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

描述（由申请人提供）：在过去两年中，我们已经证明：1)Wwox， Ap23和ErbB4是他莫昔芬耐药的独立标记物。降低Wwox表达优于PR预测耐药，特别是在高危患者中；核Ap23表达优于Her2，特别是在低危患者中；2) ErbB4缺失是他莫昔芬耐药的独立标志物；3)他莫昔芬耐药细胞的体外研究证实，Ap23在细胞质中与Wwox结合，在Wwox阴性的他莫昔芬耐药细胞中释放到细胞核中，是HER2的核激活剂；4) bbb800浸润性乳腺癌在组织微阵列（TMAs）上被划分为特定亚型：三阴性肿瘤（TN: ER、PR、HER2阴性、基底样肿瘤）EGFR、CK5/6和AP23频繁表达，Fhit和Wwox频繁缺失，提示Fhit和Wwox表达减少在乳腺癌基底分化的发病机制中起作用。Fhit和Wwox的表达改变发生在约90%的基底样/TN乳腺癌中，并可能导致DNA修复缺陷，正如在BRCA1-缺陷癌症中观察到的那样。DNA损伤反应（DDR）蛋白（3H2AX、pChk2、p53）在TN和基底样肿瘤中表达显著增加。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，以及具有原发和转移性癌症核心的tma，具有相关的治疗和结果数据，用于体内研究，将用于以下目的概述的实验：体外研究乳腺癌中的Wwox效应物：a)利用乳腺癌细胞的luminal a、HER2+和TN/Basal亚型，鉴定特定亚型的Wwox相互作用蛋白，感染AdenoWWOX后，分离出Wwox复合物，随后用质谱法鉴定复合物中的蛋白；B)检测候选相互作用物在特定乳腺癌亚型细胞系和组织中的表达；2. DNA损伤反应检查点抑制剂在体外乳腺癌中的作用：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影响；b) Chk1和Parp1抑制剂联合细胞毒性化疗药物，将测试对特定乳腺癌亚型的细胞毒性的影响；3. 乳腺癌DDR检查点激活与治疗结果的关联：基于ER、PR、HER2、EGFR、CK5、6状态将TMAs上的肿瘤划分为亚型；评估每种亚型肿瘤中DDR检查点蛋白的状态，并将标记物与治疗和结果相关联；4. 条件小鼠Wwox敲除X Fhit敲除。小鼠杂交将检查对乳腺癌的易感性以及正常和肿瘤腺体发育的机制。公共卫生相关性：我们发现三阴性/基底样乳腺癌，通常是侵袭性乳腺癌，常见于年轻女性和非洲裔女性，优先表现出活化检查点蛋白的表达证据；此外，三阴性/基底样癌症表达Fhit和Wwox减少，Fhit缺失导致检查点激活和对顺铂和紫杉醇的抵抗。Fhit和Wwox是参与保护基因组完整性的蛋白质。BRCA1和2突变型乳腺癌通常是基底样亚型，在实验室测试时，极易被DNA损伤反应检查点抑制剂杀死。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DNA修复途径的靶向抑制与DNA损伤剂（如卡铂）相结合，化疗可能成为更好的选择。

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.