Wwox and Fhit loss and the DNA damage response in breast cancer subtypes

描述(申请人提供)：在过去的两年中，我们已经证明：1)WWOX、Ap23和ErbB4是三苯氧胺抗性的独立标记。在预测耐药性方面，Wwox表达降低优于PR，尤其是在高危患者，核Ap23的表达好于Her2，尤其在低风险患者；2)当调整其他有意义的预测因子时，ErbB4丢失是他莫昔芬耐药的独立标志；3)对他莫昔芬耐药细胞的体外研究证实，在Wworx阴性的他莫昔芬耐药细胞中，Ap23与Wworx结合，释放到细胞核中，是HER2的核激活因子；4)&GT；对800例浸润性乳腺癌组织芯片(TMAS)进行亚型分析：三阴性肿瘤(TN：ER、PR、HER2阴性、基底细胞样癌)EGFR、CK5/6和AP23表达频繁，FHIT和WWOX表达缺失，提示FHIT和WWOX表达降低在乳腺癌基础分化的发病机制中起一定作用。FHIT和WWOX的表达改变发生在约90%的基底细胞样型/TN乳腺癌中，并可能导致DNA修复缺陷，就像在BRCA1缺陷的乳腺癌中观察到的那样。DNA损伤反应(DDR)蛋白(3H_2AX、pChk2、P53)在TN和基底细胞样瘤中的表达显著增加。因此，DDR检查点蛋白可能成为治疗这些癌症的靶点。用于机制研究的特定亚型乳腺癌细胞系，以及用于体内研究的具有关联治疗和结果数据的来自原发和转移癌的核心的TMA将被用于下列目标概述的实验：1.乳腺癌的体外WWOX效应：a)使用管腔A、HER2+和TN/Basal亚型的乳腺癌细胞，鉴定特定亚型中的WWOX相互作用蛋白，在感染AdenoWWOX后，分离WWOX复合体，然后对复合体中的蛋白质进行质谱鉴定；b)检测特定乳腺癌亚型中候选相互作用分子在细胞系和组织中的表达；2.体外乳腺癌中的DNA损伤反应检查点抑制物：a)Chk1和PARP1抑制剂将在组织培养中测试对特定乳腺癌亚型的作用；b)Chk1和PARP1抑制剂将与细胞毒性化疗药物一起测试对特定乳腺癌亚型的细胞毒性的影响；3.乳腺癌中被激活的DDR检查点与治疗结果的关系：根据ER、PR、HER2、EGFR、CK5、6状态将TMA上的肿瘤分成亚型；评估每个亚型肿瘤中DDR检查点蛋白的状态，并与治疗和结果相关的标记；4.有条件的小鼠Wwox基因敲除FX基因敲除。将检测小鼠杂交对乳腺癌的易感性，以及正常和肿瘤腺体的发育机制。1与公共卫生相关：我们发现，三阴性/基底样癌是通常困扰年轻女性和非洲裔妇女的典型侵袭性乳腺癌，优先表现出激活的检查点蛋白表达的证据；三阴性/基底样癌也表达减少的FHIT和WWOX，这是参与保护基因组完整性的蛋白质，FHIT丢失导致强烈的检查点激活和对顺铂和紫杉醇的耐药性。BRCA1和BRCA2突变乳腺癌通常是基底样亚型，在实验室检测时，非常容易被DNA损伤反应检查点的抑制剂杀死。我们将确定基底样乳腺癌细胞和其他FHIT/WWOX缺陷乳腺癌亚型是否像BRCA1/2缺陷细胞一样，容易被检查点抑制剂杀死。化疗是目前治疗这些癌症的唯一选择，如果靶向抑制DNA修复途径与DNA损伤剂(如卡铂)相结合，可能会成为更好的选择。

DESCRIPTION (provided by applicant): In the last two years we have shown that: 1) Wwox, Ap23, and ErbB4 are independent markers of tamoxifen resistance. Reduced Wwox expression was better than PR in prediction of resistance, especially in high-risk patients, and nuclear Ap23 expression was better than Her2, especially in low-risk patients; 2) ErbB4 loss was an independent marker of tamoxifen resistance when adjusted for other significant predictors; 3) in vitro studies of tamoxifen resistant cells confirmed that Ap23 was bound by Wwox in the cytoplasm, released into the nucleus in Wwox negative, tamoxifen resistant cells and was a nuclear activator of HER2; 4) >800 invasive breast cancers on Tissue Micro Arrays (TMAs) were classified into specific subtypes: triple negative tumors (TN: ER, PR, HER2 negative, basal-like tumors) showed frequent expression of EGFR, CK5/6 and AP23 and frequent loss of Fhit and Wwox, suggesting that reduced Fhit and Wwox expression have roles in pathogenesis of basal differentiation in breast cancer. Alteration of expression of Fhit and Wwox occured in ~90% of the basal-like/TN breast cancers and may contribute to defects in DNA repair, as observed in BRCA1- deficient cancers. DNA damage response (DDR) proteins (3H2AX, pChk2, p53) were expressed highly significantly more in TN and basal-like tumors. Thus, DDR checkpoint proteins could be targets for treatment of these cancers. Specific subtype breast cancer cell lines, for mechanistic studies, and TMAs with cores from primary and metastatic cancers with linked treatment and outcome data, for in vivo studies, will be used in experiments outlined in the following Aims: 1. Wwox effectors in breast cancer in vitro: a) using breast cancer cells of luminal A, HER2+ and TN/Basal subtypes, identify Wwox interactor proteins in specific subtypes, after infection with AdenoWWOX, isolation of the Wwox complex, followed by mass spectrometry identification of proteins in the complex; b) examine expression of candidate interactors in specific breast cancer subtypes in cell lines and tissues; 2. DNA damage response checkpoint inhibitors in breast cancer in vitro: a) Chk1 and Parp1 inhibitors will be tested for effect on specific breast cancer subtypes in tissue culture; b) Chk1 and Parp1 inhibitors, in combination with cytotoxic chemotherapeutic drugs, will be tested for effect on cytotoxicity of specific breast cancer subtypes; 3. Association of activated DDR checkpoint in breast cancers with treatment outcome: divide tumors on TMAs into subtypes based on ER, PR, HER2, EGFR, CK5,6 status; assess status of the DDR checkpoint proteins in tumors of each subtype and correlate markers with treatment and outcome; 4. Conditional mouse Wwox knockdown X Fhit knockout. The mouse cross will be examined for susceptibility to mammary gland cancer and mechanisms involved i development of normal and neoplastic glands. 1 PUBLIC HEALTH RELEVANCE: We find that triple-negative/basal-like breast cancers, the typically aggressive breast cancers that commonly afflict young women and women of African origin, preferentially show evidence of expression of activated checkpoint proteins; also triple-negative/basal-like cancers express reduced Fhit and Wwox, proteins involved in protection of genome integrity, with Fhit loss resulting in strong checkpoint activation and resistance to cisplatin and paclitaxel. BRCA1 and 2 mutant breast cancers are usually of the basal-like subtype and when tested in the laboratory, are highly susceptible to killing by inhibitors of the DNA damage response checkpoint. We will determine if basal-like breast cancer cells and other Fhit/Wwox-deficient breast cancer subtypes are, like BRCA1/2-deficient cells, susceptible to killing by checkpoint inhibitors. Chemotherapy is currently the only option for these cancers and might become a better option if targeted inhibition of DNA repair pathways were combined with DNA damaging agents, such as carboplatin.