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

项目总结/摘要 在过去的两年中，我们已经表明：1）Wwox，Ap 2和ErbB 4是他莫昔芬的独立标记物 阻力Wwox表达降低在预测耐药方面优于PR，尤其是在高危人群中。 患者，且核Ap 2 â表达优于Her 2，尤其是在低风险患者中; 2）ErbB 4缺失 当调整其他重要预测因素时，是他莫昔芬耐药的独立标志物; 3）体外研究 他莫昔芬耐药细胞的研究证实，Ap 2？在细胞质中被Wwox结合，释放到细胞质中， 在Wwox阴性、他莫昔芬抗性细胞中，细胞核中的细胞核，并且是HER 2的细胞核激活剂; 4）>800侵袭性 组织微阵列（TMAs）上的乳腺癌被分为特定的亚型：三阴性肿瘤 (TN：ER、PR、HER 2阴性，基底细胞样肿瘤）显示EGFR、CK 5/6和AP 2？频繁表达， Fhit和Wwox的频繁丢失，表明Fhit和Wwox表达的减少在 乳腺癌基底分化的发病机制。Fhit和Wwox表达的改变发生在 ~90%的基底细胞样/TN乳腺癌，可能导致DNA修复缺陷，如BRCA 1- 缺陷型癌症DNA损伤反应（DDR）蛋白（<$H2AX，pChk 2，p53）在细胞内高表达 在TN和基底样肿瘤中显著更多。因此，DDR检查点蛋白可以成为治疗 这些癌症。用于机制研究的特定亚型乳腺癌细胞系，以及具有来自 对于体内研究，将使用具有相关治疗和结局数据的原发性和转移性癌症， 实验概述如下目的：1.体外乳腺癌中的WWOX效应物：a）使用乳腺癌细胞， 管腔A、HER 2+和TN/基底亚型的癌细胞，鉴定特异性表达的Wwox相互作用蛋白， 亚型，在用AdenoWWOX感染后，分离Wwox复合物，然后进行质谱分析 鉴定复合物中的蛋白质; B）检查特定乳腺癌中候选相互作用物的表达， 细胞系和组织中的癌症亚型; 2.乳腺癌中的DNA损伤反应检查点抑制剂 体外：a）将测试Chk 1和Parp 1抑制剂对组织中特定乳腺癌亚型的作用 培养; B）Chk 1和Parp 1抑制剂与细胞毒性化疗药物联合使用， 对特定乳腺癌亚型细胞毒性的影响; 3.已启动的复员方案检查站 乳腺癌与治疗结果：根据ER、PR、HER 2将TMA上的肿瘤分为亚型， EGFR、CK 5、6状态;评估每种亚型肿瘤中DDR检查点蛋白的状态， 治疗和结果的标志物; 4.条件性小鼠Wwox敲除X Fhit敲除。的 将检查小鼠杂交对乳腺癌的易感性以及与乳腺癌相关的机制。 正常腺体和肿瘤腺体的发育。 1

PROJECT SUMMARY / ABSTRACT In the last two years we have shown that: 1) Wwox, Ap2¿, 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 Ap2¿ 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 Ap2¿ 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 AP2¿ 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 (¿H2AX, 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 in development of normal and neoplastic glands. 1