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

项目摘要/摘要 近两年的研究表明：1)WWOX、AP2和ErbB4是三苯氧胺的独立标记 抵抗。Wwox表达降低在预测耐药性方面优于PR，尤其是在高危人群中 2)ErbB4缺失率明显高于Her2； 他莫昔芬耐药性的一个独立标记，当调整其他重要预测因素时；3)体外研究 证实AP2在细胞质中与WWOX结合，释放到 WWOX阴性、三苯氧胺耐药细胞中的核，是HER2；4)&GT；800侵袭性的核活化子 在组织微阵列(TMA)上，乳腺癌被分类为特定的亚型：三阴性肿瘤 (TN：ER，PR，HER2阴性，基底样瘤)EGFR、CK5/6和AP2表达频繁。 FHIT和WWOX的频繁丢失，表明FHIT和WWOX的表达减少在 乳腺癌基底分化的发病机制。FHIT和WWOX的表达发生了变化 ~90%的基底细胞样/TN乳腺癌，并可能导致DNA修复缺陷，如在BRCA1- 有缺陷的癌症。DNA损伤反应(DDR)蛋白(H_2AX、pChk2、P53)高表达 在TN和基底细胞样瘤中明显更多。因此，DDR检查点蛋白可能成为治疗的靶点。 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 具有相关治疗和结果数据的原发和转移癌症，将用于活体研究， 实验概述如下：1.WWOX效应器在体外乳腺癌中的作用：a)使用乳房 鲁米那A、HER2+和Tn/Basal亚型癌细胞鉴定特异性WWOX相互作用蛋白 亚型，感染AdenoWWOX后，分离Wwox复合体，然后进行质谱分析 鉴定复合体中的蛋白质；b)检测候选相互作用因子在特定乳房中的表达 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物 在体外：a)将测试Chk1和PARP1抑制剂对组织中特定乳腺癌亚型的影响 培养；b)将检测Chk1和PARP1抑制剂与细胞毒性化疗药物的结合 特定乳腺癌亚型对细胞毒作用的影响；3.激活的DDR检查点在 乳腺癌的治疗结果：根据ER、PR、HER2、 EGFR、CK5、6状态；评估DDR检查点蛋白在各亚型肿瘤中的状态及其相关性 标记与治疗和结果；4.有条件的小鼠WWOX基因敲除X FHIT基因敲除。这个 将检测小鼠杂交对乳腺癌的易感性和参与的机制 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