CRY2 is suppressed by FOXM1 mediated promoter hypermethylation in breast cancer.

Loss of CRY2 confers aggressive phenotypes to breast cancer. However, the mechanism of its downregulation and its prognostic value in breast cancer are still not clear. Our data mining in TCGA breast cancer cohort (TCGA-BRCA) showed that the luminal A subtype of breast cancer had the highest CRY2 expression, while the basal-like subtype had the lowest CRY2 expression. The ER+ group had significantly higher CRY2 expression than the ER- group. Demethylation treatment using 5-AZA-dC significantly restored CRY2 expression in MDA-MB-231 and BT549 cells. Co-expression analysis in TCGA-BRCA showed a strong negative correlation between CRY2 and FOXM1 (Pearson's r = -0.62). FOXM1 overexpression in MCF-7 cells reduced CRY2 expression, while FOXM1 knockdown in MDA-MB-231 cells increased CRY2 expression. Demethylation significantly abrogated FOXM1 induced CRY2 suppression in MCF-7 cells. Bioinformatic scanning predicted a common FOXM1 binding site in CRY2 transcript 1 and transcript 2 promoter. The following studies confirmed that through binding with DNMT3b, FOXM1 can bind to CRY2 promoter and enhance methylation in this region. Univariate analysis based on Cox proportional hazards model and the following NPI and AOL adjusted studies in bc-GenExMiner 4.0 showed that high CRY2 expression was an independent indicator of reduced risk of metastatic relapse (MR) in ER+ breast cancer patients, but not in ER- breast cancer. With these findings, we infer that FOXM1 is a negative regulator of CRY2 in breast cancer via enhancing methylation in CRY2 promoter and its high expression is an independent predictor of favorable MR-free survival in ER+ breast cancer patients.