Chemoprevention and therapeutic treatment of BRCA1 associated mammary tumors

I.染料木黄酮对BRCA 1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的大豆消费量高有关。大豆的活性抑制成分被认为是植物雌激素，如染料木素和大豆苷元。染料木黄酮已被证明可以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性激酶（Cdk）抑制剂p21、细胞周期蛋白B1和磷酸化p34。染料木黄酮还可以通过抑制拓扑异构酶II活性来抑制DNA的去连环化，并通过稳定共价拓扑异构酶IDNA切割复合物来产生DNA双链断裂（DSB）来诱导DNA损伤。此外，染料木黄酮已显示通过降低Bcl 2的表达和上调Bax来诱导细胞凋亡。 目前，还没有关于染料木黄酮对BRCA 1相关乳腺癌的影响的报道。鉴于染料木黄酮抑制大多数不同类型的癌症研究到目前为止，我们假设染料木黄酮可能抑制BRCA 1相关的乳腺癌的生长。为了研究这一点，我们用染料木素处理Brca 1突变乳腺肿瘤细胞。我们发现，染料木黄酮治疗耗尽G1细胞群，这是伴随着在G2细胞的积累。一些染料木黄酮处理的细胞进入有丝分裂，然而，他们表现出染色体异常，并保持四倍体由于有丝分裂退出流产。部分G2期细胞发生核内复制，成为多倍体，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，Brca 1突变细胞对染料木黄酮比其他类型的癌细胞更敏感，突出了染料木黄酮对BRCA 1相关乳腺癌的良好治疗潜力。 二. PARP-1抑制对BRCA 1突变癌细胞的影响。 BRCA 1和PARP 1都参与DNA损伤反应和DNA损伤修复。最近的研究表明，抑制PARP 1代表了一种有前途的化学预防/治疗方法，用于特异性治疗BRCA 1和BRCA 2相关的乳腺癌。为了研究Brca 1和Parp 1之间的遗传相互作用，我们将携带全长Brca 1杂合缺失（Brca 1（+/Delta 11））的小鼠与Parp 1缺失小鼠杂交。我们发现Brca 1（Delta 11/Delta 11）; Parp 1（-/-）胚胎在胚胎（E）第6.5天之前死亡，而Brca 1（Delta 11/Delta 11）胚胎在E12.5天之后死亡，这表明Parp 1的缺乏显著加速了Brca 1缺乏引起的致死性。令人惊讶的是，Parp 1在Brca 1（Delta 11/Delta 11）胚胎中的单倍不足诱导严重的染色体畸变，中心体扩增和端粒功能障碍，导致细胞凋亡和加速胚胎致死。值得注意的是，Brca 1（Delta 11/Delta 11）; Parp 1（+/-）MEFs中端粒缩短与Ku 70表达减少相关，Ku 70在端粒维持中起重要作用。因此，Parp 1的单倍体丢失足以诱导Brca 1缺陷细胞的致死性，这表明部分抑制PARP 1可能代表了BRCA 1相关乳腺癌的一种实用的化学预防/治疗方法。 接下来，我们通过使用PARP-1抑制剂AG 14361测试PARP-1抑制对BRCA 1突变细胞的影响。我们发现BRCA 1缺陷的ES细胞对AG 14361的处理非常敏感。培养的BRCA 1突变癌细胞也显示出对AG 14361的一些敏感性。然而，在携带BRCA 1-/-乳腺肿瘤的小鼠的同种异体移植物中，我们仅观察到BRCA 1-/-和BRCA 1 +/+肿瘤中肿瘤生长的部分抑制。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BRCA 1-/-乳腺癌细胞对PARP-1抑制剂耐药的确切原因仍然难以捉摸。这些发现表明，PARP抑制可作为预防BRCA相关乳腺癌的方法，并可与其他化疗药物联合治疗乳腺癌。目前，我们正在筛选其他可以与AG 14361协同杀死BRCA 1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.