I. Effects of genistein on BRCA1 mutant cancer cells In Eastern countries like China and Japan, the risk for breast cancer is much lower than in Western countries. Various studies have shown that this reduced risk may be associated with a high amount of soy consumption in these countries. The active inhibitory components of soy are thought to be isoflavone phytoestrogens, such as genistein and daidzein. Genistein has been shown to inhibit cell growth by inducing cell cycle arrest, possibly through upregulation of cell cycle-dependent kinase (Cdk) inhibitor p21, cyclin B1, and phospho-p34. Genistein can also inhibit decatenation of DNA by inhibiting topoisomerase II activity and induce DNA damage by generating DNA double-strand breaks (DSBs) through stabilizing the covalent topisomerase IIDNA cleavage complex. Furthermore, genistein has been shown to induce apoptosis through decreased expression of Bcl2 and upregulation of Bax. Currently, there has been no report about the effects of genistein on BRCA1-associated breast cancer. Given the observation that genistein inhibits most different types of cancers studied so far, we hypothesize that genistein may inhibit the growth of BRCA1-associated breast cancer. To investigate this, we treated Brca1 mutant mammary tumor cells with genistein. We showed that genistein treatment depleted the G1 population of cells, which was accompanied by an accumulation of cells at G2. Some genistein-treated cells entered mitosis;however, they exhibited chromosome abnormalities and maintained tetraploidy owing to abortive mitotic exit. A fraction of G2 cells underwent endoreduplication and became polyploid, which was accompanied by increased cell death through activating DNA damage response. Furthermore, our data indicated that Brca1 mutant cells were more sensitive to genistein than some other types of cancer cells, highlighting a good therapeutic potential of genistein for BRCA1-associated breast cancer. II. Effects of PARP-1 inhibition on BRCA1 mutant cancer cells. BRCA1 and PARP1 are both involved in DNA-damage response and DNA-damage repair. Recent investigations have suggested that inhibition of PARP1 represents a promising chemopreventive/therapeutic approach for specifically treating BRCA1- and BRCA2-associated breast cancer. To study the genetic interactions between Brca1 and Parp1, we interbred mice carrying a heterozygous deletion of full-length Brca1 (Brca1(+/Delta11)) with Parp1-null mice. We show that Brca1(Delta11/Delta11);Parp1(-/-) embryos die before embryonic (E) day 6.5, whereas Brca1(Delta11/Delta11) embryos die after E12.5, indicating that absence of Parp1 dramatically accelerates lethality caused by Brca1 deficiency. Surprisingly, haploinsufficiency of Parp1 in Brca1(Delta11/Delta11) embryos induces a severe chromosome aberrations, centrosome amplification, and telomere dysfunction, leading to apoptosis and accelerated embryonic lethality. Notably, telomere shortening in Brca1(Delta11/Delta11);Parp1(+/-) MEFs was correlated with decreased expression of Ku70, which plays an important role in telomere maintenance. Thus, haploid loss of Parp1 is sufficient to induce lethality of Brca1-deficient cells, suggesting that partial inhibition of PARP1 may represent a practical chemopreventive/therapeutic approach for BRCA1-associated breast cancer. Next, we tested effect of PARP-1 inhibition on BRCA1 mutant cells by using a PARP-1 inhibitor, AG14361. We found that BRCA1-deficient ES cells are very sensitive to the treatment of AG14361. Cultured BRCA1 mutant cancer cells also show some sensitivities to AG14361. However, in allografts of mouse carrying BRCA1-/- mammary tumors, we only observe a partial inhibition of tumor growth in both the BRCA1-/- and BRCA1+/+ tumors. Our study indicated that additional mutations occurring during cancer progression may be a culprit, although the exact cause for the resistance of BRCA1-/- breastcancer cells to PARP-1 inhibitors remains elusive. These findings suggest that PARP inhibition may serve as an approach for the prevention of BRCA related breast cancer and may be useful in combination with other chemotherapeutic agents in the treatment of breast cancer. Currently we are screening other chemicals that can synergistically kill BRCA1 mutant cancer cells with AG14361.

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