Elucidating the function of WNT4 in endocrine response and resistance in invasive lobular carcinoma

 描述(申请人提供)：应聘者：Matthew J.Sikora博士在密歇根大学接受药理学本科和研究生培训期间开始了他的研究生涯。在此期间，西科拉博士对将实验室发现转化为临床应用的转化研究产生了兴趣。此外，他还对癌症研究产生了热情，并致力于乳腺癌研究事业。在匹兹堡大学博士后培训期间，西科拉博士开始研究浸润性小叶癌(ILC)的内分泌反应和耐药性，ILC是一种鲜为人知的乳腺癌亚型。这项研究让西科拉博士发展了他在药理学和内分泌治疗方面的优势，同时也发掘了他对个体化医学的兴趣，并开发了转化型研究项目。西科拉博士一直在努力实现成为一流学术机构和综合性癌症中心的首席研究员的目标，最终目标是将他的研究进展转化为改善癌症治疗和患者结果的方法。 培训：该研究项目包括重要的培训方面，既能增强西科拉博士目前的研究实力，又能让他从导师那里脱颖而出。研究项目的总体重点(如下所述)将为西科拉博士提供在阐明信号转导途径方面的丰富经验，这在以前并不是他培训的组成部分。这种经验对于理解和靶向治疗耐药的机制是至关重要的。此外，他对Wnt信号的关注是他目前的导师Steffi Oester reich博士的一个新的研究领域。与乳腺WNT信号专家卡罗琳·亚历山大博士共同指导，将为西科拉博士的独立研究生涯提供独特的方向。该项目还将利用新的模型系统，使西科拉博士能够直接在患者肿瘤组织中快速测试治疗策略。通过与Karen Knudsen博士的合作，Sikora博士将开发体外组织培养模型，以评估Wnt信号抑制物在患者来源的ILC组织中的功能。在BOT信号转导和新型模型系统方面的培训，再加上西科拉博士在药理学和内分泌疗法方面的独特背景，将使西科拉博士在乳腺癌研究方面拥有独特的利基市场，这将使他能够影响患者的护理。 研究：该项目的总体假设是，浸润性小叶癌细胞利用由WNT4驱动的独特信号通路来调节ILC的内分泌反应，WNT4代表假定的靶点。这一假说将在三个目标中得到验证：目标1：确定调节雌激素诱导的ILC细胞生长的WNT4受体复合体。WNT配体结合并激活卷曲(FZD)受体，后者随后激活杂乱(DVL)蛋白以启动信号传递；DVL蛋白也受到直接抑制。为了鉴定WNT4受体和相关蛋白，我们将使用新的免疫沉淀方法(IFAST)来鉴定WNT4和受体蛋白之间的蛋白质-蛋白质相互作用。这将与siRNA和小分子抑制剂结合，以识别雌激素诱导生长所必需的受体复合蛋白。受体复合体的活性也将在异种细胞系肿瘤的体外培养中作为ILC的新的3D模型进行评估。目的2：确定ILC细胞中非典型的WNT4信号级联反应。ILC的一个显著特征是E-钙粘附素的丢失，这在ILC细胞中是独一无二的，这也会导致β-连环蛋白的丢失和典型的Wnt信号的功能障碍。WNT4可能在ILC中利用了新的非正则途径。我们将使用蛋白质阵列研究WNT4介导的信号转导级联的激活。此外，我们将通过CRISPR介导的WNT4基因和WNT4基因上的ER结合位点的敲除来定义WNT4信号下游的特定转录程序。非正则信号在调节ILC细胞活性和增殖中的作用也将在ILC的3D模型中进行评估。目的3：确定Wnt4是否在ILC中促进雌激素非依赖性和内分泌抵抗。这一目标将启动该项目向临床应用的转化。为了研究Wnt4信号在内分泌耐药中的作用，我们将使用siRNA和Wnt4途径抑制剂来靶向ILC细胞系的新的雌激素非依赖性变体。此外，Wnt4通路基因的表达将在ILC患者样本中进行测量，并进行长期随访。患者来源的ILC肿瘤组织的体外培养将被用于确定能够有效抑制WNT4信号并改善ILC患者内分泌反应的治疗策略。ILC代表了一个研究不足的患者群体，是个体化治疗以改善患者结果的理想选择。我们假设靶向Wnt4及其下游信号通路将改善ILC细胞的内分泌反应。该项目将阐明以前未知的途径，并将确定预防或逆转ILC内分泌耐药的假定治疗靶点。通过在新模型、存档患者样本和患者衍生组织中测试假说，该项目将迅速将结果转化为临床应用 最终改善ILC患者的预后。

 DESCRIPTION (provided by applicant): Candidate: Dr. Matthew J. Sikora began his research career during his undergraduate and graduate training in pharmacology at the University of Michigan. During this time, Dr. Sikora developed an interest in translational research that moves laboratory findings to clinical application. Also, he developed a passion for cancer research, and committed to a career in breast cancer research. During his post-doctoral training at the University of Pittsburgh, Dr. Sikora began to study endocrine response and resistance in invasive lobular carcinoma (ILC), a poorly understood subtype of breast cancer. This research has allowed Dr. Sikora to develop his strengths in pharmacology and endocrine therapy, while also exploring his interest in individualized medicine and developing translational research projects. Dr. Sikora has been working toward the goal of becoming a principle investigator at a top-tier academic institution and comprehensive cancer center, with the ultimate goal of translating his research progress to improve cancer therapy and patient outcomes. Training: This research project includes important training aspects that will both augment Dr. Sikora's current research strengths and allow him to differentiate himself from his mentor. The overall focus of the research project (described below) will provide Dr. Sikora with extensive experience in elucidating signal transduction pathways, which has not previously been a component of his training. This experience is critical in understanding and targeting mechanisms of therapeutic resistance. Additionally, his focus on Wnt signaling is a new area of research for his current mentor, Dr. Steffi Oesterreich. A co-mentorship with Dr. Caroline Alexander, an expert in Wnt signaling in the mammary gland, will provide a unique direction for Dr. Sikora's independent research career. This project will also utilize novel model systems that will allow Dr. Sikora to rapidly test therapeutic strategies directly in patient tumor tissue. Throgh collaboration with Dr. Karen Knudsen, Dr. Sikora will develop the ex vivo tissue culture model to assess the function of Wnt signaling inhibitors in patient-derived ILC tissues. The training in bot signal transduction and novel model systems, in conjunction with Dr. Sikora's unique background in pharmacology and endocrine therapies, will provide Dr. Sikora with a unique niche in breast cancer research that will allow him to impact patient care. Research: The overall hypothesis of this project is that invasive lobular carcinoma cells utilize unique signaling pathways driven by WNT4 that represent putative targets to modulate endocrine response in ILC. This hypothesis will be tested in three aims: Aim 1: Identify the WNT4 receptor complex that regulates estrogen-induced growth in ILC cells. Wnt ligands bind and activate frizzled (FZD) receptors which subsequently activate dishevelled (DVL) proteins to initiate signaling; DVL proteins are also subject to direct inhibition. To identify the WNT4 receptor and associated proteins, we will use novel immunoprecipitation methods (IFAST) to identify protein-protein interactions among WNT4 and receptor proteins. This will couple with siRNA and small molecule inhibitors to identify receptor complex proteins necessary for estrogen-induced growth. Receptor complex activity will also be assessed in ex vivo cultures of cell line xenograft tumors as a novel 3D model of ILC. Aim 2: Identify the non-canonical WNT4 signaling cascade in ILC cells. A defining feature of ILC is loss of E-cadherin; uniquely in ILC cells, this also causes β-catenin protein loss and dysfunction of canonical Wnt signaling. WNT4 likely utilizes novel non-canonical pathways in ILC. We will examine WNT4-mediated activation of signal transduction cascades using protein arrays. Additionally, we will define the specific transcriptional program downstream of WNT4 signaling via CRISPR-mediated knockout of the WNT4 gene and the ER binding site at the WNT4 locus. The role of non-canonical signaling in regulating ILC cell viability and proliferation will also be assessed in 3D models of ILC. Aim 3: Determine whether WNT4 drives estrogen-independence and endocrine resistance in ILC. This aim will initiate translation of this project toward clinical application. To examine the role of WNT4 signaling in endocrine resistance, we will use siRNA and WNT4 pathway inhibitors to target novel estrogen independent variants of ILC cell lines. Further, WNT4 pathway gene expression will be measured in ILC patient specimens with long-term follow-up data. Ex vivo cultures of patient-derived ILC tumor tissue will be used to identify therapeutic strategies that can effectively inhibit WNT4 signaling and improve endocrine response for ILC patients. ILC represents an understudied patient population, ideal for individualized therapies to improve patient outcomes. We hypothesize that targeting WNT4 and downstream signaling pathways will improve endocrine response in ILC cells. This project will elucidate previously uncharacterized pathways, and will identify putative targets for therapy to prevent or reverse endocrine resistance in ILC. By testing hypotheses in novel models, archival patient samples, and patient-derived tissues, this project will rapidly translate results to clinical application to ultimately improve ILC patient outcomes.