Woo Chan Shin

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.

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.

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.

Characteristic molecular and proteomic signatures of drug-induced liver injury in a rat model.

Drug‐induced liver injury (DILI) is a major safety concern during drug development and remains one of the main reasons for withdrawal of drugs from the market. Although it is crucial to develop methods that will detect potential hepatotoxicity of drug candidates as early and as quickly as possible, there is still a lack of sensitive and specific biomarkers for DILI that consequently leads to a scarcity of reliable hepatotoxic data. Hence, in this study, we assessed characteristic molecular signatures in rat liver treated with drugs (pyrazinamide, ranitidine, enalapril, carbamazepine and chlorpromazine) that are known to cause DILI in humans. Unsupervised hierarchical clustering analysis of transcriptome changes induced by DILI‐causing drugs resulted in three different subclusters on dendrogram, i.e., hepatocellular, cholestatic and mixed type of DILI at early time points (2 days), and multiclassification analysis suggested 31 genes as discernible markers for each DILI pattern. Further analysis for characteristic molecular signature of each DILI pattern provided a molecular basis for different modes of DILI action. A proteomics study of the same rat livers was used to confirm the results, and the two sets of data showed 60 matching classifiers. In conclusion, the data of different DILI‐causing drug treatments from genomic analysis in a rat model suggest that DILI‐specific molecular signatures can discriminate different patterns of DILI at an early exposure time point, and that they provide useful information for mechanistic studies that may lead to a better understanding of the molecular basis of DILI. Copyright

HDAC6 sustains growth stimulation by prolonging the activation of EGF receptor through the inhibition of rabaptin-5-mediated early endosome fusion in gastric cancer

The aberrant regulation of histone deacetylase 6 (HDAC6) contributes to malignant progression in various types of cancer, but the mechanism underlying gastric carcinogenesis remains unknown. Aberrant HDAC6 overexpression was observed in a subset of human gastric cancer cells. HDAC6 knockdown caused the significant inhibition of gastric cancer cell growth without affecting the transition of cell cycles or the processing of cell death. We demonstrate that an increase in epidermal growth factor receptor (EGFR) signaling through decreased EGFR degradation was mediated by HDAC6 in gastric carcinogenesis. These results establish a molecular mechanism responsible for oncogenic HDAC6, explaining how EGFR signaling induced by the growth factor is sustained during the malignant progression of gastric cancer.

Abstract 4810: Histone deacetylase 2 is a potential determinant of the sensitivity of hepatocellular carcinoma cells to sorafenib

Hepatocellular carcinoma (HCC) is the fifth frequently diagnosed cancer and is the second leading cause of cancer death worldwide. Many groups have suggested possible clinical applications for liver cancer therapy but sorafenib, an orally-available kinase inhibitor, is the only standard systematic therapeutics for treatment of hepatocellular carcinoma. However, survival benefit of sorafenib is unsatisfactory due to high level heterogeneity of individual response. We previously reported that histone deacetylase 2 (HDAC2) was deregulated in HCC, and thereby contributed to liver tumorigenesis by enhancing mitotic and metastatic potential of transformed cells. Targeted-disruption of HDAC2 suppressed in vitro and in vivo tumorgigenic potential of HDAC2 in liver cancer. The goal of this study was to investigate whether both sorafenib treatment and HDAC2 inhibition elicit synergistic anti-tumor effect against HCC cells. Here, we showed that sorafenib effectively blocked ERK/MEK and AKT signaling pathway upon EGF stimulation in liver cancer cell lines. Also, we found that valproic acid (VPA), a selective inhibitor of histone deacetylase family I, and sorafenib treatment synergistically evoked liver cancer cell growth retardation. This effect was recapitulated by HDAC2 knockdown with sorafenib treatment and exhibited synergistic effect on apoptotic cell death of liver cancer cell lines, Hep3B and Huh7, compared to single treatment of sorafenib. Our data suggest that combined sorafenib treatment with HDAC2 targeting may provide more benefits toward HCC therapy providing a novel approach for future application in patients with advanced HCC. Citation Format: Hyung Seok Kim, Jung Woo Eun, Qingyu Shen, Woo Chan Shin, Hee Doo Yang, Sang Yeon Kim, Suk Woo Nam. Histone deacetylase 2 is a potential determinant of the sensitivity of hepatocellular carcinoma cells to sorafenib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4810.

Abstract 3111: JNK/c-Jun- and NF-κB-mediated microRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver cancer

Many clinical studies of cancer patients have established that the most common reason behind changes in HDAC function is its overexpression. However, among HDACs in liver cancer, HDAC6, a cytoplasmic tubulin deacetylase, is uniquely endowed with a tumor suppressor, but the mechanism underlying HDAC6 inactivation has yet to be uncovered. In this study we show that genetic and epigenetic dysregulation was not part of the HDAC6 inactivation mechanism. Instead, microRNA-221-3p (miR-221) directly suppressed HDAC6 in hepatocellular carcinoma (HCC) cells. A comprehensive miRNA profiling analysis identified seven putative endogenous miRNAs that are significantly upregulated in HCC. Ectopic expression of miRNA mimics in Dicer knockdown cells evidenced miR-221 to suppress HDAC6 in HCC cells. Notably, targeted-disruption of miR-221 repressed cancer cell growth and proliferation, and it recapitulated HDAC6 overexpression effects in HCC cells. Our results also demonstrate that c-Met-mediated c-Jun NH2-terminal kinase (JNK)/c-Jun signaling induced miR-221 to suppress HDAC6 and was sustained in liver cancer cells but not in normal hepatic cells. In addition, cytokine-induced NF-κBp65 independently regulated miR-221, thereby suppressing HDAC6 expression in liver cancer cells. HCC tissues derived from chemical-induced rat liver cancer models validated that JNK/c-Jun activation and NF-kBp65 nuclear translocation are essential for the transcriptional activation of oncogenic miR-221 to repress tumor suppressor HDAC6 in HCC. In conclusion, our finding suggest that the functional loss or suppression of the tumor suppressor HDAC6 is caused by 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. Citation Format: Hyung Seok Kim, Qingyu Shen, Jung Woo Eun, Woo Chan Shin, Hee Doo Yang, Won Sang Park, Joung Young Lee, Suk Woo Nam. JNK/c-Jun- and NF-κB-mediated microRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2015-3111

Identification of aberrant overexpression of long non-coding RNA MALAT1 and role as a regulatory microRNA in liver cancer

MALAT1 is deregulated in various cancers. The underlying mechanisms of MALA T1-mediated tumorigenesis are unclear. We found that MALAT1 was highly overexpressed and its overexpression was significantly associated with poor prognosis of liver cancer patients analyzed from the TCGA Liver Hepatocellular Carcinoma and Gene Expression Omnibus databases of the National Center for Biotechnology Information. Microarray analysis to identify the miRNAs deregulated by silencing MALAT1 in two liver cancer cell lines, HepG2 and Hep3B, revealed the common deregulation of 16 miRNAs including miR-574 and miR-20b in both cell lines. The predicted targets of miR-574 and miR-20b were cancer-related pathways including the RAS, MAPK and Wnt/β-catenin pathways. Aberrant expression of MALAT1 might contribute to liver cancer tumorigenesis by deregulation of cancer-associated miRNAs.

Abstract 5261: Aberrant expression of HDAC6 sustains malignant progression by preventing epidermal growth factor receptor degradation in gastric cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cytoplasmic deacetylase, histone deacetylase 6 (HDAC6) is a critical component of the lysomal protein degradation pathways. HDAC6 has been shown to be involved in carcinogenic transformation and to modulate the epithelial-mesenchymal transition in several cancers by way of the regulations of several critical cellular functions, but the biological roles in gastric carcinogenesis remains to be elucidated. In this study, we observed that HDAC6 is aberrantly over-expressed in a subset of gastric cancer, and that targeted-inactivation of HDC6 caused cell cycle and cell death pathways-independent growth inhibition. Additional experiments evidenced that ectopic overexpression of HDAC6 sustains epidermal growth factor receptor (EGFR) degradation in EGF-stimulated gastric cancer cells. In addition, we found that HDAC6 inactivation recovered rabaptin 5, a Rab5 interacting protein potentiating Rab5, a small GTPase that regulates early endocytic events. These findings suggest that aberrant overexpression of HDAC6 confers gastric cancer cells to sustain EGF-signaling by retardation of EGFR degradation by way of repression of rababtin-5, and HDAC6 is a bona fide target for therapeutic intervention in gastric cancers. Note: This abstract was not presented at the meeting. Citation Format: Se Jin Park, Hyun Jin Bae, Jung Woo Eun, Qinngyu Shen, Hyung Seok Kim, Woo Chan Shin, Won Sang Park, Jung Young Lee, Suk Woo Nam. Aberrant expression of HDAC6 sustains malignant progression by preventing epidermal growth factor receptor degradation in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2014-5261