Young Gyoon Chang

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.

HDAC2 overexpression confers oncogenic potential to human lung cancer cells by deregulating expression of apoptosis and cell cycle proteins

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses, and is often significantly overexpressed in solid tumors, but little is known of its role in human lung cancer. In this study, we demonstrated the aberrant expression of HDAC2 in lung cancer tissues and investigated oncogenic properties of HDAC2 in human lung cancer cell lines. HDAC2 inactivation resulted in regression of tumor cell growth and activation of cellular apoptosis via p53 and Bax activation and Bcl2 suppression. In cell cycle regulation, HDAC2 inactivation caused induction of p21WAF1/CIP1 expression, and simultaneously suppressed the expressions of cyclin E2, cyclin D1, and CDK2, respectively. Consequently, this led to the hypophosphorylation of pRb protein in G1/S transition and thereby inactivated E2F/DP1 target gene transcriptions of A549 cells. In addition, we demonstrated that HDAC2 directly regulated p21WAF1/CIP1 expression in a p53‐independent manner. However, HDAC1 was not related to p21WAF1/CIP1 expression and tumorigenesis of lung cancer. Lastly, we observed that sustained‐suppression of HDAC2 in A549 lung cancer cells attenuated in vitro tumorigenic properties and in vivo tumor growth of the mouse xenograft model. Taken together, we suggest that the aberrant regulation of HDAC2 and its epigenetic regulation of gene transcription in apoptosis and cell cycle components play an important role in the development of lung cancer. J. Cell. Biochem. 113: 2167–2177, 2012.

HDAC1 inactivation induces mitotic defect and caspase-independent autophagic cell death in liver cancer

Histone deacetylases (HDACs) are known to play a central role in the regulation of several cellular properties interlinked with the development and progression of cancer. Recently, HDAC1 has been reported to be overexpressed in hepatocellular carcinoma (HCC), but its biological roles in hepatocarcinogenesis remain to be elucidated. In this study, we demonstrated overexpression of HDAC1 in a subset of human HCCs and liver cancer cell lines. HDAC1 inactivation resulted in regression of tumor cell growth and activation of caspase-independent autophagic cell death, via LC3B-II activation pathway in Hep3B cells. In cell cycle regulation, HDAC1 inactivation selectively induced both p21WAF1/Cip1 and p27Kip1 expressions, and simultaneously suppressed the expression of cyclin D1 and CDK2. Consequently, HDAC1 inactivation led to the hypophosphorylation of pRb in G1/S transition, and thereby inactivated E2F/DP1 transcription activity. In addition, we demonstrated that HDAC1 suppresses p21WAF1/Cip1 transcriptional activity through Sp1-binding sites in the p21WAF1/Cip1 promoter. Furthermore, sustained suppression of HDAC1 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Taken together, we suggest the aberrant regulation of HDAC1 in HCC and its epigenetic regulation of gene transcription of autophagy and cell cycle components. Overexpression of HDAC1 may play a pivotal role through the systemic regulation of mitotic effectors in the development of HCC, providing a particularly relevant potential target in cancer therapy.

Aberrant Regulation of HDAC2 Mediates Proliferation of Hepatocellular Carcinoma Cells by Deregulating Expression of G1/S Cell Cycle Proteins

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses and is often significantly overexpressed in solid tumors; but little is known about its role in human hepatocellular carcinoma (HCC). In this study, we showed that targeted-disruption of HDAC2 resulted in reduction of both tumor cell growth and de novo DNA synthesis in Hep3B cells. We then demonstrated that HDAC2 regulated cell cycle and that disruption of HDAC2 caused G1/S arrest in cell cycle. In G1/S transition, targeted-disruption of HDAC2 selectively induced the expression of p16INK4A and p21WAF1/Cip1, and simultaneously suppressed the expression of cyclin D1, CDK4 and CDK2. Consequently, HDAC2 inhibition led to the down-regulation of E2F/DP1 target genes through a reduction in phosphorylation status of pRb protein. In addition, sustained suppression of HDAC2 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Further, we found that HDAC2 suppresses p21WAF1/Cip1 transcriptional activity via Sp1-binding site enriched proximal region of p21WAF1/Cip1 promoter. In conclusion, we suggest that the aberrant regulation of HDAC2 may play a pivotal role in the development of HCC through its regulation of cell cycle components at the transcription level providing HDAC2 as a relevant target in liver cancer therapy.

Histone deacetylase 6 functions as a tumor suppressor by activating c-Jun NH2-terminal kinase-mediated beclin 1-dependent autophagic cell death in liver cancer†

Ubiquitin‐binding histone deacetylase 6 (HDAC6) is uniquely endowed with tubulin deacetylase activity and plays an important role in the clearance of misfolded protein by autophagy. In cancer, HDAC6 has become a target for drug development due to its major contribution to oncogenic cell transformation. In the present study we show that HDAC6 expression was down‐regulated in a large cohort of human hepatocellular carcinoma (HCC) patients, and that low expression of HDAC6 was significantly associated with poor prognosis of HCC patients in 5‐year overall, disease‐free, and recurrence‐free survival. Notably, we observed that ectopic overexpression of HDAC6 suppressed tumor cell growth and proliferation in various liver cancer cells, and elicited increased LC3B‐II conversion and autophagic vacuole formation without causing apoptotic cell death or cell cycle inhibition. In addition, the sustained overexpression of HDAC6 reduced the in vivo tumor growth rate in a mouse xenograft model. It was also found that HDAC6 mediated autophagic cell death by way of Beclin 1 and activation of the LC3‐II pathway in liver cancer cells, and that HDAC6 overexpression activated c‐Jun NH2‐terminal kinase (JNK) and increased the phosphorylation of c‐Jun. In contrast, the induction of Beclin 1 expression was blocked by SP600125 (a specific inhibitor of JNK) or by small interfering RNA directed against HDAC6. Conclusion: Our findings suggest that loss of HDAC6 expression in human HCCs and tumor suppression by HDAC6 occur by way of activation of caspase‐independent autophagic cell death through the JNK/Beclin 1 pathway in liver cancer and, thus, that a novel tumor suppressor function mechanism involving HDAC6 may be amenable to nonepigenetic regulation. (HEPATOLOGY 2012)

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.

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 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.

Characteristic Molecular Signature for Early Detection and Prediction of Persistent Organic Pollutants in Rat Liver

Persistent organic pollutants (POPs) are degradation-resistant anthropogenic chemicals that accumulate in the food chain and in adipose tissue, and are among the most hazardous compounds ever synthesized. However, their toxic mechanisms are still undefined. To investigate whether characteristic molecular signatures can discriminate individual POP and provide prediction markers for the early detection of POPs exposure in an animal model, we performed transcriptomic analysis of rat liver tissues after exposure to POPs. The six different POPs (toxaphene, hexachlorobenzene, chlordane, mirex, dieldrin, and heptachlor) were administered to 11-week-old male Sprague-Dawley rats, and after 48 h of exposure, RNAs were extracted from liver tissues and subjected to rat whole genome expression microarrays. Early during exposure, conventional toxicological analysis including changes in the body and organ weight, histopathological examination, and blood biochemical analysis did not reflect any toxicant stresses. However, unsupervised gene expression analysis of rat liver tissues revealed in a characteristic molecular signature for each toxicant, and supervised analysis identified 2708 outlier genes that discerned the POPs exposure group from the vehicle-treated control. Combination analysis of two different multiclassifications suggested 384 genes as early detection markers for predicting each POP exposure with 100% accuracy. The data from large-scale gene expression analysis of a different POP exposure in rat model suggest that characteristic expression profiles exist in liver hepatic cells and multiclassification of POP-specific molecular signatures can discriminate each toxicant at an early exposure time. The use of these molecular markers may be more widely implemented in combination with more traditional techniques for assessment and prediction of toxicity exposure to POPs from an environmental aspect.