Wwox as a Critical Signal Mediator in Breast Cancer

描述(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的腺体中表达缺失或降低，这表明WWOX缺失在乳腺癌中是一种早期的、可能是易感的事件。Wwox蛋白通过其第一个WW结构域与转录因子p73、Ap2pha和Gamma、ErbB4相互作用，并影响它们的转录活性。抑制MCF7乳腺癌细胞中WWOX的表达可以消除体外他莫昔芬的反应，相反，耐他莫昔芬的MCF7克隆显示WWOX表达降低。这项建议的近期目标是：确定癌症衍生细胞系中他莫昔芬耐药的机制，确认癌症组织中的途径，从而确定可能决定哪些癌症对他莫昔芬治疗敏感的标记物；绘制整个乳腺癌切除标本中WWOX启动子甲基化的程度，作为乳腺癌现场癌变的标记物。这项拟议的研究基于相互关联的假设，即WWOX的表达水平是乳腺癌对ERAlpha阳性肿瘤中他莫昔芬反应的关键决定因素，WWOX缺失是乳腺癌的早期决定因素。长期目标是建立WWOX作为乳腺癌激素反应和治疗的中心媒介，一种可以通过表观遗传疗法重新表达的媒介，以激活或维持他莫昔芬的敏感性。这些研究的目的是：1.建立WWOX表达与他莫昔芬反应的关系，方法是：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；b)在类似的实验中使用WWOX WW域突变体，取消WWOX与相互作用的转录因子的结合；c)在WWOX阳性和阴性细胞中恢复ERα的表达，并检测他莫昔芬的作用；d)检测PKA和PKA-Rlα的表达水平与WWOX表达水平的关系；e)通过去甲基化试剂确定WWOX重新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WWOX阴性乳腺癌可以通过表观遗传疗法重建他莫昔芬的敏感性。2.通过a)检测已知的Wworx结合伙伴在三苯氧胺敏感和难治性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Wworx表达缺失与PKA-Rlpha表达降低之间的机制联系；c)通过免疫共沉淀鉴定新的相互作用因子，并检测在他莫昔芬反应中的作用，如果已知的Wworx相互作用因子与耐药性无关。这一目标将确定介导他莫昔芬反应的关键WWOX结合蛋白。3.通过测定ERpha、PR、WWOX、ErbB2、Ap2pha、Ap2Gamma、p73、ErbB4、PKA和PKA-Rlpha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小组，并重新评估ER、PR和ErbB2状态；b)免疫组织化学评估WWOX相互作用蛋白的表达和亚细胞定位。这一目标将在乳腺癌体内证实WWOX在调节ErbB2等蛋白的表达中的作用，以及WWOX的表达和亚细胞定位，其功能控制他莫昔芬的反应。4.绘制手术切除的乳腺癌组织中WWOX启动子甲基化的范围。这一目的将决定WWOX沉默是否是野场癌变效应的标志。

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.