Wwox as a Critical Signal Mediator in Breast Cancer

描述（由申请人提供）：Wwox（一种可能的乳腺癌肿瘤抑制基因）的表达在大多数浸润性乳腺癌和DCIS以及1/3乳腺癌的一些正常腺体中丢失或减少，表明Wwox丢失是乳腺癌的早期，可能是诱发事件。Wwox蛋白通过其第一个WW结构域与转录因子p73，Ap 2 α和γ，ErbB 4相互作用，并影响其转录活性。MCF 7乳腺癌细胞中Wwox表达的敲低消除了体外他莫昔芬应答，相反，他莫昔芬抗性MCF 7克隆显示出降低的Wwox表达。该提案的直接目标是：定义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志物;绘制整个癌性乳房切除术标本中WWOX启动子甲基化的程度，作为乳腺癌领域癌变的标志物。该研究基于以下相互关联的假设：Wwox表达水平是ER α阳性肿瘤中乳腺癌对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。长期目标是将Wwox确立为激素反应和乳腺癌治疗的中心介体，该介体可以通过表观遗传疗法重新表达，以激活或维持他莫昔芬敏感性。目的是：1.通过以下步骤建立Wwox表达与他莫昔芬应答的关系：a）在乳腺癌细胞中上调和下调Wwox并检查对他莫昔芬应答的影响; B）在类似实验中使用消除Wwox与相互作用的转录因子结合的Wwox WW结构域突变体; c）恢复Wwox阳性和阴性细胞中的ER α表达并检查他莫昔芬的影响; d）检测PKA和PKA-R1 α相对于Wwox表达水平的表达水平; e）确定通过去甲基化剂进行的Wwox再活化对他莫昔芬应答的影响。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.通过以下步骤确定Wwox介导他莫昔芬信号的机制：a）在他莫昔芬敏感性和难治性乳腺癌中检查已知Wwox结合配偶体的表达和亚细胞定位; B）寻找Wwox表达丧失和PKA-R1 α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物，并检查在他莫昔芬应答中的作用，如果已知Wwox相互作用物不涉及抗性。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ER α、PR、Wwox、ErbB 2、Ap 2 α、Ap 2 γ、p73、ErbB 4、PKA和PKA-R1 α在他莫昔芬敏感性和耐药性乳腺癌中的表达水平和亚细胞定位，建立Wwox及其结合配偶体在乳腺癌中的体内相关性：a）通过选择他莫昔芬敏感性和难治性乳腺癌组并重新评估ER、PR和ErbB 2状态; B）Wwox相互作用蛋白的表达和亚细胞定位的免疫组织化学评价。这一目标将证实，在体内乳腺癌中，Wwox在介导ErbB 2等蛋白质的表达中的作用，以及其功能控制他莫昔芬反应的Wwox相互作用物的表达和亚细胞定位。4.绘制整个手术切除的癌性乳腺组织中WWOX启动子甲基化的程度。这一目标将确定WWOX沉默是否是田间癌变效应的标志。

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.