Abstract B78: RAD51 sensitizes pancreatic cancer cells to AKT inhibition
Authors: Srinivasan Surpiya , Shi Chanjuan , Roberts Casey , Dai Xizi , Messaggio Fanuel , Kovacs Krisztina , VanSaun Michael , Merchant Nipun , Nagathihalli Nagaraj 
Published: December 2016

Introduction: Cell survival after DNA damage relies on DNA repair processes to protect the integrity of the genome. The repair process involves DNA homologous recombination system that requires numerous factors including the recombinase RAD51 and BRCA2, which co-localize to replication centers within the damaged cell nucleus. The defective DNA repair mechanisms in cancer cells can be exploited for therapy, when abrogated DNA double-strand breaks (DSB) repair causes genomic instability and increase in cancer cell sensitivity to consequential cellular apoptosis. Targeted therapies are challenged by emergence of tumor cell resistance associated with downstream components of KRAS signaling pathways in pancreatic ductal adenocarcinoma (PDAC). It is, therefore, essential to develop novel therapeutic strategies to overcome chemoresistance. The PI3K/AKT pathway is activated in human PDAC and mouse models of KRAS-driven PDAC. We sought to determine the effects of RAD51 on therapeutic sensitivity and resistance to AKT inhibition.

Experimental procedure: RAD51 expression was determined in human pancreatic tissues from a tissue microarray (TMA) and analyzed for overall survival (OS). Cell lysates from mouse cell lines derived from the LSL-KrasG12D/+;Pdx1Cre/+ (PanIN) and LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ (PDA and LMP) genetic mouse models of PDAC were immunoblotted for RAD51 expression. IC50 values of B02 (a RAD51 inhibitor) treatment in human PDAC cell lines were determined. Orthotopic tumors were generated with direct injections of luciferase-tagged PANC1 cells and were treated with B02. Bioluminescence imaging (BLI) was utilized to monitor orthotopic tumor growth and treatment response. Colony formation, spheroid generation size and metabolic activity, cell cycle analysis and apoptosis assays were performed in human PDAC cells treated with inhibitors for RAD51 (B02) and/or AKT (MK2206). PDA (express low RAD51) and LMP (express high RAD51) cell lines from mice were treated with B02 and/or MK2206 and analyzed for their colony forming ability. The in vitro sensitivities of BRCA2 deficient Capan1 and BRCA2 proficient MiaPaCa2 cell lines to MK2206 were compared by analyzing cell proliferation and colony formation. RAD51 knockdown cells were treated with MK2206 and analyzed for cell proliferation and apoptosis using flow cytometry.

Results: RAD51 expression confirmed a stepwise increase from normal pancreas to chronic pancreatitis through advancing grade and stage of PDAC. Patients with PDAC tumors expressing high levels of RAD51 had significantly higher tumor grade, stage, and lower OS when compared with patients with tumors that had low RAD51 expression (median survival of 15 months vs 37 months, respectively; P=0.025). BRCA2 deficient Capan1 PDAC cells were sensitive to MK2206 when compared to BRCA2 proficient MiaPaCa2 cells. Bioluminescent imaging (BLI) guided tumor growth analysis confirmed that RAD51 inhibition with B02 treatment decreased tumor growth in PDAC cells. B02 and MK2206 treated PDA and LMP cells showed synergistic downregulation of colony formation in comparison to either B02 or MK2206 drugs treated cells. RAD51 knockdown cells treated with MK2206 showed enhanced cell apoptosis and attenuated cell proliferation.

Conclusions: Our findings indicate that RAD51 inhibition increases sensitivity to AKT inhibition. RAD51 expression levels in PDAC tumors may be useful in identification of PDAC patients who will benefit from this therapy.

Citation Format: Surpiya Srinivasan, Chanjuan Shi, Casey Roberts, Xizi Dai, Fanuel Messaggio, Krisztina Kovacs, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli.{Authors}. RAD51 sensitizes pancreatic cancer cells to AKT inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B78.