The forkhead box-F (FOXF) proteins, important regulators in development, function as tumor suppressors and are epigenetically silenced in breast cancer

C277 The forkhead-box (FOX) family of transcription factors have steadily garnered attention following the discovery of their critical role in the regulation of various developmental and immunological processes in eukaryotes. Although transforming genetic alterations of a few FOX gene members (e.g. FOXO subfamily) have been identified in cancers, the role of other many FOX gene members has not yet been explored and characterized. We have found frequent epigenetic silencing of the forkhead box-F (FOXF) genes, FOXF1 and FOXF2, in breast cancer cell lines and in primary tumors. The CpG-island-containing promoters of FOXF1 and FOXF2 genes were hypermethylated in 37.6% (44/117) and 71.0% (83/117) of invasive ductal carcinomas (IDC), respectively, but not in normal mammoplasty specimens and mammary epithelial organoids (0/14) examined. The methylation of FOXF1 gene correlated with tumor grade (p = 0.04) and the methylation of FOXF2 gene correlated with the patients’ age (p = 0.048), tumor size (p = 0.019) and stage (p = 0.022). mRNA and protein expression studies indicate that loss or downregulation of FOXF1 and F2 expression in breast cancer cell lines and tumors is tightly correlated with hypermethylation of CpG-island promoter sequence. Inactivation of methyltransferases in cancer cells by treatment with the demethylating agent (5-azaC) or by genetic knockout of DNMT genes restored expression of FOXF genes. The restoration of FOXF gene expression caused growth suppression of cancer cells in vitro and suppressed tumor formation in nude mice. Dependent on cell types analyzed, the growth suppression was attributable to FOXF-induced G1 arrest with or without apoptosis. FOXF factors were found to block the cell cycle at the G1-S transition by inhibiting the kinase activity of Cdk2, RB hyperphosphorylation and E2F1 transcriptional activity. Besides their involvement in regulation of developmental processes, we have shown for the first time, that FOXF genes possess functional characteristics of a tumor suppressor gene, whose function is frequently abolished in breast cancer.