Targeting bidirectional signaling in lung stroma and cancer cells

项目摘要 肺癌（LC）对酪氨酸激酶抑制剂（TKI）的耐药性通常与癌症相关的 成纤维细胞（CAF）是肿瘤微环境（TME）的主要成分。CAF可通过以下途径引起耐药性： 生长因子分泌以及与癌细胞直接接触。此外，肿瘤细胞培养TME 成纤维细胞适应CAF表型，导致癌细胞之间复杂的双向信号传导， 咖啡馆。重要的是，TKI并不简单地关闭致癌信号，而是导致细胞的适应性重新布线。 信令网此外，大多数TKI都有多个靶点，TKI脱靶会产生重要影响 对疗效和反应，无论是通过限制或提高它。这不仅限于癌细胞，但TKI可以 同时参与癌症以及基质细胞中的蛋白质和信号通路。所有这些 情景创建了癌症的高度动态的、双向的和药物特异性的适应性信号应答， 细胞/CAF系统，其导致药物敏感性的调节和耐药性"持久"的发展。 细胞群我们假设a）单个TKI引发药物和细胞特异性适应性信号传导 LC细胞/CAF系统中的响应和抗性机制，和B）破坏双向 LC细胞和CAF之间的信号传导可以增强药物敏感性并消除CAF支持的持续性 细胞使用无偏的，细胞类型特异性的蛋白质组学方法，我们将在下面测试这些假设 具体目的：1）研究癌细胞激活成纤维细胞RTK通路的机制和作用。 在CAFs内遗传激活RTK驱动的信号通路将允许表征双向的 使用基于"使用氨基酸前体的细胞类型特异性标记"（CTAP）的CAF和LC细胞的信号传导 磷酸化蛋白质组学及其如何反过来影响LC细胞增殖、侵袭和药物敏感性。相关 信号通路将通过患者来源的组织微阵列（TMA）上的邻位连接测定进行评价 以及原位异型体内模型。2)制定策略，在功能上参与TKI诱导的 CAF和LC小区中的自适应信令。使用基于CTAP的化学和磷酸化蛋白质组学，我们将 确定LC和CAF特异性适应性信号传导反应和临床TKI的靶向特征。功能 通过RNAi和拯救实验的验证将鉴定CAF靶向药物。与TKI的协同作用将是 在共培养物和患者来源的异种移植物（PDX）模型中评价。这种方法是创新的，因为它 代表了一种通过开发同时参与信号通路的策略来靶向癌症的新方法 在癌细胞以及TME中，这是通过应用最先进的蛋白质组学实现的。拟议 这项研究意义重大，因为它将改变我们对信号的动态和复杂性的理解 诱发LC细胞CAF的相互作用和这些电路在耐药性和敏化的作用。此外，本发明还 这些研究将揭示靶向组合临床开发的概念上的新机会 为有显著未满足医疗需求的患者人群提供治疗。

PROJECT ABSTRACT Resistance to tyrosine kinase inhibitors (TKI) in lung cancer (LC) is often connected to cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME). CAFs can cause drug resistance via secretion of growth factors as well as direct contact with cancer cells. Furthermore, tumor cells educate TME fibroblasts to adapt a CAF phenotype, leading to complex and bi-directional signaling between cancer cells and CAFs. Importantly, TKIs do not simply shut down oncogenic signaling, but lead to an adaptive rewiring of the signaling network. In addition, most TKIs have multiple targets, and TKI off-targets can have important effects on efficacy and response, either by restricting or boosting it. This is not limited to cancer cells, but TKIs can simultaneously engage proteins and signaling pathways in cancer as well as stromal cells. Together, these scenarios create a highly dynamic, bi-directional and drug-specific adaptive signaling response of the cancer cell/CAF system, which results in modulation of drug sensitivity and development of drug-tolerant “persister” cell populations. We hypothesize a) that individual TKIs elicit drug- and cell-specific adaptive signaling responses and resistance mechanisms in the LC cell/CAF system, and b) that disrupting bi-directional signaling between LC cells and CAFs can enhance drug sensitivity and eliminate CAF-supported persister cells. Using unbiased, cell type-specific proteomics approaches, we will test these hypotheses in the following specific aims: 1) To characterize mechanisms and roles of fibroblast RTK pathway activation by cancer cells. Genetically activating RTK-driven signaling pathways inside CAFs will allow the characterization of bi-directional signaling of CAFs and LC cells using “cell type-specific labeling using amino acid precursors” (CTAP)-based phosphoproteomics and how it in turn affects LC cell proliferation, invasion and drug sensitivity. Relevant signaling pathways will be evaluated by proximity-ligation assays on patient-derived tissue microarrays (TMAs) and in orthotopic, heterotypic in vivo models. 2) To develop strategies to functionally engage TKI-induced adaptive signaling in CAFs and LC cells. Using CTAP-based chemical and phosphoproteomics, we will determine LC- and CAF-specific adaptive signaling responses and target profiles of clinical TKIs. Functional validation by RNAi and rescue experiments will identify CAF targeting drugs. Synergy with TKI will be evaluated in co-culture and in patient-derived xenograft (PDX) models. The approach is innovative, because it represents a novel way of targeting cancer by developing strategies to simultaneously engage signaling pathways in cancer cells as well as the TME, which are enabled by application of state-of-the-art proteomics. The proposed research is significant as it will transform our understanding of the dynamics and complexity of signaling evoked by LC cell-CAF interactions and the roles of these circuits in drug resistance and sensitization. Further, these studies will reveal conceptually novel opportunities for clinical development of targeted combination therapies for a patient population with a significant unmet medical need.