Search

Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long‐term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen‐mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c‐Kit, by epigenetic silencing the tumor suppressor miR‐193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c‐KIT, but downregulated miR‐193a. Luciferase assays further confirmed that microRNA‐193a targets both E2F6 and c‐Kit. Interestingly, ChIP‐PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR‐193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR‐193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR‐193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen‐mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA‐193a activity, promoting ovarian cancer stemness and tumorigenesis.

PurposeThe purpose of this study was to identify genes that were epigenetically silenced by STAT3 in gastric cancer.MethodsMBDcap-Seq and expression microarray were performed to identify genes that were epigenetically silenced in AGS gastric cancer cell lines depleted of STAT3. Cell lines and animal experiments were performed to investigate proliferation and metastasis of miR-193a and YWHAZ in gastric cancer cell lines. Bisulfite pyrosequencing and tissue microarray were performed to investigate the promoter methylation of miR-193a and expression of STAT3, YWHAZ in patients with gastritis (n = 8) and gastric cancer (n = 71). Quantitative methylation-specific PCR was performed to examine miR-193a promoter methylation in cell-free DNA of serum samples in gastric cancer patients (n = 19).ResultsAs compared with parental cells, depletion of STAT3 resulted in demethylation of a putative STAT3 target, miR-193a, in AGS gastric cancer cells. Although bisulfite pyrosequencing and epigenetic treatment confirmed that miR-193a was epigenetically silenced in gastric cancer cell lines, ChIP-PCR found that it may be indirectly affected by STAT3. Ectopic expression of miR-193a in AGS cells inhibited proliferation and migration of gastric cancer cells. Further expression microarray and bioinformatics analysis identified YWHAZ as one of the target of miR-193a in AGS gastric cancer cells, such that depletion of YWHAZ reduced migration in AGS cells, while its overexpression increased invasion in MKN45 cells in vitro and in vivo. Clinically, bisulfite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n = 11) as compared to gastritis (n = 8, p < 0.05). Patients infected with H. pylori showed a significantly higher miR-193a methylation than those without H. pylori infection (p < 0.05). Tissue microarray also showed a positive trend between STAT3 and YWHAZ expression in gastric cancer patients (n = 60). Patients with serum miR-193a methylation was associated with shorter overall survival than those without methylation (p < 0.05).ConclusionsConstitutive activation of JAK/STAT signaling may confer epigenetic silencing of the STAT3 indirect target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted inhibition of STAT3 may be a novel therapeutic strategy against gastric cancer.

Gastric cancer is one of the leading causes of cancer death worldwide. Previous studies demonstrated that activation of STAT3 is crucial for the development and progression of gastric cancer. However, the role of STAT3 in neuronal related gene methylation in gastric cancer has never been explored. In this study, by using DNA methylation microarray, we identified a potential STAT3 target, C11orf87, showing promoter hypomethylation in gastric cancer patients with lower STAT3 activation and AGS gastric cancer cell lines depleted with STAT3 activation. Although C11orf87 methylation is independent of its expression, ectopic expression of a constitutive activated STAT3 mutant upregulated its expression in gastric cancer cell line. Further bisulfite pyrosequencing demonstrated a progressive increase in DNA methylation of this target in patient tissues from gastritis, intestinal metaplasia, to gastric cancer. Intriguingly, patients with higher C11orf87 methylation was associated with better survival. Furthermore, hypermethylation of C11orf87 was also frequently observed in other GI cancers, as compared to their adjacent normal tissues. These results suggested that C11orf87 methylation may serve as a biomarker for diagnosis and prognosis of GI cancers, including gastric cancer. We further postulated that constitutive activation of STAT3 might be able to epigenetically silence C11orf87 as a possible negative feedback mechanism to protect the cells from the overactivation of STAT3. Targeted inhibition of STAT3 may not be appropriate in gastric cancer patients with promoter hypermethylation of C11orf87.

Aims The impaired angiogenesis is one of the main factors affecting the healing of diabetic foot ulcer (DFU) wounds. Maggot debridement therapy (MDT) promotes granulation tissue growth and angiogenesis during DFU wound healing. Non-coding microRNAs can also promote local angiogenesis in DFU wounds by regulating wound repairing related gene expression. The purpose of this study was to investigate the mechanism of microRNAs in MDT promoting DFU wound angiogenesis. Methods In this study, we applied MDT to treat DFU wound tissue and detect the expression of the miR-17–92 cluster. In vitro experiments, human umbilical vein endothelial cells (HUVECs) were treated with maggot excretions/secretions (ES), the miR-17–92 cluster and the predicted target gene expression were measured. Tube formation assay and cell scratch assay were performed when inhibition of miR-18a/19a or overexpression of thrombospondin-1 (TSP-1) were used in this study. Results miR-18a/19a transcription significantly up-regulated and TSP-1 expression down-regulated in patients wound tissue and in HUVECs. Inhibition of miR-18a/19a or overexpression of TSP-1 partially blocked the migration and tube formation ability stimulated by ES. Conclusion Targeted activation of miR-18a/19a transcription levels and subsequent regulation of TSP-1 expression may be a novel therapeutic strategy for DFU.

Gastric cancer is a leading cause of cancer worldwide. Our previous studies showed that aberrant activation of JAK/STAT3 signaling confer epigenetically silences STAT3 target genes in gastric cancer. To further investigate the clinical significance of this phenomenon, we performed Illumina 850K methylation microarray analysis in AGS gastric cancer cells, and cells depleted of STAT3. Integrative computational analysis identified SPG20 as a putative STAT3 epigenetic target, showing promoter hypomethylation in STAT3-depleted AGS cells. Bisulphite pyrosequencing and qRT-PCR confirmed that SPG20 is epigenetically silenced by promoter hypermethylation in a panel of gastric cancer cell lines including AGS cells, but not in immortalized gastric epithelial GES cells. Expression of SPG20 could be restored by the treatment with a DNMT inhibitor, further suggesting that SPG20 is epigenetically silenced by promoter methylation. Clinically, a progressive increase in SPG20 methylation was observed in tissues samples from gastritis (n = 34), to intestinal metaplasia (IM, n = 33), to gastric cancer (n = 53). Importantly, SPG20 methylation could be detected in cell-free DNA isolated from serum samples of gastritis, IM and gastric cancer patients, having a progressive similar to tissues. Taken together, SPG20, a potential STAT3 target, is frequently methylated in gastric cancer, representing a novel noninvasive biomarker for early detection of this deadly disease.

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 3'UTR 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 3'UTR 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.