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

Wwox 和 Fhit 丢失以及乳腺癌亚型中的 DNA 损伤反应

• 批准号: 7729870
• 负责人: KAY HUEBNER
• 金额: $ 17.71万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-18 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729870
• 关键词: African; Agreement; BRCA1 gene; Binding; Breast; Breast Cancer Cell; Cancer cell line; Carboplatin; Cell Line; Cell Nucleus; Cells; Chromosome Fragile Sites; Cisplatin; Clinical; Complex; Cytoplasm; Cytotoxic agent; DNA Damage; DNA Repair; DNA Repair Pathway; DNA damage checkpoint; Data; Defect; Development; Disseminated Malignant Neoplasm; ERBB2 gene; Epidermal Growth Factor Receptor; ErbB4 gene; FHIT gene; Funding; Genes; Genome; Gland; Goals; In Vitro; Infection; Knock-out; Knockout Mice; Laboratories; Link; Malignant Neoplasms; Mammary Neoplasms; Mammary gland; Mass Spectrum Analysis; Membrane; Modeling; Molecular; Multivariate Analysis; Mus; Neoplasms; Nickel; Nuclear; Nude Mice; Paclitaxel; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Predisposition; Proteins; Resistance; Risk; Role; Signal Pathway; TP53 gene; Tamoxifen; Testing; Tissue Microarray; Tissues; Treatment outcome; Universities; Woman; Xenograft procedure; base; cancer therapy; chemotherapy; cytotoxic; cytotoxicity; high risk; in vivo; inhibitor/antagonist; killings; malignant breast neoplasm; mutant; neoplastic; promoter; public health relevance; research study; response; tissue culture; tumor

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.

描述（由申请人提供）：在过去的两年中，我们已经表明：1）Wwox，Ap 23和ErbB 4是他莫昔芬耐药的独立标志物。Wwox表达降低对三苯氧胺耐药的预测作用优于PR，尤其是在高危患者中，Ap 23表达降低对三苯氧胺耐药的预测作用优于Her 2，尤其是在低危患者中; 3）他莫昔芬抗性细胞的体外研究证实，Ap 23在细胞质中被Wwox结合，在Wwox阴性时释放到细胞核中，他莫昔芬抗性细胞，并且是HER 2的核激活剂; 4）在组织微阵列（TMA）上将>800个浸润性乳腺癌分类为特定亚型：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 23的频繁表达以及Fhit和Wwox的频繁丢失，表明Fhit和Wwox表达降低在乳腺癌基底分化的发病机制中具有作用。Fhit和Wwox表达的改变发生在~90%的基底样/TN乳腺癌中，并且可能导致DNA修复缺陷，如在BRCA 1缺陷型癌症中观察到的。DNA损伤反应（DDR）蛋白（3 H2 AX、pChk 2、p53）在TN和基底细胞样肿瘤中的表达显著高于正常对照组。因此，DDR检查点蛋白可以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来自原发性和转移性癌症的核心的TMA，其具有相关的治疗和结果数据，将用于以下目的中概述的实验：体外乳腺癌中的Wwox效应物：a）使用管腔A、HER 2+和TN/基底亚型的乳腺癌细胞，鉴定特定亚型中的Wwox相互作用物蛋白，在用AdenoWWOX感染后，分离Wwox复合物，随后质谱鉴定复合物中的蛋白; B）检查细胞系和组织中特定乳腺癌亚型中候选相互作用物的表达; 2.体外乳腺癌中的DNA损伤反应检查点抑制剂：a）将测试Chk 1和Parp 1抑制剂对组织培养物中特定乳腺癌亚型的影响; B）将测试Chk 1和Parp 1抑制剂与细胞毒性化疗药物组合对特定乳腺癌亚型的细胞毒性的影响; 3.乳腺癌中激活的DDR检查点与治疗结果的关联：基于ER、PR、HER 2、EGFR、CK 5、6状态将TMA上的肿瘤分为亚型;评估每种亚型肿瘤中DDR检查点蛋白的状态，并将标志物与治疗和结果相关联; 4.条件性小鼠Wwox敲除X Fhit敲除。将检查小鼠杂交对乳腺癌的易感性以及涉及正常和肿瘤腺体发育的机制。1.公共卫生相关性：我们发现，三阴性/基底样乳腺癌，通常困扰年轻女性和非洲裔女性的典型侵袭性乳腺癌，优先显示出活化检查点蛋白表达的证据;三阴性/基底样癌症也表达减少的Fhit和Wwox，参与保护基因组完整性的蛋白质，Fhit损失导致强烈的检查点活化和对顺铂和紫杉醇的抗性。BRCA 1和2突变乳腺癌通常属于基底样亚型，当在实验室中进行测试时，非常容易被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缺陷型乳腺癌亚型是否像BRCA 1/2缺陷型细胞一样容易被检查点抑制剂杀死。化疗目前是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，化疗可能会成为更好的选择。