Jora M. J. Lin

Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis.

Aberrant JAK/STAT Signaling Suppresses TFF1 and TFF2 through Epigenetic Silencing of GATA6 in Gastric Cancer

Aberrant Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling is crucial to the development of gastric cancer. In this study, we examined the role of STAT3 in the expression and methylation of its targets in gastric cancer patients. Results from RNA sequencing identified an inverse correlation between the expression of STAT3 and GATA6 in 23 pairs of gastric cancer patient samples. We discovered that the expression of GATA6 is epigenetically silenced through promoter methylation in gastric cancer cell lines. Interestingly, the inhibition of STAT3 using a novel STAT3 inhibitor restored the expression of GATA6 and its targets, trefoil factors 1 and 2 (TFF1/2). Moreover, disruption of STAT3 binding to GATA6 promoter by small hairpin RNA restored GATA6 expression in AGS cells. A clinically significant correlation was also observed between the expression of GATA6 and TFF1/2 among tissue samples from 60 gastric cancer patients. Finally, bisulfite pyrosequencing revealed GATA6 methylation in 65% (39/60) of the patients, and those with higher GATA6 methylation tended to have shorter overall survival. In conclusion, we demonstrated that aberrant JAK/STAT signaling suppresses TFF1/2 partially through the epigenetic silencing of GATA6. Therapeutic intervention of STAT3 in reversing the epigenetic status of GATA6 could benefit the treatment of gastric cancer and is worthy of further investigation.

Abstract 74: E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer

Ovarian cancer is one of the most lethal cancers in the female reproductive system. Previous study suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To decipher this complicated process, we generated a mathematical model and found that estrogen-mediated up-regulation of E2F6 could upregulate the ovarian cancer stem/initiating marker, c-kit by two means one through epigenetic silencing of their co-targeted miR193a by binding of E2F6 which subsequently recruit EZH2 to miR-193a promoter; and second, by competing endogenous (ceRNA) mechanism. To confirm this model, treatment of E2 or environmental hormone, BPA resulted in upregulation of both E2F6 and c-kit but down-regulation of miR-193a in immortalized ovarian surface epithelial cells. Further bisulfite pyrosequencing, ChIP-qPCR and epigenetic treatment found that miR193a was epigenetically silenced by DNA methylation and H3K27me3 in CP70 but not HeyC2 ovarian cancer cells. Overexpression of miR193a inhibited tumor growth in vitro and in vivo. Depletion of EZH2 or E2F6 in CP70 restored miR-193a expression and decreased the number of “ovo” spheroid by reversing the repressive chromatin status of miR-193a promoter. To further explore the biological significance of this E2F6 ceRNA network, integrative RNA-Seq and computational analysis found that PBX1, a miR-193a target and transcriptional activator of the immunosuppressive cytokine IL-10, was down-regulated in E2F6 and EZH2 knockdown CP70 cells. Overexpression of E2F6 39UTR containing miR-193a MRE but not MRE mutant increased the expression of PBX1 and IL10 in ovarian cancer cells. Importantly, co-culture of conditional media from E2F6 39UTR overexpressing CP70 cells inhibited the differentiation of THP-1 monocytes into dendritic cell and the T-cell activating function of this THP-1 derived DC. This phenomenon can be rescued by incubation of anti-IL-10 antibody or pretreatment of CP70 cells with EZH2 inhibitor. Finally, clinical studies demonstrated that patients with higher promoter methylation of miR193a were associated with poor survival. Serum IL10 level was found to be higher in high staged ovarian cancer patients and patients with higher E2F6 mRNA level. Additional analysis from TCGA ovarian cancer expression microarray dataset demonstrated that ovarian cancer patients with low expression of EZH2, showed a positive correlation between E2F6, c-KIT and PBX1 resembling the ceRNA phenomenon between these mRNAs. Taken together, our results showed that estrogen-mediated E2F6 ceRNA network can regulate cancer stemness and anti-tumor immunity of DC through epigenetic silencing of miR-193a. Anti-estrogen therapy together with the EZH2 inhibitor may be a novel strategy against this deadly cancer. Citation Format: Frank Hsueh-Che Cheng, Hon-Yi Lin, Yin-Chen Chen, Tzy-Wei Hwang, Rui-Lan Huang, Chia-Bin Chang, Ru-Inn Lin, Ching-Wen Lin, Gary C.W. Chen, Jora M. J. Lin, Yu-Ming Chuang, Jian-Liang Chou, Chin Li, Alfred S.L. Cheng, Hung-Cheng Lai, Shu-Fen Wu, Je-Chiang Tsai, Michael W.Y. Chan. E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 74.

Abstract 1501: Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer

Gastric cancer is the fourth leading cause of cancer-related death worldwide. Previous studies demonstrated that activation of the JAK/STAT3 signaling pathway is frequently observed in H. pylori infected gastric cancer. However, the role of aberrant JAK/STAT signaling in the global epigenetic changes remains unclear. In this regard, we compared the global methylomic changes in AGS gastric cancer cells showing constitutive activation of STA3 vs its STAT3 knock-down subclone by MBDcap-Seq followed by PrEMeR-CG analysis. Together with RNA-Seq, we identified 97 targets showing concomitant hypomethylation and over-expression while 76 targets showing concomitant hypermethylation and down-regulation after STAT3 knock down. Genes showing hypomethylaton/over-expression were subjected to transcription factor binding site analysis by MEME CentriMo. Interestingly, the transcriptional repressors binding site for ETS1 (p = 2.90E-06) and EHF (p = 3.50E-06) were overrepresented in those identifed STAT3 targets suggesting the cooperative binding with STAT3 in the epigenetic silencing of the targets. Further gene ontology analysis by DAVID showed that genes involved in cell cycle and apoptosis were significantly enriched in the hypomethylated/over-expressed targets while genes involved in protein degradation and ubiquitination were found among the hypermethylation/down-regulated targets. To experimentally confirm our result, we analyzed the functional role of one of the hypomethylated targets, miR-193a in gastric cancer. Concomitant with MBDcap-Seq, bisulphite pyrosequencing confirmed that promoter region of miR-193a was hypomethylated in AGS cells depleted with STAT3 but hypermethylated in MKN28 gastric cancer cells overexpressed with constitutive activated STAT3. Cell lines studies also found that promoter region of miR-193a was hypermethylated in gastric cancer cells which did not express miR-193a. Over-expression of miR-193a in AGS cells inhibited cell proliferation (p Citation Format: Jora M.j. Lin, Jiang-Liang Chou, David E. Frankhouser, Yu-Ming Chuang, Alex Liang-Yu Chang, Li-Han Zeng, Szu-Shan Chen, Ru-Inn Lin, Cheng-Shyong Wu, Kuo-Liang Wei, Enders K.W. Ng, Pearlly S. Yan, Alfred S.L. Cheng, Chin Li, Michael W. Y. Chan. Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer. [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 1501.

Abstract 3314: DNA methylome analysis identifies epigenetic silencing of FHIT as a determining factor for radiosensitivity in oral cancer and its implication in treatment and outcome prediction

Radioresistance is still an emerging problem for radiotherapy of oral cancer. Aberrant epigenetic alterations play an important role in cancer development, yet the role of such alterations in radioresistance of oral cancer is not fully explored. Using Illumina 27K methylation BeadChip microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation (5-Gy by 10 fractions) of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation and H3K27me3 as demonstrated by MBDcap-PCR, bisulfite pyrosequencing and ChIP-PCR. These phenomenon were partially attributed to overexpression of EZH2 and DNMT3a, 3b in OML1-R cells. In consistent with these observations, treatment of 5-azaDC, EZH2 inhibitor (GSK343) or depletion of EZH2 by lentiviral knockdown restored FHIT expression in OML1-R cells. Interestingly, knockdown of EZH2 also reversed histone modifications (increased of H3K4me3 and decreased of H3K27me3) and reduced promoter methylation of FHIT thus suggesting that H3K27me3 linked to DNA methylation in this loci. We also analyzed the expression of FHIT in primary human oral keratinocyte (HOK) and four other oral cancer cell lines (OCSL, SCC25, SAS, and SCC4). FHIT expression demonstrated a tight inverse relationship with its promoter methylation. Ectopic expression of FHIT restored radiosensitivity (single fraction, 10-Gy) in OML1-R cells and oral cancer cells (SAS, SCC25) showing epigenetic silencing of FHIT. These phenomenon may be due to restoration of Chk2 phosphorylation, induction of apoptosis and G2/M check point. Reciprocal experiments also showed that depletion of FHIT in OSCL cells, which highly express FHIT, slightly enhanced radioresistance. Clinically, bisulfite pyrosequencing and iummnohistochemistry revealed that promoter hypermethylation of FHIT inversely correlated with its expression. Patients with higher FHIT methylation (methylation>10%, n = 22) are associated with lower locoregional control (P Citation Format: Hon-Yi Lin, Shih-Kai Hung, Moon-Sing Lee, Wen-Yen Chiou, Tze-Ta Huang, Chih-En Tseng, Liang-Yu Shih, Ru-Inn Lin, Jora Lin, Yi-Hui Lai, Chia-Bin Chang, Feng-Chun Hsu, Liang-Cheng Chen, Shiang-Jiun Tsai, Yu-Chieh Su, Szu-Chi Li, Hung-Chih Lai, Wen-Lin Hsu, Dai-Wei Liu, Chien-Kuo Tai, Shu-Fen Wu, Michael W. Chan. DNA methylome analysis identifies epigenetic silencing of FHIT as a determining factor for radiosensitivity in oral cancer and its implication in treatment and outcome prediction. [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 3314. doi:10.1158/1538-7445.AM2015-3314

Abstract 1128: TET1-mediated epigenetic reprogramming switches metabolism and promotes malignant phenotypes of ovarian cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epigenetic dysregulation is one of the mechanisms involved in ovarian cancer carcinogenesis. Recently identified a new epigenetic modulator ten-eleven translocation protein 1 (TET1), a DNA dioxygenase which is believed in DNA demethylation through a 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) conversion, plays an important role in regulating self-renewal and specification in embryonic stem cells. In addition, it works as a tumor suppressor gene by inhibition of cell invasion, migration and tumor growth in breast and prostate cancer. However, the role of TET1 in ovarian cancer remains unknown. Thus we examed the expression level of TET1 in ovarian cancer tissues. We found that TET1 expression level correlated with cancer staging (p = 0.03) in 88 ovarian cancer from our biobank and poor survival (p = 0.012) in TCGA database. High expression of TET1 was observed in advanced stage, high-grade primary tumor specimens in comparison with normal ovarian surface epithelium (OSE) brushings (p = 0.0005) by NCBI database (GSE18520). These results suggested that TET1 may play some roles in ovarian cancer development, which is different from those previously published in other cancers. To study the function of TET1 in ovarian cancer, we generated TET1 over-expressing and knockdown cell lines model, the expression level and enzymatic activities of TET1 were confirmed by real time PCR, western and 5hmC stain. Here we found that TET1 increases the abilities of cell migration, anchorage-independent growth and promotes tumor growth. In addition, TET1 actives cancer stem markers and enhances the abilities of spheroid formation. Ovarian cancer stem cells (OCSCs) from cell line express high level of TET1 while the differentiated progenies suppress TET1 expression. Moreover, seven of eight patient-derived OCSCs revealed high expressing of TET1 in comparison with its parental cancer cells by quantitative PCR. To further examine the TET1 regulation network, we combined the expression array and MethCap-seq to analyze the epigomic changes. We found that a cluster of target genes which were up-regulated through DNA demethylation were enzymes responsible for oxidative phosphorylation. We investigated metabolic status by Extracellular Flux Analyzer (seahourse) on the TET1-overexpressing cells. Compared with control cells, TET1-overexpressing cells revealed 1.7 fold (p = 0.011) increase of oxygen consumption rate (OCR); while the extracellular acidification rate (ECAR) showed no difference (p = 0.856). This bioenergetic metabolism shift may be due to demethylation of subunits of mitochondria complexs. Taken together, TET1 reprograms the epigenome, shifts the metabolism, increases the malignant phenotypes and confers a poor prognosis of ovarian cancer. Targeting mitochondria on TET1-expressing ovarian cancer patients may provide a new way of personalized therapy. Citation Format: Lin-yu Chen, Rui-Lan Huang, Pearlly S. Yan, Tien-Shuo Huang, Yu-Ping Liao, Jian-Liang Chou, Jora M.J. Lin, Tai-Kuang Chao, Michael W.Y. Chan, Wun-Shaing Wayne Chang, Hung-Cheng Lai. TET1-mediated epigenetic reprogramming switches metabolism and promotes malignant phenotypes of ovarian 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 1128. doi:10.1158/1538-7445.AM2015-1128

Abstract 2318: The role of EZH2 as an epigenetic switch of the TGF-β/SMAD4 targets in regulating EMT in ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The TGF-β signaling regulates numerous cellular processes, including cell proliferation, cell differentiation, apoptosis, migration and cell fate. It was also demonstrated that TGF-β functions as a tumor suppressor in normal ovarian surface epithelium (OSE) cells but promotes tumor proliferation and epithelial-mesenchymal transition (EMT) during ovarian cancer progression. Nevertheless, the molecular mechanisms leading to this divert role of TGF-β signaling in ovarian cancer remains to be elucidated. Our previous studies demonstrated that several TGF-β/SMAD4 regulated targeted are epigenetically silenced by DNA methylation and histone modification including H3K27me3. We therefore hypothesized that the histone-lysine N-methyltransferase, EZH2 may act as an epigenetic switch to facilitate the TGF-β mediated EMT in ovarian cancer. In this study, we utilized our previously identified TGF-β responsive targets using combined ChIP-chip and expression arrays in an immortalized ovarian surface epithelial (IOSE) cell which showed TGF-β growth inhibition. Bioinformatics analysis using ENCODE ChiP-Seq data identified that several of these TGF-β targets are marked by EZH2. To investigate the effect of TGF-β signaling on the methylome of ovarian cancer, we performed MBDcap-Seq in SMAD4 knockdown CP70 cells. Our result showed that there are 99 and 73 TGF-β targets showing hyper- or hypo-methylation respectively after SMAD4 knockdown. Analysis of the gene functions on DAVID has uncovered that some of these hypermethylated genes are associated with EMT process and the TGF-β signaling pathway. On the other hand, the hypomethylated genes are associated with transcription repressor activity. We selected one of the hypermethylated target, LTBP2 which was previously found to be a tumor suppressor for further experiment. This gene was expressed in IOSE but down regulated in a panel of ovarian cancer cells showing overexpression of EZH2. Except for IOSE, promoter hypermethylation of LTBP2 was found in ovarian cancer cells as revealed by bisulphite pyrosequencing. In consistent with deep sequencing result, knock-down of SMAD4 further increased the promoter methylation in CP70 cells. Treatment of demethylation agent, 5azaDC partially restored its expression in these cancer cells. Interestingly, synergistic treatment of 5azaDC and EZH2 inhibitor, GSK343 resulted in a dramatic increase of LTBP2 expression in MCP3 and CP70 cells. Taken together, our result suggested that EZH2 may be involved in the epigenetic silencing of TGF-β/SMAD4-regulated tumor suppressors in ovarian cancer. The differential occupancy of EZH2 into these SMAD4 loci may act as an epigenetic switch to turn the function of TGF-β from a tumor suppressor into an EMT regulator. The therapeutic potential of targeting EZH2 in the inhibition of EMT in ovarian cancer deserves further investigation. Citation Format: Jora Meng-Ju Lin, Jacqueline Shay, Jian-Liang Chou, Pearlly S. Yan, Tim H.-M. Huang, Hung Cheng Lai, Michael W.y. Chan. The role of EZH2 as an epigenetic switch of the TGF-β/SMAD4 targets in regulating EMT in ovarian 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 2318. doi:10.1158/1538-7445.AM2014-2318