Ovarian cancer is the fifth leading cause of cancer death and the most deadly gynecological malignancy in women. Epigenetic modifications play an important role in regulating gene transcription. Specifically, aberrant promoter hypermethylation has been implicated as a hallmark of cancer. In order to identify genes that are differentially methylated in ovarian cancer, we performed meDIP-chip in various ovarian cancer cell lines using Agilent 244K CpG island microarray. One of the targets, ARNTL which is a core component of the circadian clock is methylated in a sub-set of ovarian cancer cell lines. Combined bisulfite restriction analysis (COBRA) confirmed the results of the microarray. Additional analysis using ChIP-PCR revealed that promoter of ARNTL is enriched with the repressive histone mark H3K27me3 in CP70 and MCP2 ovarian cancer cells. Treatment with the EZH2 inhibitor (GSK126) significantly restored ARNTL expression in these cells (CP70 and MCP2). Further functional analysis demonstrated that overexpression of ARNTL inhibited cell growth and enhanced chemosensitivity of cisplatin in ovarian cancer cells. Finally, overexpression of ARNTL restored the rhythmic activity of c-MYC in ovarian cancer cells. These results suggested that ARNTL may be a tumor suppressor and is epigenetically silenced in ovarian cancer.

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The deregulation of annexin A1 (ANXA1), a regulator of inflammation and immunity, leads to cancer growth and metastasis. However, whether ANXA1 is involved in cancer immunosuppression is still unclear. Here, we report that ANXA1 knockdown (i) dramatically downregulates programmed cell death-ligand 1 (PD-L1) expression in breast cancer, lung cancer, and melanoma cells; (ii) promotes T cell-mediated killing of cancer cells in vitro; and (iii) inhibits cancer immune escape in immune-competent mice via downregulating PD-L1 expression and increasing the number and killing activity of CD8+ T cells. Mechanistically, ANXA1 functioned as a sponge molecule for interaction of PARP1 and Stat3. Specifically, binding of ANXA1 to PARP1 decreased PARP1’s binding to Stat3, which reduced poly(ADP-ribosyl)ation and dephosphorylation of Stat3 and thus, increased Stat3’s transcriptional activity, leading to transcriptionally upregulated expression of PD-L1 in multiple cancer cells. In clinical samples, expression of ANXA1 and PD-L1 was significantly higher in breast cancer, non-small cell lung cancer, and skin cutaneous melanoma compared to corresponding normal tissues and positively correlated in cancer tissues. Moreover, using both ANXA1 and PD-L1 proteins for predicting efficacy of anti-PD-1 immunotherapy and patient prognosis were superior to using individual proteins. Our data suggest that ANXA1 promotes cancer immune escape via binding PARP1 and upregulating Stat3-induced expression of PD-L1, that ANXA1 is a potential new target for cancer immunotherapy, and combination of ANXA1 and PD-L1 expression is a potential marker for predicting efficacy of anti-PD-1 immunotherapy in multiple cancers.