Targeting GRB7 Signaling in Solid Tumors

靶向实体瘤中的 GRB7 信号传导

• 批准号: 8820512
• 负责人: Shiuh-Wen Luoh
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8820512
• 关键词: 17q11; Aggressive behavior; American; Animal Model; Apoptosis; Area; Biological Assay; Biology; Breast Cancer Cell; Breast Cancer cell line; Cancer Biology; Cancer Cell Growth; Cancer Etiology; Catalysis; Cell Cycle; Cell Line; Cell Proliferation; Cells; Chromosomes; Chromosomes, Human, Pair 17; Clinical; Clinical Trials; Colon Carcinoma; Coupled; Data; Dependency; Development; Disease; ERBB2 gene; Esophageal; Extracellular Domain; FDA approved; GRB7 gene; Genes; Genetic; Genetic Screening; Human; Image Analysis; Incidence; Investigation; Lead; Libraries; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Metabolism; Methods; Military Personnel; Mission; Mus; Oncogenic; Outcome; Pathway interactions; Patients; Phase; Phenotype; Phosphotransferases; Preparation; Printing; Prognostic Factor; Prognostic Marker; Protein Overexpression; Research Priority; Resistance; Role; Signal Transduction; Signaling Molecule; Slide; Small Interfering RNA; Solid Neoplasm; Spottings; Technology; Therapeutic; Time; Transfection; Translations; Tumor Suppression; Virulent; Woman; Women' s Health; Work; Xenograft Model; base; bench to bedside; cancer type; cell growth; cell type; clinically significant; combinatorial; comparative; design; drug development; high throughput screening; human GRB7 protein; improved; inhibitor/antagonist; innovation; interest; knock-down; mRNA Differential Displays; malignant breast neoplasm; migration; new therapeutic target; novel; novel therapeutics; outcome forecast; public health relevance; screening; small hairpin RNA; small molecule; targeted treatment; therapeutic target; triple-negative invasive breast carcinoma; tumor

DESCRIPTION (provided by applicant): GRB7 is amplified and over-expressed in breast cancer with chromosome 17q11-12 amplification. GRB7 can interact with multiple signaling molecules and is implicated in breast cancer biology. GRB7 can facilitate HER-2 mediated signaling and tumor formation. Studies, including that of ours, have shown GRB7 protein overexpression, rather than that of HER-2, is an adverse prognostic factor in breast cancer. Moreover, GRB7 protein is the only adverse prognostic marker, from among 374 studied, in triple negative breast cancer. We hypothesize that GRB7 directed signaling is functionally important in breast cancer with chromosome 17q11-12 amplification. Targeting GRB7 signaling therefore represents an important and novel therapeutic strategy for HER-2 positive and triple negative breast cancers that are otherwise clinically aggressive. The objectives are: (1) to investigate the dependency of breast cancer cells on GRB7 signaling by determining if inhibition of GRB7 signaling functions leads to suppression of breast cancer cell growth; (2) to develop isogenic pairs of breast cancer cell lines with shRNA mediated GRB7 knockdown and perform ""Induced Essentiality"" screening with small molecule inhibitor libraries; (3) to perform screening with a siRNA library that covers the whole "actionable" kinome with cell spot microarrays and a novel reverse transfection technology to identify and validate targets whose knock down displays GRB7-dependent effects on proliferation, apoptosis, and/or migration/invasion; and (4) to investigate the functional involvement of candidate cell cycle kinase(s) in GRB7 signaling and their roles as therapeutic targets. Our approaches are the following: GRB7 knock down has been achieved by transient siRNA transfection in multiple breast cancer cell lines with chromosome 17q11-12 amplification. GRB7 protein depletion decreases cell proliferation in cell lines irrespective of their sensitivityto currently available HER-2 targeted therapies. Stable GRB7 knock down has been achieved by shRNA expression in breast cancer cell lines and its effect on cell growth in culture and in orthotopic mouse xenograft models will be assessed. Stable GRB7 knock down creates a 'sensitized' cellular context to screen for therapeutic targets and molecules whose signaling functions depend on GRB7 expression. We will screen for signaling molecules whose inhibition is sensitized by GRB7 over-expression- i.e. induced essentiality. In addition, signaling molecules whose inhibition displays differential sensitivity according to their GRB7 status will unravel the functional complexities of GRB7 signaling in breast cancer. The novel cell spot microarray coupled with siRNA reverse transfection will be used to screen actionable targets of the whole human kinome. We are able to expand this cell based functional genetic screen to include more siRNA constructs, allow combinatorial siRNA analyses, and obtain multi-parametric phenotypic readouts and comparative analysis of multiple cell types simultaneously. We will also perform screening of small molecule inhibitor libraries that comprise of compounds that are well characterized and undergoing early clinical development. With screening of small molecule inhibitor and siRNA libraries made up by "actionable" targets, we are looking for candidates whose inhibition produces distinct phenotypes that depend on the GRB7 expression status. Screen positives will be validated with independent functional assays and in additional cell lines and animal models. Since screen positives are molecules with actionable features or compounds already undergoing early phase clinical development, they can be readily moved forwards in drug development expeditiously. Thorough investigation of these GRB7 dependent signaling molecules and therapeutic targets will produce new therapies and reveal why GRB7 expression predicts adverse prognosis. High throughout screening of actionable siRNA and small molecule inhibitor libraries represents a novel paradigm to elucidate the function of an adaptor molecule and, more importantly, to effectively produce clinically useful, non-toxic therapeutics specifically directed against an oncogenic signaling molecule that is otherwise "difficult to target".

描述（由申请人提供）： GRB7在17q11-12扩增的乳腺癌中放大并表达了过表达。 GRB7可以与多个信号分子相互作用，并与乳腺癌生物学有关。 GRB7可以促进HER-2介导的信号传导和肿瘤形成。包括我们的研究表明，GRB7蛋白过表达而不是HER-2的研究是乳腺癌的不良预后因素。此外，GRB7蛋白是在三个阴性乳腺癌中研究的唯一不良预后标记。 我们假设GRB7定向信号在具有17q11-12染色体扩增的乳腺癌中在功能上很重要。因此，针对GRB7信号传导代表了HER-2阳性和三重负乳腺癌的重要且新颖的治疗策略，否则在临床上是侵略性的。 目的是：（1）通过确定抑制GRB7信号传导功能是否导致抑制乳腺癌细胞的生长来研究乳腺癌细胞对GRB7信号传导的依赖性； （2）开发乳腺癌细胞系的等生成对 使用SHRNA介导的GRB7敲低，并执行“诱导的本质性”筛选，并用小分子抑制剂文库进行筛选； （3）用siRNA库进行筛查，该库涵盖了整个“可起作用”的Kinome，并具有细胞点微阵列和一种新型的反向转染技术，以识别和验证其敲除显示GRB7依赖性依赖于增殖，凋亡和/或迁移/入侵的目标； （4）研究候选细胞周期激酶在GRB7信号传导中的功能参与及其作为治疗靶标的作用。 我们的方法如下：通过在具有17q11-12染色体染色体的多个乳腺癌细胞系中的瞬时siRNA转染来实现GRB7敲低。 GRB7蛋白耗竭可降低细胞系中细胞的增殖，而与目前可用的Her-2靶向疗法的敏感性无关。将评估稳定的GRB7敲低通过在乳腺癌细胞系中的ShRNA表达来实现的，并且将评估其对培养物和原位小鼠异种移植模型中细胞生长的影响。稳定的GRB7敲低会产生“敏化”的细胞上下文，以筛选其信号函数取决于GRB7表达的治疗靶标和分子。我们将筛选信号分子，其抑制作用被grb7过表达敏感 - 即诱导的本质。此外，抑制作用根据其GRB7状态显示差异敏感性的信号分子将揭示乳腺癌中GRB7信号的功能复杂性。新型的细胞点微阵列与siRNA反向转染相结合，将用于筛选整个人类动物组的可操作靶标。我们能够扩展基于细胞的功能遗传筛选，以包括更多的siRNA构建体，允许组合siRNA分析，并同时获得多种细胞类型的多参数表型读数和比较分析。我们还将对小分子抑制剂文库进行筛查，其中包括具有良好特征并经历早期临床发育的化合物。通过筛选小分子抑制剂和siRNA文库由“可行”靶标组成，我们正在寻找候选者，其抑制作用会产生取决于GRB7表达状态的不同表型。屏幕阳性将通过独立的功能测定以及其他细胞系和动物模型来验证。由于屏幕呈阳性是具有早期临床开发的可起作特征或化合物的分子，因此很容易在药物开发中迅速转移它们。对这些GRB7依赖的信号分子和治疗靶标的彻底研究将产生新的疗法，并揭示为什么GRB7表达能够预测不良预后。在整个筛选可起作用的siRNA和小分子抑制剂文库中，都代表了一种新型范式，以阐明适配器分子的功能，更重要的是，可以有效地产生针对针对致癌信号分子的临床上有用的，无毒的疗法，否则难以靶向靶向”。