65 independently regulated miR-221, thereby suppressing HDAC6 expression in HCC cells. HCC tissues derived from chemical-induced rat and H-ras12V transgenic mice liver cancer models validated that JNK/c-Jun activation and NF-κBp65 nuclear translocation are essential for the transcription of miR-221 leading to repression of HDAC6 in HCC. CONCLUSIONS Our findings suggest that the functional loss or suppression of the tumor suppressor HDAC6 is caused by induction of miR-221 through coordinated JNK/c-Jun- and NF-κB-signaling pathways during liver tumorigenesis, providing a novel target for the molecular treatment of liver malignancies.

MiR-101 functions as a tumor suppressor by directly targeting nemo-like kinase in liver cancer

Nemo-like kinase (NLK), an evolutionarily conserved MAP kinase-related kinase, has been reported to be involved in the development of hepatocellular carcinoma (HCC), but the underlying mechanisms leading to oncogenic NLK are poorly understood. A comprehensive microRNA (miRNA) profiling analysis on human HCC tissues identified four downregulated miRNAs that may target NLK. Ectopic expression of miRNA mimics suggested that miR-101 could suppress NLK in HCC cells. Notably, ectopic miR-101 expression repressed cancer cell growth and proliferation and imitated NLK knockdown effect on HCC cells. In conclusion, we suggest that miR-101 functions as a tumor suppressor by regulating abnormal NLK activity in liver.

Targeted inactivation of HDAC2 restores p16INK4a activity and exerts antitumor effects on human gastric cancer.

Aberrant regulation of histone deacetylase 2 (HDAC2) was reported for gastric cancers. However, responsive cancer genes in disease onset and progression are less understood. HDAC2 expression was studied by quantitative RT-PCR and Western blotting. The functional consequences of HDAC2 knockdown on cell-cycle regulation, programmed cell death, and gene target identification was investigated by flow cytometry, Western blotting, electron microscopy, anchorage-independent colony formation, and cell migration assay and by whole-genome microarray. Therapeutic efficacy of HDAC2 knockdown was determined in nude mice with small hairpin expressing human gastric cancer cells. Epigenetic regulation of p16INK4a was studied by methylation-specific PCR and chromatin-IP to evidence HDAC2 or acetylated-histone-H4 binding at gene specific promoter sequences. HDAC2 gene and protein expression was significantly upregulated in different histopathologic grades of human gastric cancers and cancer cell lines. HDAC2 inactivation significantly reduced cell motility, cell invasion, clonal expansion, and tumor growth. HDAC2 knockdown-induced G1–S cell cycle arrest and restored activity of p16INK4a and the proapoptotic factors. This treatment caused PARP cleavage and hypophosphorylation of the Rb-protein, repressed cyclinD1, CDK4, and Bcl-2 expression and induced autophagic phenotype, that is, LC3B-II conversion. Some gastric tumors and cancer cells displayed p16INK4a promoter hypermethylation but treatment with 5-aza-deoxycitidine restored activity. With others the methylation status was unchanged. Here, chromatin-IP evidenced HDAC2 binding. Nonetheless, expression of p16INK4a was restored by HDAC2 knockdown with notable histone-H4-acetylation, as determined by chromatin-IP. Thus, p16INK4a is regulated by HDAC2. HDAC2 is a bona fide target for novel molecular therapies in gastric cancers. Mol Cancer Res; 11(1); 62–73. ©2012 AACR.

HDAC2 Provides a Critical Support to Malignant Progression of Hepatocellular Carcinoma through Feedback Control of mTORC1 and AKT

Aberrant regulation of histone deacetylase 2 (HDAC2) contributes to malignant progression in various cancers, but the underlying mechanism leading to the activation of oncogenic HDAC2 remains unknown. In this study, we show that HDAC2 expression is upregulated in a large cohort of patients with human hepatocellular carcinoma, and that high expression of HDAC2 was significantly associated with poor prognosis of patients with hepatocellular carcinoma. We found that mTORC1/NF-κBp50 signaling is necessary for the growth factor-induced HDAC2 and is sustained in hepatocellular carcinoma, but not in normal hepatic cells. Growth factor-induced mTORC1 activates the nuclear translocation of NF-κBp50, where it binds to the intragenic sequences of the HDAC2 gene and promotes its transcription. Hepatocellular carcinoma tissues derived from chemical-induced mouse and rat liver cancer models validated that mTORC1 activation and NF-κBp50 nuclear translocation are essential for the transcriptional activation of oncogenic HDAC2 in hepatocellular carcinoma. In addition, we demonstrate that HDAC2 is required to maintain mTORC1 activity by stabilizing the mTOR/RAPTOR complex. Elevated expression of HDAC2 triggers a positive feedback loop that activates AKT phosphorylation via the transcriptional modulation of phosphoinositide signaling molecules. Bioinformatics analysis of HDAC2 signature and immunoblot analysis of mesenchymal genes also evidenced that HDAC2 plays a role in the malignant behavior of tumor cells by Snail induction and simultaneously E-cadherin suppression in hepatocellular carcinoma cells. These findings establish a molecular mechanism responsible for the activation of oncogenic HDAC2, which explains how growth factor-induced HDAC2 maintains mitogenic signaling and function during hepatocellular malignant progression and provide a novel strategy for therapeutic intervention in liver cancer. Cancer Res; 74(6); 1728-38. ©2014 AACR.

Characteristic molecular signature for the early detection and prediction of polycyclic aromatic hydrocarbons in rat liver

Predictions of toxicity are central for the assessment of chemical toxicity, and the effects of environmental toxic compounds are still a major issue for predicting potential human health risks. Among the various environmental toxicants, polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutant, and many PAHs are known or suspected carcinogens. In the present study, to investigate whether characteristic expression profiles of PAHs exist in rat liver and whether a characteristic molecular signature can discriminate and predict among different PAHs at an early exposure time, we analyzed the genome-wide expression profiles of rat livers exposed to PAHs [benzo[a]anthracene (BA), benzo[a]pyrene (BP), phenanthrene (PA) and naphthalene (NT)]. At early time-point PAH exposure, large-scale gene expression analysis resulted in characteristic molecular signatures for each PAH, and supervised analysis identified 1183 outlier genes as a distinct molecular signature discerning PAHs from the normal control group. We identified 158 outlier genes as early predictive and surrogate markers for predicting each tested PAH by combination of two different multi-classification algorithms with 100% accuracy through a leave-one out cross-validation method. In conclusion, the characteristic gene expression signatures from a rat model system could be used as predictable and discernible gene-based biomarkers for the detection and prediction of PAHs, and these molecular markers may provide insights into the underlying mechanisms for genotoxicity of exposure to PAHs from environmental aspect.

Oncogenic potential of histone-variant H2A.Z.1 and its regulatory role in cell cycle and epithelial-mesenchymal transition in liver cancer.

H2A.Z is a highly conserved H2A variant, and two distinct H2A.Z isoforms, H2A.Z.1 and H2A.Z.2, have been identified as products of two non-allelic genes, H2AFZ and H2AFV. H2A.Z has been reported to be overexpressed in breast, prostate and bladder cancers, but most studies did not clearly distinguish between isoforms. One recent study reported a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. Here we first report that H2A.Z.1 plays a pivotal role in the liver tumorigenesis by selectively regulating key molecules in cell cycle and epithelial-mesenchymal transition (EMT). H2AFZ expression was significantly overexpressed in a large cohort of hepatocellular carcinoma (HCC) patients, and high expression of H2AFZ was significantly associated with their poor prognosis. H2A.Z.1 overexpression was demonstrated in a subset of human HCC and cell lines. H2A.Z.1 knockdown suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins and caused apoptotic cell death of HCC cells. We also observed that H2A.Z.1 knockdown reduced the metastatic potential of HCC cells by selectively modulating epithelial-mesenchymal transition regulatory proteins such as E-cadherin and fibronectin. In addition, H2A.Z.1 knockdown reduced the in vivo tumor growth rate in a mouse xenograft model. In conclusion, our findings suggest the oncogenic potential of H2A.Z.1 in liver tumorigenesis and that it plays established role in accelerating cell cycle transition and EMT during hepatocarcinogenesis. This makes H2A.Z.1 a promising target in liver cancer therapy.

Oncogenic potential of CK2α and its regulatory role in EGF-induced HDAC2 expression in human liver cancer.

Histone deacetylase 2 (HDAC2) is aberrantly regulated and plays a pivotal role in the development of hepatocellular carcinoma (HCC) through regulation of cell‐cycle components at the transcriptional level, but the underlying mechanism leading to oncogenic HDAC2 remains unknown. In this study, we show that expression of CK2α (casein kinase II α subunit) was up‐regulated in a large cohort of human HCC patients, and that high expression of CK2α was significantly associated with poor prognosis of HCC patients in terms of five‐year overall survival. It was also found that CK2α over‐expression positively correlated with HDAC2 over‐expression in a subset of HCCs. We observed that treatment with epidermal growth factor (EGF) elicited an increase in CK2α expression and Akt phosphorylation, causing induction of HDAC2 expression in liver cancer cells. It was also observed that ectopic expression of dominant‐negative CK2α blocked EGF‐induced HDAC2 expression, and that ectopic CK2α expression attenuated the suppressive effect of Akt knockdown on HDAC2 expression in liver cancer cells. Targeted disruption of CK2α influenced the cell cycle, causing a significant increase in the number of liver cancer cells remaining in G2/M phase, and suppressed growth via repression of Cdc25c and cyclin B in liver cancer cells. Taken together, our findings suggest the oncogenic potential of CK2α in liver tumorigenesis. Furthermore, a regulatory mechanism for HDAC2 expression is proposed whereby EGF induces transcriptional activation of HDAC2 by CK2α/Akt activation in liver cancer cells. Therefore, this makes CK2α a promising target in cancer therapy.

Epigenetic reader BRD4 inhibition as a therapeutic strategy to suppress E2F2-cell cycle regulation circuit in liver cancer

Deregulation of the epigenome component affects multiple pathways in the cancer phenotype since the epigenome acts at the pinnacle of the hierarchy of gene expression. Pioneering work over the past decades has highlighted that targeting enzymes or proteins involved in the epigenetic regulation is a valuable approach to cancer therapy. Very recent results demonstrated that inhibiting the epigenetic reader BRD4 has notable efficacy in diverse cancer types. We investigated the potential of BRD4 as a therapeutic target in liver malignancy. BRD4 was overexpressed in three different large cohort of hepatocellular carcinoma (HCC) patients as well as in liver cancer cell lines. BRD4 inhibition by JQ1 induced anti-tumorigenic effects including cell cycle arrest, cellular senescence, reduced wound healing capacity and soft agar colony formation in liver cancer cell lines. Notably, BRD4 inhibition caused MYC-independent large-scale gene expression changes in liver cancer cells. Serial gene expression analyses with SK-Hep1 liver cancer cells treated with JQ1 to delineate the key player of BRD4 inhibition identified E2F2 as the first line of downstream direct target of BRD4. Further experiments including chromatin immunoprecipitation (ChIP) assay and loss of function study confirmed E2F2 as key player of BRD4 inhibition. Overexpressed E2F2 is a crucial center of cell cycle regulation and high expression of E2F2 is significantly associated with poor prognosis of HCC patients. Our findings reveal BRD4-E2F2-cell cycle regulation as a novel molecular circuit in liver cancer and provide a therapeutic strategy and innovative insights for liver cancer therapies.