Jian-Liang Chou

Promoter hypermethylation and silencing of tissue factor pathway inhibitor-2 in oral squamous cell carcinoma

BackgroundThe treatment of oral squamous cell carcinoma (OSCC) following early detection is associated with good outcomes. Therefore, the survival and prognosis of OSCC patients could be hugely improved by identifying reliable biomarkers for the early diagnosis of the disease. Our previous methylation microarray analysis results have suggested that the gene encoding tissue factor pathway inhibitor-2 (TFPI-2) is a potential clinical predictor as well as a key regulator involved in OSCC malignancy.MethodsMethylation of the TFPI-2 promoter in oral tissue specimens was evaluated by bisulfite sequencing assay, quantitative methylation-specific PCR, and pyrosequencing assay. The differences in methylation levels among the groups were compared using the Mann-Whitney U test. The area under the receiver operating characteristic curve (AUROC) was used to evaluate the discrimination ability for detecting OSCC. Cellular TFPI-2 expression was analyzed by quantitative reverse-transcription PCR before and after treatment with 5′-aza-2′-deoxycytidine and trichostatin A, to confirm whether TFPI-2 was epigenetically silenced in OSCC cells. We investigated whether TFPI-2 plays a role as a tumor suppressor by establishing TFPI-2-overexpressing OSCC cells and subjecting them to in vitro cellular proliferation, migration, and invasion assays, as well as an in vivo metastasis assay.ResultsTFPI-2 was hypermethylated in OSCC tissues versus normal oral tissues (P < 0.0001), with AUROC = 0.91, when using a pyrosequencing assay to quantify the methylation level. TFPI-2 silencing in OSCC was regulated by both DNA methylation and chromatin histone modification. Restoration of TFPI-2 counteracted the invasiveness of OSCC by inhibiting the enzymatic activity of matrix metalloproteinase-2, and consequently interfered with OSCC metastasis in vivo.ConclusionsOur data suggest strongly that TFPI-2 is a down-regulated tumor suppressor gene in OSCC, probably involving epigenetic silencing mechanisms. The loss of TFPI-2 expression is a key event for oral tumorigenesis, especially in the process of tumor metastasis.

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 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

Abstract 1476: Hypermethylation of a TGF-β regulated micro-RNA, miR-193a, predicts prognosis in ovarian cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is one of the most lethal cancers in the female reproductive system. One of the hypotheses suggests that ovarian cancer arises from cancer stem/progenitor cell with surface antigen CD117 (or c-kit). In order to identify microRNAs (miRNAs) that can regulate the expression of c-kit, bioinformatic prediction was performed and identified miR-193a as one of the regulators. Expression analysis showed that miR-193a was up-regulated upon TGF-β treatment in immortalized ovarian surface epithelial cells (IOSE) cells. On the contrary, expression of miR-193a cannot be detected in a panel of ovarian cancer cell lines (SKOV3, A2780, CP70, MCP2, MCP3). Bisulphite pyrosequencing revealed that promoter CpG island of miR-193a was not methylated in IOSE cell but hypermethylated in the ovarian cancer cells showing down-regulation of miR-193a. Treatment of 5azaDC, a DNMT inhibitor, partially restored miR-193a expression in the ovarian cancer cells, thus suggesting DNA hypermethylation may be responsible for the down-regulation of miR-193a in ovarian cancer. Further clinical studies using bisulphite pyrosequencing found that promoter region of miR-193a was heavily methylated in 109 ovarian cancer patient samples but not in normal OSE samples. Kaplan-meier analysis revealed that patients with higher methylation of miR-193a was significantly associated with shorter overall survival and progression-free survival. Taken together, miR-193a may be a TGF-β regulated tumor suppressor micro-RNA and is epigenetically silenced in ovarian cancer. Methylation of miR-193a may be able to predict prognosis in ovarian cancer patients. Citation Format: Frank Hsueh-Che Cheng, Gary C.W. Chen, Jian-Liang Chou, Ya-Wen Lin, Lin-Yu Chen, Hung-Cheng Lai, Chin Li, Michael W.Y. Chan. Hypermethylation of a TGF-β regulated micro-RNA, miR-193a, predicts prognosis 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 1476. doi:10.1158/1538-7445.AM2014-1476

Abstract 2310: Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlying mechanism in the gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothesized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 target genes in gastric cancer. To test this hypothesis, a constitutively activated mouse STAT3 mutant (STAT3c) was transfected into MKN28 gastric cancer cells in which the JAK/STAT signaling pathway is inactive. STAT3c stable transfectant (S16) showing hyperphosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control (C9). Integrative expression microarray coupled with bioinformatic analysis identified putative STAT3 targets, NR4A3 that are down-regulated in S16 cells. In association with up-regulation of DNMT1, NR4A3 exhibited increased promoter methylation in S16 but not C9 cells as demonstrated by bisulphite sequencing and demethylation treatment. Interestingly, NR4A3 was also found to be epigenetically silenced in AGS cells where JAK/STAT signaling is constitutively activated. ChIP-PCR experiment revealed that STAT3 bound to the putative STAT3 binding site in NR4A3 promoter of AGS cells. Depletion of STAT3 by lenti-viral knockdown restored NR4A3 expression in this cell. Interestingly, luciferase reporter assay using the NR4A3 promoter containing putative STAT3 binding site exhibited a further 1.6 fold increment after deleting the STAT3 binding region (P < 0.005). Ectopic expression of NR4A3 in AGS cells reduced cancer cell growth in colony formation assay (P < 0.001). In clinical specimens, quantitative MSP demonstrated a significant correlation between the degree of NR4A3 methylation and STAT3 nuclear translocation in 72 gastric tumor samples (P < 0.05). Importantly, methylation of NR4A3 was significantly associated with patients with shorter survival (P < 0.05). In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling confers epigenetic silencing of a potential tumor suppressor, NR4A3 in gastric cancer. Methylation of NR4A3 may be able to serve as a prognostic indicator in gastric cancer patients. Citation Format: Michael W. Y. Chan, Li-Han Zeng, Liang-Yu Chang, Claudia Dittner, Jian-Liang Chou, Yao-Ting Huang, Alfred S.L. Cheng, Jiayuh Lin, Kun-Tu Yeh. Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis 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 2310. doi:10.1158/1538-7445.AM2014-2310

Abstract 83: Hypomethylation of TGF-beta target gene, ABCA1 in ovarian cancer and cancer initialing cell and is associated with poor prognosis in cancer patients

Dysregulation of TGF-β signaling plays a crucial role in ovarian carcinogenesis and maintaining cancer stem cell properties. We have previously identified the TGF-β responsive targets in immortalized ovarian surface epithelial cells (IOSE) by expression microarray and found that ABCA1, a family members of ATP-binding cassette is up-regulated following TGF-β treatment (Qin et al, BMC Syst Biol 2009). Thus, we hypothesize that ABCA1 may be involved in ovarian cancer and its initiation. First, we compared the expression level of ABCA1 in IOSE and a panel of ovarian cancer cell lines and found that ABCA1 was up-regulated in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but down-regulated in A2780 and CP70 ovarian cancer cells. The down-regulation in A2780 and CP70 cells was associated with promoter hypermethylation as demonstrated by bisulphite sequencing and demethylation treatment. To investigate if ABCA1 is involved in ovarian cancer initiating cells (OCIC), we compared the methylation level of ABCA1 in ovarian tumor samples and their corresponding spheroids which is previously found to be enriched with OCIC. Lower methylation of ABCA1 could be detected in 2/6 OCIC samples as compared to their original tumors. We further analyzed the expression and methylation level of ABCA1 in CP70 spheroid (CP70sp) derived from suspension culture of CP70 ovarian cancer cells. Comparing to CP70, up-regulation of stem cell markers Nanog (3.8 fold) and Oct4 (12 fold) was detected in CP70sp cells suggesting that CP70sp may contain OCIC. Interestingly, up-regulation of ABCA1 (5.2 fold) concomitant with promoter hypomethylation was detected in CP70sp cells. Additionally, active demethylation may be involved in the hypomethylation of ABCA1 as expression of the methylcytosine dioxygenase, TET1 was up-regulated in the CP70sp cells (5.3 fold). Furthermore, ABCA1 was involved in drug resistance of ovarian cancer which is a feature of cancer initiating cells, as lenti-viral knockdown of ABCA1 in a platinum resistant MCP2 ovarian cancer cells resensitized the cells to cisplatin (IC50: shABCA1 vs shGFP: 0.267 ug/ml vs 0.402 ug/ml). We further analyzed the methylation level of ABCA1 in 97 ovarian cancer, 46 benign, and 4 normal samples using real-time quantitative MSP assay and found that higher methylation level of ABCA1 was detected in cancer than benign (P=0.051) and normal tissues (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 83. doi:10.1158/1538-7445.AM2011-83

Abstract 3072: Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Refractory to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in aberrant expression of downstream target genes in the disease. We hypothesized that aberrant expression of TGF-β/SMAD4 targets are mediated through epigenetic mechanism and also contribute to resistance to TGF-β meditated growth inhibition. Our previous report using chromatin immunoprecipitation microarray (ChIP-chip) identified FBXO32 as one of SMAD4 targets in immortalized ovarian surface epithelial cell (IOSE) (Qin et al., BMC Syst Biol, 17: 73, 2009). In the present study, we investigated the mechanism conferring FBXO32 down-regulation, its clinical significance, and its function in ovarian cancer. Our result showed that expression of FBXO32 was observed in normal ovarian surface epithelium but not in ovarian cancer cell lines (HeyC2, SKOV3, CP70, A2708, MCP2, MCP3) using real time RT-PCR. Promoter methylation of FBXO32 was seen in ovarian cancer cell lines, HeyC2 and SKOV3, that display constitutive TGF-β/SMAD4 signaling. Moreover, our finding that epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggested that epigenetic regulation of FBXO32 in ovarian cancer may be a common event. Re-expression of FBXO32 markedly impeded proliferation of a platinum-resistant ovarian cancer cell lines, HeyC2 and CP70 (colony number: HeyC2: 19.33 ± 3.06 vs 1 ± 0, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3072.