Abstract 3317: Autophagy protects ovarian cancer cells from olaparib-induced toxicity
Authors: Santiago-O'Farrill Janice M. , Essien Safia , Figueroa Mary , Pang Lan , Amaravadi Ravi , Lu Zhen , Bast Robert C. 
Published: July 2017

Abstract
Ovarian cancer (OC) is the most lethal gynecologic malignancy. Persistence of dormant, drug resistant cancer cells is one of the major factors that contribute to poor outcomes. The Poly (ADP) ribose polymerase (PARP) inhibitor, olaparib, has shown promising anti-cancer activity and has been approved by the FDA for the treatment of ovarian cancer with BRCA1/2 mutations. Olaparib targets DNA repair mechanisms within cancer cells and induces apoptosis. Both preclinical and clinical data indicate that efficacy of olaparib can be decreased by acquired resistance. One mechanism of resistance involves autophagy. Autophagy can degrade organelles and long-lived proteins to provide energy for cancer cells under nutrient poor conditions or in the presence of stress. The energy provided from autophagy is used to fuel the DNA repair process and metabolic needs in cancer cells. Autophagy can protect cancer cells from chemotherapy or can enhance the response to certain drugs. In this study, we have asked whether autophagy protects OC cells against the cytotoxic effects of olaparib or augments damage.
We first determined PARP activity using a modified PARP activity assay in different OC cell lines. Olaparib-induced autophagy was determined by punctate localization of GFP-LC3, conversion of LC3I to LC3II on protein blots and electron microscopy. To test whether autophagy contributes to the resistance of OC cells to olaparib, we determined the impact of autophagy inhibition on the sensitivity of OC cells to olaparib by either using the pharmacologic inhibitors chloroquine (CQ), LYS05 or short interfering RNA (siRNA) against ATG7. Upon investigation of a potential mechanism of olaparib-induced autophagy we evaluated the production of reactive oxygen species (ROS) and the expression of JNK, Ataxia-telangiectasia mutated (ATM), mTOR and p70S6K.
Inhibition of PARP by olaparib treatment, decreased PARP activity in a dose- dependent manner, leading to DNA damage judged by phosphorylation of γ-H2AX. Olaparib treatment decreased cell growth, caused cell cycle arrest and significantly enhanced apoptosis in 5 of 5 OC cell lines. Olaparib induced autophagy in 8 of 8 OC cell lines. Both pharmacologic and genetic inhibition of autophagy increased OC cells sensitivity to olaparib and induced apoptosis in 5 of 6 OC cell lines. Furthermore, flow cytometry and western blot analysis demonstrated that olaparib treatment increased ROS production and increased the phosphorylation of JNK and ATM while decreasing the phosphorylation of mTOR and p70S6K.
Taken together, our data described autophagy as a potential mechanism of olaparib resistance in OC that may depend upon the activation of ATM and JNK by ROS downregulating pmTOR and p70S6K. Combination of olaparib with CQ, LYS05 or other autophagy inhibitor may provide a novel strategy to increase the efficacy of olaparib in OC. Current studies are underway to test our hypothesis in vivo.
Citation Format: Janice M. Santiago-O'Farrill, Safia Essien, Mary Figueroa, Lan Pang, Ravi Amaravadi, Zhen Lu, Robert C. Bast. Autophagy protects ovarian cancer cells from olaparib-induced toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2017-3317