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)的耐药常与癌症相关 成纤维细胞(CAF)是肿瘤微环境的主要组成部分。CAF可通过以下途径引起耐药性 分泌生长因子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，导致癌细胞和 CAF。重要的是，TKI不是简单地关闭致癌信号，而是导致自适应地重新连接 信令网络。此外，大多数TKI有多个目标，而TKI偏离目标可能会产生重要影响 在疗效和反应上，无论是通过限制还是加强它。这不仅限于癌细胞，但TKI可以 同时参与癌症和间质细胞中的蛋白质和信号通路。加在一起，这些 情景创造了癌症的高度动态、双向和药物特异性的适应性信号反应 细胞/CAF系统，导致药物敏感性的调节和耐药“持久性”的发展 细胞群。我们假设a)单个TKI诱导药物和细胞特异性的适应性信号 在LC细胞/CAF系统中的响应和抵抗机制，以及b)破坏双向 LC细胞和CAF之间的信号转导可以增强药物敏感性并消除CAF支持的持久性 细胞。使用无偏见的、细胞类型特定的蛋白质组学方法，我们将在以下方面检验这些假设 具体目的：1)研究癌细胞激活成纤维细胞RTK途径的机制和作用。 在CAF中通过基因激活RTK驱动的信号通路将允许双向 基于CTAP的细胞类型特异性氨基酸前体标记在CAF和LC细胞中的信号传递 磷酸蛋白质组学及其如何反过来影响LC细胞的增殖、侵袭和药物敏感性。相关 信号通路将通过患者来源的组织芯片(TMA)的邻近连接分析来评估。 在原位、异型的活体模型中。2)制定战略，从功能上参与由TKI引发的活动 CAF和LC细胞中的适应性信号。使用基于CTAP的化学和磷酸蛋白质组学，我们将 确定LC和CAF特定的适应性信号反应和临床TKI的靶向分布。功能性 通过RNAi和救援实验的验证将确定CAF靶向药物。与将军澳的协同效应将是 在共培养和患者来源的异种移植(PDX)模型中进行评估。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 在癌细胞和TME中，这是通过应用最先进的蛋白质组学实现的。建议数 研究意义重大，因为它将改变我们对信号动力学和复杂性的理解 由LC细胞-CAF相互作用以及这些回路在耐药和增敏中的作用。此外， 这些研究将在概念上揭示靶向联合的临床开发的新机会 针对有重大未得到满足的医疗需求的患者群体的治疗。

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.