Structure-Functions Studies of Twist1-induced Radioresistance in Lung Cancer

描述（由申请人提供）：不可切除的局部晚期非小细胞肺癌同时接受含铂化疗和胸部放疗，导致>35-80%的放射抵抗性肿瘤原发性肿瘤复发。因此，旨在了解辐射抗性机制和克服这一问题的策略的研究对于更好的肺癌治疗结果至关重要。我们发现TWIST 1在人肺癌组织中普遍过表达，并且Twist 1在体内可赋予肺上皮细胞、肺癌细胞和原位小鼠肺肿瘤放射抗性。Twist 1是一种基本的螺旋-环-螺旋转录因子。Twist 1的结构域和Twist 1的转录靶点是辐射抗性所需的未知。为了最大限度地发挥抑制Twist 1对肺癌的治疗潜力，我们必须精确定义Twist 1的蛋白结构域和/或Twist 1的下游转录靶点，以实现抗辐射。在目前的提案中，我们有三个具体的目标来测试两个假设，这两个假设将为Twist 1和肺癌辐射抗性的结构-功能关系提供更多的机制见解。这个提议的一个中心假设是，Twist 1的特定蛋白质结构域是辐射抗性所必需的。我们已经开发了一种新的肺上皮特异性Twist 1小鼠模型和Twist 1的各种突变版本，以促进这些研究。我们的第二个可验证的假设是，有一个定义的“核心”转录靶点的Twist 1所需的辐射抗性。在我们的建议中概述的研究将提供有价值的知识，在肺癌和Twist 1诱导的放射抗性的病理生理作用。最后，验证一种新的临床前诱导型肺癌辐射抗性模型将创建一个模型，以测试这种致命疾病的新治疗方法与辐射相结合。具体目标#1：表征体外辐射抗性所需的Twist 1结构域。我们将使用小鼠胚胎成纤维细胞（MEFs）和表达Twist 1和Twist 1突变体的同基因人肺癌细胞来研究Twist 1的哪些结构域是放射抗性表型所必需的。我们将在体外确定Twist 1诱导的辐射抗性的细胞自主机制。具体目标#2：表征KrasG 12 D诱导的自体肺肿瘤的放射抗性所需的Twist 1结构域。我们将用一系列非侵入性成像来验证这一假设，并用标准的分子组织学方法来证实我们的发现，以跟踪我们新型Twist 1转基因肺肿瘤模型对辐射的反应。我们还将研究Twist 1依赖的辐射反应调制在体内的主导机制。具体目标#3：使用基因表达和ChIP芯片分析确定与Twist 1诱导的辐射抗性相关的Twist 1转录靶点。我们将使用整合的全基因组生物信息学方法对MEFs和人肺癌细胞分离Twist 1诱导的辐射抗性所需的“核心”基因靶点。

DESCRIPTION (provided by applicant): Unresectable locally advanced non-small cell lung cancer treated with concurrent platinum-based chemotherapy and thoracic radiation results in primary tumor recurrence in >35-80% of cases from radioresistant tumors. Therefore, research aimed at understanding mechanisms of radioresistance and strategies to overcome this problem are paramount to better lung cancer treatment outcomes. We found TWIST1 was commonly overexpressed in human lung cancers and Twist1 could confer radiation resistance to lung epithelial cells, lung cancer cells and autochthonous mouse lung tumors in vivo. Twist1 is a basic helix- loop-helix transcriptional factor. The domains of Twist1 and transcriptional targets of Twist1 that are required for radioresistance are unknown. In order to maximize the therapeutic potential of inhibiting Twist1 for lung cancer, we must precisely define the protein domains of Twist1 and/or the downstream transcriptional targets of Twist1 required for radioresistance. In the present proposal we have three specific aims to test two hypotheses that will provide more mechanistic insight into the structure-function relationships of Twist1 and lung cancer radioresistance. One central hypothesis of this proposal is that specific protein domains of Twist1 are required for radioresistance. We have developed a novel lung epithelium specific Twist1 mouse model and various mutant versions of Twist1 to facilitate these studies. Our second testable hypothesis is there is a defined set of "core" transcriptional targets of Twist1 required for radioresistance. The studies outlined in our proposal will provide valuable knowledge to the pathophysiologic role of Twist1 in lung cancer and Twist1- induced radioresistance. Finally, validating a novel preclinical inducible lung cancer model of radioresistance will create a model to test new treatments for this fatal disease in combination with radiation. Specific Aim #1: Characterize the domains of Twist1 required for radioresistance in vitro. We will use mouse embryo fibroblasts (MEFs) and isogenic human lung cancer cells expressing Twist1 and Twist1 mutants to examine which domains of Twist1 are necessary for a radioresistant phenotype. We will determine the cell autonomous mechanisms of Twist1-induced radioresistance in vitro. Specific Aim #2: Characterize the domains of Twist1 required for radioresistance of KrasG12D-induced autochthonous lung tumors. We will test this hypothesis with serial non-invasive imaging and confirm our findings with standard molecular-histologic methods to follow the response to radiation in our novel Twist1 transgenic lung tumor model. We will also examine the dominant mechanisms for Twist1-dependent radiation response modulation in vivo. Specific Aim #3: Determine Twist1 transcriptional targets correlated with Twist1-induced radioresistance using gene expression and ChIP-chip profiling. We will use integrative genome-wide bioinformatic approaches on MEFs and human lung cancer cells to isolate the "core" gene targets required for Twist1-induced radioresistance.