In this paper, we report the identification of FOXF1 gene as a novel epigenetic target in breast cancer. DNA hypermethylation was identified as the predominant underlying mechanism for silencing FOXF1 gene expression. Our studies showed that FOXF1 re-expression in breast cancer cell lines inhibited their growth and tumorigenicity, mainly by blocking G1-S transition of cell cycle, whereas inactivation of FOXF1 resulted in DNA over-replication, a risk factor leading to genomic instability. Our findings support the hypothesis that aberrant epigenetic inactivation of FOXF1 in mammary epithelial cells could contribute to mammary tumorigenesis.
Gene expression analysis in silico using the microarray database revealed that, compared to normal tissues, FOXF1 is underexpressed in a variety of cancers such as lung, prostate, bladder, ovarian, and breast. Hypermethylation of the FOXF1 promoter was found to be frequent (80% in cell lines, 40% in primary tumors) and is, in all likelihood, the predominant mechanism accounting for loss or downregulation of FOXF1 expression in breast cancer. FOXF1 promoter hypermethylation positively correlated with the tumor grade and appears to be an early event across both ductal and lobular breast cancers with the same frequency (data not shown). Therefore, FOXF1 gene is a common epigenetic target among various types of breast cancer. It will be important to decipher whether epigenetic mechanisms are also implicated in downregulating FOXF1 expression in other types of cancers.
Our studies have shed light on the mechanisms underlying FOXF1-mediated growth suppression. We found that enforced overexpression of FOXF1 led to G1 arrest concurrently with or without apoptosis dependent on the cell type examined. Inhibition of CDK2-RB-E2F signaling cascade by FOXF1 is most likely the mechanism accounting for the G1-arrest activity of FOXF1. Although mechanisms for FOXF1-mediated inactivation of the CDK2-RB-E2F cascade are still unclear, our siRNA knockdown studies provided some mechanistic clues. We found that knockdown of FOXF1 in cells led to an increase in DNA re-replication, a sign of a defect in the stringent control of DNA replication initiation. The stringency of DNA replication initiation is finely controlled by a sequential process involving DNA replication origin licensing, activation of origin to replicate DNA and disassembly of pre-replication complexes (47
). Deregulation of these processes permits repeated firing of DNA replication origins, leading to DNA rereplication (also called over-replication) (48
). Expression profiling analysis of cell cycle regulatory genes suggested that FOXF1 inhibits DNA replication initiation by downregulating expression of DNA replication initiation factor genes (e.g. MCM3
) and by upregulating expression of mitotic genes (e.g. ANAPC2, ANAPC4
) involved in negatively regulating G1-S transition. In addition, other FOXF1-regulated cell cycle genes such as CDK6
might also contribute to this effect. These findings, taken together, suggest that gain or loss of FOXF1 function alters expression of these cell cycle regulatory genes, which in turn leads to these observed outcomes. In addition to MCM3
, expression of five other E2F-induced genes was also elevated in cells depleted of FOXF1, suggesting that E2F transcriptional activity is activated upon silencing of FOXF1. These results are consistent with the data showing that FOXF1 suppresses E2F transcriptional activity through abrogation of the CDK2-RB-E2F cascade.
In addition to cell-cycle functions, the identified FOXF1-regulated genes are also implicated in multiple biological processes such as DNA repair, transcriptional regulation, nuclear transportation and cell survival control. For example, we found that BRCA1
is the downstream target of FOXF1 signaling. BRCA1
is a well-known tumor suppressor gene that plays multiple roles in transcription and DNA repair. BRCA1
is inactivated through genetic mutation or aberrant downregulation of its mRNA expression (50
). It is tempting to speculate that epigenetic inactivation of FOXF1
is one of mechanisms leading to downregulation of BRCA1 expression in breast cancer. Collectively, our findings link the molecular roles of FOXF1 to cell cycle regulation and other biological processes.
In summary, our studies support a novel role for FOXF1 transcription factor in cell cycle regulation; a function that is abrogated through epigenetic silencing of FOXF1 during tumorigenesis. Therefore, FOXF1 and its downstream effectors might serve as novel molecular targets for detection and treatment of breast cancer and other human cancers.