Functional analysis of the Rad51d (E233G) breast cancer associated polymorphism and a pharmacogenetic evaluation of RAD51D status

by Nadkarni, Aditi A.

RAD51D performs functions in homologous recombination and telomere maintenance, two genome stabilization mechanisms. A gene variant in the human RAD51D gene that replaced glutamic acid (E) to glycine (G) at amino acid position 233 was identified as a low penetrance allele in high-risk familial breast cancer cases not associated with either BRCA1 or BRCA2 mutations. This dissertation describes the effects of this variant allele on RAD51D protein interactions and function. Surprisingly, the Rad51d (E233G) polymorphism increased cellular resistance to the DNA damaging agents, mitomycin C, cisplatin, UV light and methyl methane sulfonate as well as to the microtubule inhibitor taxol in mouse cells. Additionally, the Rad51d (E233G) variant reduced the anaphase bridge index, a telomere dysfunction correlate, and conferred increased cellular proliferation, suggesting that this variant may affect telomere function. Protein interaction, measured by yeast-2-hybrid analysis, demonstrated a two-fold decrease in interaction between RAD51C and RAD51D-E233G, while normal levels of interaction between XRCC2 and the variant were maintained. Molecular modeling suggested that the glutamic acid-233 forms a salt bridge with lysine-23 in the N-terminal domain of RAD51D, and the E233G substitution may disrupt an inter-domain interaction. This indicated that the breast cancer associated E233G amino acid change affected RAD51D protein interactions and function. The effect of this variant was further tested in three human breast cancer cell lines, BT- 20, T47D and MCF-7. Interestingly, Rad51d (E233G) conferred increased cisplatin resistance and cell growth in breast cancer cells with a mutant p53 (BT20 and T47D) but not with a functional p53 (MCF-7). Inhibition of p53 by pifithrin alpha treatment rescued this phenotype in the MCF-7 cell lines suggesting that the effect was p53-dependent. Additionally, Rad51d (E233G) conferred an increase in the cisplatin resistance selectively of the MCF-7 cells in which p53 expression was stably knocked down by siRNA, further supporting the p53-dependent effect of the variant. These studies indicate that the E233G variant affects RAD51D protein interactions and functions and confers p53-dependent cisplatin resistance in breast cancer cell lines. This study is the first to analyze the functional effects of a clinically relevant RAD51D amino acid substitution. Since RAD51D functions in at least two possible mechanisms of chemoprotection, HR and telomere protection further study of this variant will provide insight into which cellular mechanism involving RAD51D functions in cellular response to anticancer agents. Ellagic acid is a dietary polyphenol currently being investigated for its anticarcinogenic as well as chemoprotective effects. A study described in the Results section of this dissertation describes the effects of mammalian Rad51d-gene status on cellular response to ellagic acid. Selective cytotoxicity of ellagic acid towards Rad51d-deficient cells with an HR defect and chromosomal instability provides preliminary evidence of a potential anticancer effect of ellagic acid that involves HR repair. The findings detailed in this dissertation characterize a role for RAD51D in chemoresistance and propose the potential of this HR player as a molecular target for anticancer therapy.