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.

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 a methylation microarray, we identified promoter hypermethylation of FHIT (fragile histidine triad) in radioresistant OML1-R cells, established from hypo-fractionated irradiation of parental OML1 radiosensitive oral cancer cells. Further analysis confirmed that transcriptional repression of FHIT was due to promoter hypermethylation, H3K27me3 and overexpression of methyltransferase EZH2 in OML1-R cells. Epigenetic interventions or depletion of EZH2 restored FHIT expression. Ectopic expression of FHIT inhibited tumor growth in both in vitro and in vivo models, while also resensitizing radioresistant cancer cells to irradiation, by restoring Chk2 phosphorylation and G2/M arrest. Clinically, promoter hypermethylation of FHIT inversely correlated with its expression and independently predicted both locoregional control and overall survival in 40 match-paired oral cancer patient samples. Further in vivo therapeutic experiments confirmed that inhibition of DNA methylation significantly resensitized radioresistant oral cancer cell xenograft tumors. These results show that epigenetic silencing of FHIT contributes partially to radioresistance and predicts clinical outcomes in irradiated oral cancer. The radiosensitizing effect of epigenetic interventions warrants further clinical investigation.

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.

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.

Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.