Investigating the Role of Autophagy in Pancreatic Cancer Radiation Resistance

描述（由申请人提供）：研究自噬在胰腺癌放射治疗耐药中的作用。胰腺导管腺癌（PDAC）是美国第四大癌症死亡原因，其5年生存率仅为3-5%。这种侵袭性癌症的一个特点是它对包括化疗和放疗在内的现有疗法具有耐药性。我们探讨了这种耐药性的一个组成部分是否归因于细胞生存/死亡途径的改变。在这些研究中，我们发现几乎所有的人类PDAC原发肿瘤和细胞系都有升高的基础自噬。我们发现，通过药理或遗传手段抑制PDAC的自噬会导致活性氧（ROS）的显著升高和DNA双链断裂的增加，从而导致体外明显的生长停滞和体内肿瘤消退。因此，通过防止高水平DNA损伤的积累，自噬是PDAC生长所必需的。重要的是，自噬抑制引起的DNA损伤与辐射具有协同作用。因此，我们假设自噬在PDAC对化疗和放疗的临床耐药中起关键作用。由于有效的自噬抑制剂，如氯喹（CQ）和羟氯喹（HCQ），是可用的和FDA批准的，这项工作可以立即临床应用于治疗PDAC和其他自噬升高的肿瘤类型。由于对放疗的耐药性和随后的局部失败是PDAC患者发病率和死亡率的重要原因，这些研究具有变革性影响的潜力。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细胞系和异种移植物的放射增敏剂；2)利用基因工程小鼠模型确定自噬在原发性PDAC的组织发病过程中的作用，以及在原生肿瘤对放疗和化疗的敏感性中的作用。3)评估自噬抑制在放射治疗和自噬抑制剂羟氯喹治疗的PDAC患者肿瘤中作为放射增敏剂的潜力。Aim 1将使用bbb70小鼠和人类PDAC细胞系的集合来确定各种基因型（KRAS、p53、Ink4a、Smad4、Lkb1和PTEN突变的组合）、整体基因拷贝数和mRNA表达谱是否可以预测对HCQ作为放射增敏剂的反应。目的2将采用经过验证的PDAC进展小鼠模型，自噬报告者，以及药理学和遗传学方法来灭活体内进化肿瘤中的自噬，阐明自噬对肿瘤在其原生环境中产生的发展和放射耐药的贡献。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放射增敏剂的疗效，以验证肿瘤的体内自噬抑制，评估组织学反应，并验证Aim 1在患者样本中的分子预测因子。

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. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is highly lethal and current treatments such as chemotherapy and radiation are only minimally effective due to its profound therapeutic resistance. This proposal seeks to build on data from our laboratory showing that a process called autophagy may be important in the resistance of these tumors to radiation therapy.