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

描述(由申请人提供)：不能切除的局部晚期非小细胞肺癌，同时接受以铂为基础的化疗和胸部放射治疗，会导致35-80%的放射抵抗肿瘤患者的原发肿瘤复发。因此，旨在了解辐射抵抗的机制和克服这一问题的策略的研究对于改善肺癌的治疗结果至关重要。我们发现Twist1在人类肺癌中普遍过表达，并且Twist1在体内对肺上皮细胞、肺癌细胞和小鼠原位肺癌具有辐射抗性。Twist1是一种基本的螺旋-环-螺旋转录因子。Twist1的结构域和Twist1的转录靶点是辐射抗性所需的，目前尚不清楚。为了最大限度地发挥抑制Twist1对肺癌的治疗作用，我们必须准确地确定Twist1的蛋白结构域和/或Twist1的下游转录靶点。在目前的提案中，我们有三个具体的目标来检验两个假说，这两个假说将为Twist1和肺癌放射抗性的结构-功能关系提供更多的机械性见解。这一建议的一个中心假设是Twist1的特定蛋白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Twist1小鼠模型和Twist1的各种突变版本来促进这些研究。我们的第二个可测试假设是，Twist1有一组确定的“核心”转录靶点，这是辐射抗性所必需的。我们建议中概述的研究将为Twist1在肺癌和Twist1诱导的辐射抵抗中的病理生理学作用提供有价值的知识。最后，验证一种新的临床前诱导性肺癌放射抵抗模型将创建一个模型，以测试这种致命疾病与辐射相结合的新治疗方法。具体目标#1：确定Twist1在体外抗辐射所需的结构域。我们将使用小鼠胚胎成纤维细胞(MEF)和表达Twist1和Twist1突变体的同基因人肺癌细胞来检查Twist1的哪些区域是辐射抗性表型所必需的。我们将在体外确定Twist1诱导的辐射抗性的细胞自主机制。特定目标#2：鉴定KrasG12D诱导的自体肺肿瘤放射抗性所需的Twist1结构域。我们将用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，以跟踪我们新的Twist1转基因肺肿瘤模型对辐射的反应。我们还将研究体内Twist1依赖的辐射反应调节的主要机制。具体目标#3：利用基因表达和芯片图谱确定与Twist1诱导的辐射抗性相关的Twist1转录靶点。我们将在MEF和人类肺癌细胞上使用整合的全基因组生物信息学方法来分离Twist1诱导辐射抵抗所需的“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.