Investigating the Role of Autophagy in Pancreatic Cancer Radiation Resistance

描述(申请人提供)：研究自噬在胰腺癌放射治疗抵抗中的作用。胰腺导管腺癌(PDAC)是美国第四大癌症死亡原因，5年生存率低达3-5%。这种侵袭性癌症的一个特点是对包括化疗和放射在内的现有治疗方法具有抵抗力。我们探索了这种耐药性的一个组成部分是否可归因于细胞生存/死亡途径的改变。在这些研究中，我们有了新的发现，几乎所有的人PDAC原发肿瘤和细胞系都有基础自噬升高。我们发现，通过药物或遗传手段抑制PDAC的自噬会导致明显的ROS升高和DNA双链断裂，导致明显的体外生长停滞和体内肿瘤的消退。因此，自噬是PDAC生长所必需的，因为它可以防止高水平的DNA损伤积累。重要的是，自噬抑制引起的DNA损伤与辐射是协同作用的。因此，我们假设在临床环境中，自噬在PDAC对化疗和放疗的抵抗中起着关键作用。由于已有有效的自噬抑制剂，如氯喹(CQ)和羟基氯喹(HCQ)，并得到FDA的批准，这项工作立即应用于PDAC和其他自噬升高的肿瘤类型的治疗。由于放射治疗抵抗继发的局部失败是PDAC患者发病率和死亡率的重要原因，这些研究有可能产生变革性的影响。基于此，我们建议1)定义自噬抑制反应的分子预测因子，作为PDAC细胞和异种移植的放射增敏剂；2)利用基因工程小鼠模型，确定自噬在原发PDAC的组织病理机制中的作用以及天然肿瘤对放射和化疗的敏感性；以及3)评估自噬抑制作为放射增敏剂在接受放射治疗和自噬抑制剂羟基氯喹治疗的人PDAC患者的肿瘤中的可能性。Aim 1将使用&gt；70小鼠和人类PDAC株的集合来确定不同的基因类型(KRAS、P53、Ink4a、Smad4、Lkb1和PTEN突变的组合)、全球基因拷贝数和mRNA表达谱是否可以预测作为放射增敏剂的HCQ的反应。目的2将使用经过验证的PDAC进展的小鼠模型、自噬报告以及药理学和遗传学方法来灭活体内进化的肿瘤中的自噬，阐明自噬在肿瘤的发生和辐射抵抗中的作用。AIM 3将利用参加临床试验的患者的肿瘤，评估自噬抑制剂HCQ作为PDAC放射增敏剂的有效性，以验证体内对肿瘤的自噬抑制，评估组织学反应，并在患者样本中验证AIM 1的分子预测因子。

DESCRIPTION (provided by applicant): Investigation of the role of autophagy in the resistance of pancreatic cancer to radiation therapy. Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the U.S. with a dismal 5-year survival rate of 3-5%. A hallmark of this aggressive cancer is its resistance to existing therapies including chemotherapy and radiation. We explored whether a component of this resistance was attributable to altered cell survival/death pathways. During these studies we made the novel finding that virtually all human PDAC primary tumors and cell lines have elevated basal autophagy. We found that inhibition of autophagy in PDAC by pharmacological or genetic means causes pronounced elevation of reactive oxygen species (ROS) and increased DNA double strand breaks, leading to marked growth arrest in vitro as well as tumor regression in vivo. Thus, autophagy is required for PDAC growth by preventing accumulation of high levels of DNA damage. Importantly, the DNA damage caused by autophagy inhibition is synergistic with radiation. We therefore hypothesize that autophagy has a critical role in the resistance of PDAC to chemotherapy and radiation in the clinical setting. Since potent autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), are available and FDA approved, this work has immediate clinical application to the treatment of PDAC and other tumor types with elevated autophagy. As resistance to radiotherapy with subsequent local failure is a significant cause of morbidity and mortality in PDAC patients, these studies have the potential for a transformative impact. Based on this, we propose to 1) define molecular predictors for response to autophagy inhibition as a radiosensitizer in PDAC cell lines and xenografts, 2) establish the role of autophagy in the histopathogenesis primary PDAC and in sensitivity of native tumors to radiation and chemotherapy using genetically engineered mouse models, and 3) to assess the potential of autophagy inhibition as a radiosensitizer in tumors from human PDAC patients treated with radiation and the autophagy inhibitor hydroxychloroquine. Aim 1 will use a collection of > 70 mouse and human PDAC lines to determine if various genotypes (combinations of KRAS, p53, Ink4a, Smad4, Lkb1, and PTEN mutations), global gene copy number and mRNA expression profiles can predict response to HCQ as a radiosensitizer. Aim 2 will employ validated mouse models of PDAC progression, autophagy reporters, and pharmacologic and genetic approaches to inactivate autophagy in evolving tumors in vivo elucidate the contribution of autophagy to the development and radioresistance of tumors arising in their native setting. Aim 3 will utilize tumors from patients enrolled on a clinical trial assessing the efficacy of the autophagy inhibitor HCQ as a radiosensitizer in PDAC to validate in vivo autophagy inhibition in tumors, assess histologic response, and validate molecular predictors from Aim 1 in patient samples.