Wwox as a Critical Signal Mediator in Breast Cancer

描述（由申请人提供）：Wwox是一种可能的乳腺癌抑癌基因，在大多数浸润性乳腺癌和DCIS以及1/3的乳腺癌中一些正常腺体中表达缺失或减少，表明Wwox缺失是乳腺癌的早期，可能是易感事件。Wwox蛋白通过其第一个WW结构域与转录因子p73、ap2 α和γ、ErbB4相互作用，并影响其转录活性。在MCF7乳腺癌细胞中，敲低Wwox的表达消除了体外他莫昔芬的反应，相反，他莫昔芬耐药MCF7克隆显示Wwox的表达降低。本提案的直接目标是：确定癌症来源细胞系的他莫昔芬耐药机制，确认癌症组织中的途径，从而确定可能确定哪些癌症对他莫昔芬治疗敏感的标志物；绘制乳腺癌切除标本中WWOX启动子甲基化的程度，作为乳腺癌现场癌变的标志。提出的研究基于两个相互关联的假设，即在erα阳性肿瘤中，Wwox表达水平是乳腺癌对他莫昔芬反应的关键决定因素，而Wwox缺失是乳腺癌的早期决定因素。长期目标是建立Wwox作为激素反应和乳腺癌治疗的中心介质，一个可以通过表观遗传治疗重新表达的介质，激活或维持他莫昔芬的敏感性。其目的是：1。通过：a)上调和下调乳腺癌细胞中Wwox的表达并检测其对他莫昔芬反应的影响，建立Wwox表达与他莫昔芬反应的关系；b)在类似实验中使用Wwox WW结构域突变体，使Wwox与相互作用的转录因子的结合失效；c)恢复Wwox阳性和阴性细胞中erα的表达，并检测他莫昔芬的作用；d)检测PKA和PKA- rα相对于Wwox表达水平的表达水平；e)通过去甲基化剂确定Wwox再激活对他莫昔芬反应的影响。这一目标将证实Wwox在调节他莫昔芬反应中的关键作用，并表明可以通过表观遗传疗法在Wwox阴性乳腺癌中重建他莫昔芬敏感性。2. 通过a)检测已知Wwox结合伙伴在他莫昔芬敏感和难治乳腺癌中的表达和亚细胞定位，确定Wwox介导他莫昔芬信号的机制；b)寻找Wwox表达缺失与pka - rl - alpha表达降低之间的机制联系；c)如果已知的Wwox相互作用物与耐药性无关，则通过共免疫沉淀鉴定新的相互作用物，并检查其在他莫昔芬反应中的作用。这一目标将确定介导他莫昔芬反应的关键Wwox结合蛋白。3. 通过确定erα、PR、Wwox、ErbB2、Ap2alpha、Ap2gamma、p73、ErbB4、PKA和PKA- rα在他莫昔芬敏感和耐药乳腺癌中的表达水平和亚细胞定位，建立Wwox及其结合伙伴在乳腺癌中的体内相关性：a)通过选择他莫昔芬敏感和耐药乳腺癌组，重新评估ER、PR和ErbB2状态；b)免疫组化评价Wwox相互作用蛋白的表达和亚细胞定位。这一目的将证实，在乳腺癌体内，Wwox在介导ErbB2等蛋白的表达以及Wwox相互作用物的表达和亚细胞定位中的作用，其功能控制他莫昔芬的反应。4. 绘制手术切除的癌性乳腺组织中WWOX启动子甲基化的程度。这一目的将确定WWOX沉默是否是现场癌变效应的标志。

DESCRIPTION (provided by applicant): Expression of Wwox, a likely breast cancer tumor suppressor gene, is lost or reduced in the majority of invasive breast cancers and DCIS, as well as in some normal appearing glands in 1/3 of breast cancers, suggesting that Wwox loss is an early, perhaps predisposing event in breast cancer. Wwox protein, through its first WW domain, interacts with transcription factors p73, Ap2alpha and gamma, ErbB4, and affects their transcriptional activity. Knockdown of Wwox expression in MCF7 breast cancer cells abrogates the in vitro tamoxifen response and, conversely, tamoxifen resistant MCF7 clones show reduced Wwox expression. Immediate objectives of this proposal are to: define the mechanism of tamoxifen resistance in cancer-derived cell lines, confirm the pathway in cancer tissues, and thus identify markers that may determine which cancers will be sensitive to tamoxifen therapy; map the extent of WWOX promoter methylation throughout cancerous mastectomy specimens, as a marker for breast cancer field cancerization. The proposed research is based on the interconnected hypotheses that Wwox expression level is a critical determinant of the response of breast cancers to tamoxifen in ERalpha positive tumors and that Wwox loss is an early determinant of breast cancer. The long-term objective is to establish Wwox as a central mediator of hormone response and therapy of breast cancer, a mediator that can be reexpressed through epigenetic therapy, to activate or maintain tamoxifen sensitivity. The aims are to: 1. establish the relationship of Wwox expression to tamoxifen response by: a) up- and down-modulation of Wwox in breast cancer cells and examination of effect on tamoxifen response; b) use of Wwox WW domain mutants that abolish Wwox binding to interacting transcription factors in similar experiments; c) restore ERalpha expression in Wwox positive and negative cells and examine effect of tamoxifen; d) examine expression levels of PKA and PKA-Rlalpha relative to Wwox expression level; e) determine effect of Wwox reactivation, by demethylating agents, on tamoxifen response. This aim will confirm the critical role of Wwox in modulating the tamoxifen response and show that tamoxifen sensitivity can be reestablished in Wwox negative breast cancers by epigenetic therapies. 2. Determine mechanism of Wwox mediation of tamoxifen signals by a) examining expression and subcellular localization of known Wwox binding partners, in tamoxifen sensitive and refractory breast cancers; b) seeking a mechanistic connection between loss of Wwox expression and reduced PKA-Rlalpha expression; c) identifying novel interactors by coimmunoprecipitation and examining role in tamoxifen response, if known Wwox interactors are not implicated in resistance. This aim will identify the critical Wwox binding proteins that mediate tamoxifen response. 3. Establish the in vivo relevance of Wwox and its binding partners in breast cancer by determining expression levels and subcellular localization of ERalpha, PR, Wwox, ErbB2, Ap2alpha, Ap2gamma, p73, ErbB4, PKA and PKA-Rlalpha in tamoxifen sensitive and resistant breast cancers: a) by selection of panels of tamoxifen sensitive and refractory breast cancers and reassessment of ER, PR and ErbB2 status; b) immunohistochemical evaluation of expression and subcellular localization of Wwox interactor proteins. This aim will confirm, in vivo in breast cancers, the role of Wwox in mediating expression of such proteins as ErbB2, and expression and subcellular localization Wwox interactors whose function controls tamoxifen response. 4. Map the extent of WWOX promoter methylation throughout surgically removed cancerous breast tissues. This aim will determine if WWOX silencing is a marker of a field cancerization effect.