Humans and mice with alterations in Fanconi Anemia genes (BRCA2/FANCD1 and Fancd2, respectively) are predisposed to development of epithelial malignancies, including ovarian carcinomas. Further, loss of Fanconi genes during carcinogenesis predisposes to genetic instability in evolving clones. Specifically, epigenetic suppression of FANCF has been described in sporadic ovarian cancers (Taniguchi, Nat. Med. 9:568, 2003). Because Fanconi Anemia (FA) proteins have been shown to inhibit apoptosis in other cell types (hematopoietic cells and embryonic fibroblasts), we hypothesized that ovarian epithelial cells with inactivating mutations in FA genes would be: (a) similarly hypersensitive to apoptotic cues and (b) incapable of clonally evolving as neoplasms unless the effector pathway of apoptosis was first interdicted by somatic mutation and clonal selection. Flow cytometric analyses (Annexin V binding and dye exclusion viability) carried out on primary ovarian surface epithelial (OSE) cells exposed in vitro to hydrogen peroxide and to apoptosis-inducing cytokines confirmed this model. Specifically, cytokine hypersensitivity was revealed in experiments in which OSE cells were exposed to a combination of IFNgamma, TNFalpha, and fas-ligand. No hypersensitivity was noted with TRAIL or TGF-beta exposure. Additionally, steroid hormone deprivation through charcoal stripping of serum, exacerbated the hypersensitivity of FA mutant cells to cytokines. Taken together with the preponderance of tumors in female mice due to loss of FA proteins, our findings were compatible with an important role for estrogen in modulating apoptotic responses in OSE. Recognizing that inhibition of Trp53 abrogates the TNF-hypersensitivity of FA-C cells (Freie, Blood 102:4146, 2003) and that developmental anomalies in FANCD2 deficient zebrafish are also p53 dependent (Liu, TX, Dev. Cell, 5:903, 2004), we sought to test the capacity of p53 suppression in abrogating the cytokine hypersensitive phenotype. We transformed FANCC mutant OSE cells using SV40 large T-antigen. Transformed FANCC cells were unresponsive to cytokines but retained their hypersensitivity to hydrogen peroxide and to mitomycin C, confirming our prior work on hematopoietic cells demonstrating that FA proteins modulate at least two distinct apoptotic pathways. Additional work using dominant negative mutants forms of p53 will be described. We conclude that because loss of FA gene function in ovarian epithelial cells results in an apoptotic phenotype, loss of FA gene function is a later event than somatic mutations that interdict effector pathways for programmed cell death. Moreover, the apoptotic pathways that require modulation must be precisely those that would otherwise be activated by the specific FA gene mutation.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]