Chemoprevention and therapeutic treatment of BRCA1 associated mammary tumors

I.金雀异黄素对BRCA1突变癌细胞的作用 在中国和日本等东方国家，患乳腺癌的风险比西方国家低得多。各种研究表明，这种风险的降低可能与这些国家大量消费大豆有关。大豆的活性抑制成分被认为是异黄酮类植物雌激素，如染料木素和大豆苷元。金雀异黄素已被证明通过诱导细胞周期停滞来抑制细胞生长，可能是通过上调细胞周期依赖激酶(CDK)抑制物p21、细胞周期蛋白B1和磷酸化p34。金雀异黄素还可以通过抑制拓扑异构酶II的活性来抑制DNA的降解，并通过稳定共价拓扑异构酶IIDNA裂解复合体而产生DNA双链断裂(DSB)来诱导DNA损伤。此外，金雀异黄素已被证明通过减少bcl2的表达和上调bax的表达来诱导细胞凋亡。 目前，还没有关于金雀异黄素对BRCA1相关乳腺癌的作用的报道。鉴于到目前为止所研究的金雀异黄素抑制大多数不同类型的癌症的观察，我们假设金雀异黄素可能抑制BRCA1相关乳腺癌的生长。为了研究这一点，我们用染料木素处理BRCA1突变的乳腺肿瘤细胞。我们发现金雀异黄素处理耗尽了G1期细胞，并伴随着G2期细胞的聚集。一些染料木素处理的细胞进入有丝分裂，但由于流产的有丝分裂退出，它们表现出染色体异常并保持四倍体。部分G2细胞发生内复制，形成多倍体，并通过激活DNA损伤反应增加细胞死亡。此外，我们的数据表明BRCA1突变细胞比其他类型的癌细胞对金雀异黄素更敏感，这突显了金雀异黄素对BRCA1相关乳腺癌的良好治疗潜力。 II.抑制PARP-1对BRCA1突变癌细胞的影响 BRCA1和PARP1均参与DNA损伤反应和DNA损伤修复。最近的研究表明，抑制PARP1是一种有希望的化学预防/治疗方法，用于特异性治疗BRCA1和BRCA2相关的乳腺癌。为了研究BRCA1和PARP1之间的遗传相互作用，我们将携带BRCA1全长杂合缺失(BRCA1(+/Delta11))的小鼠与PARP1缺失的小鼠进行杂交。我们发现BRCA1(Delta11/Delta11)；PARP1(-/-)胚胎在胚胎6.5天之前死亡，而BRCA1(Delta11/Delta11)胚胎在E12.5之后死亡，这表明PARP1的缺失显著加速了BRCA1缺乏引起的死亡。令人惊讶的是，BRCA1(Delta11/Delta11)胚胎中PARP1的单倍性不足会导致严重的染色体异常、中心体扩增和端粒功能障碍，导致细胞凋亡和加速胚胎死亡。值得注意的是，BRCA1(Delta11/Delta11)中端粒缩短；PARP1(+/-)MEF与Ku70表达降低相关，Ku70在端粒维持中起重要作用。因此，PARP1的单倍体缺失足以诱导BRCA1缺陷细胞的死亡，提示部分抑制PARP1可能是BRCA1相关乳腺癌的一种实用的化学预防/治疗方法。 接下来，我们用PARP-1抑制剂AG14361检测了PARP-1对BRCA1突变细胞的抑制作用。我们发现BRCA1缺陷的ES细胞对AG14361非常敏感。培养的BRCA1突变癌细胞对AG14361也表现出一定的敏感性。然而，在携带BRCA1-/-乳腺肿瘤的小鼠的同种异体移植中，我们只观察到BRCA1-/-和BRCA1+/+肿瘤的肿瘤生长受到部分抑制。我们的研究表明，在癌症进展过程中发生的额外突变可能是罪魁祸首，尽管BRCA1-/-乳腺癌细胞对PARP-1抑制剂产生耐药性的确切原因仍不清楚。这些发现表明，抑制PARP可能是预防BRCA相关乳腺癌的一种方法，并可能与其他化疗药物联合用于乳腺癌的治疗。目前，我们正在筛选其他可以与AG14361协同杀死BRCA1突变癌细胞的化学物质。

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.