Hypoxia, DNA repair, and gene silencing

描述（由申请人提供）：缺氧、DNA修复和基因沉默。 缺氧是实体肿瘤的一个关键特征，它赋予放射抵抗性，刺激血管生成，促进转移，并与不良预后有关。在这项资助的支持下，我们已经证明缺氧也是通过下调关键DNA修复基因而导致遗传不稳定的驱动因素。在过去的资助期间，我们发现缺氧也可以通过特定的表观遗传因子诱导BRCA 1和MLH 1启动子的持久沉默。在最近的初步研究中，我们进一步确定缺氧可导致肺癌细胞中促凋亡BIM基因的沉默和对EGFR抑制剂吉非替尼的抗性。这项更新申请的广泛，长期目标是阐明缺氧应激对致癌作用和癌症生物学的影响，重点是DNA修复和基因沉默。 在目标1中，我们将剖析低氧胁迫驱动表观遗传变化导致基因沉默的分子机制，重点是MLH 1启动子。我们将确定介导沉默的启动子元件和调控因子，并将使用基于选择的shRNA筛选来确定逆转这一过程的关键靶点。我们还将分析缺氧肿瘤微环境对体内肿瘤生长过程中基因沉默的影响。接下来，由于重金属是已知的人类致癌物，可以诱导缺氧相关的途径，在目标2中，我们将询问暴露于重金属是否也可以驱动基因沉默和/或下调DNA修复。在目标3中，我们将建立在新的初步结果表明，肺癌细胞在缺氧中的生长可以促进对表皮生长因子受体（EGFR）抑制剂吉非替尼的耐药性，同时沉默促凋亡因子BIM。我们将测试有关潜在机制的特定假设，并在目标1的指导下，我们将确定预防或逆转这种耐药性的策略。 拟议的工作将阐明基因沉默和DNA修复调节的关键途径，以应对缺氧（可能是致癌的重金属），这可能是致癌，遗传不稳定， 肿瘤进展以及对放射和其它癌症疗法的抗性。确定预防或逆转这些途径的策略可能为癌症预防和治疗的新方法提供基础。

DESCRIPTION (provided by applicant): Hypoxia, DNA repair, and gene silencing. Hypoxia is a key feature of solid tumors that confers radiation resistance, stimulates angiogenesis, promotes metastasis, and is linked to poor prognosis. With the support of this grant, we have shown that hypoxia is also a driver of genetic instability via down-regulation of critical DNA repair genes. In the past funding period, we have discovered that hypoxia can also induce durable silencing of the BRCA1 and MLH1 promoters via specific epigenetic factors. In recent preliminary studies, we have further determined that hypoxia can lead to silencing of the pro-apoptotic BIM gene and resistance to the EGFR inhibitor, gefitinib, in lung cancer cells. The broad, long-term goal of this renewal application is to elucidate the impact of hypoxic stress on carcinogenesis and cancer biology, with a focus on DNA repair and gene silencing. In Aim 1, we will dissect the molecular mechanisms by which hypoxic stress drives epigenetic change to cause gene silencing, with a focus on the MLH1 promoter. We will determine promoter elements and regulatory factors that mediate silencing, and we will use a facile selection-based shRNA screen to identify key targets for reversal of this process. We will also assay for the impact of the hypoxic tumor microenvironment on gene silencing during tumor growth in vivo. Next, since heavy metals are known human carcinogens that can induce hypoxia-related pathways, in Aim 2 we will ask whether exposure to heavy metals can also drive gene silencing and/or down-regulate DNA repair. In Aim 3, we will build on novel preliminary results suggesting that growth of lung cancer cells in hypoxia can promote resistance to the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, in conjunction with silencing of the pro-apoptotic factor, BIM. We wil test specific hypotheses regarding the underlying mechanisms, and, guided by Aim 1, we will identify strategies to prevent or reverse this resistance. The proposed work will elucidate key pathways of gene silencing and DNA repair regulation in response to hypoxia (and possibly to carcinogenic heavy metals) that may underlie critical steps in carcinogenesis, genetic instability, tumor progression, and resistance to radiation and other cancer therapies. Identification of strategies to prevent or reverse these pathways may provide the basis for new approaches to cancer prevention and therapy.