Investigating the Role of Autophagy in Pancreatic Cancer Radiation Resistance

描述（由申请人提供）：研究自噬在胰腺癌对放射治疗的抵抗中的作用。胰腺导管腺癌（PDAC）是美国癌症死亡的第四大原因，5年生存率为3- 5%。这种侵袭性癌症的一个标志是它对现有疗法（包括化疗和放疗）的抵抗力。我们探讨了这种耐药性的一个组成部分是否可归因于细胞存活/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原发性肿瘤和细胞系都具有升高的基础自噬。我们发现，通过药理学或遗传学手段抑制PDAC中的自噬会导致活性氧（ROS）的显著升高和DNA双链断裂的增加，从而导致体外显著的生长停滞以及体内肿瘤消退。因此，自噬是PDAC生长所必需的，通过防止高水平DNA损伤的积累。重要的是，自噬抑制引起的DNA损伤与辐射是协同的。因此，我们假设自噬在PDAC对化疗和放疗的抵抗中起着关键作用。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，这项工作有立即临床应用到PDAC和其他肿瘤类型的治疗与提高自噬。由于对放疗的抵抗以及随后的局部失败是PDAC患者发病率和死亡率的重要原因，因此这些研究有可能产生变革性影响。基于此，我们提出1）定义对PDAC细胞系和异种移植物中作为放射增敏剂的自噬抑制的响应的分子预测因子，2）使用基因工程小鼠模型建立自噬在组织发生原发性PDAC和天然肿瘤对放射和化学疗法的敏感性中的作用，和3）评估自噬抑制作为来自用放射和自噬抑制剂羟氯喹治疗的人PDAC患者的肿瘤中的放射增敏剂的潜力。目的1将使用> 70个小鼠和人PDAC系的集合来确定各种基因型（KRAS、p53、Ink 4a、Smad 4、Lkb 1和PTEN突变的组合）、总体基因拷贝数和mRNA表达谱是否可以预测对作为放射增敏剂的HCQ的响应。目的2将采用经验证的PDAC进展小鼠模型、自噬报告基因以及药理学和遗传学方法来研究体内肿瘤中的自噬，阐明自噬对肿瘤发生和放射抗性的贡献。目标3将利用来自参加临床试验的患者的肿瘤，该临床试验评估自噬抑制剂HCQ作为PDAC中的放射增敏剂的功效，以验证肿瘤中的体内自噬抑制，评估组织学反应，并验证患者样品中来自目标1的分子预测因子。 公共卫生相关性：胰腺癌是高度致命的，目前的治疗，如化疗和放疗，由于其深刻的治疗抗性，只有最低限度的有效性。该提案旨在建立在我们实验室的数据基础上，这些数据表明，一种称为自噬的过程可能在这些肿瘤对放射治疗的抵抗中很重要。

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.