P-47 Inhibition of phosphoprotein phosphatase 2A (PP2A) sensitizes pancreatic cancer to PARP inhibitors by modulation of homologous recombination repair

Abstract

Pancreatic cancer remains one of the most difficult to treat cancers with a 5-years survival of less than 6%, representing one of the most lethal types of cancer, recent research has shown that PP2A inhibitors (LB-100) sensitize pancreatic cancer to chemotherapy and radiation. In addition, the finding from clinical trial study demonstrated that the effectiveness of single-agent Talazoparib for treatment of patients with and without germline BRCA1/2 mutation in ovarian, breast, small cell lung and Pancreatic cancers. The aim of this work was to Ι. investigate activity of LB-100 against pancreatic cancer cells as monotherapy and in combination with PARP inhibitions (Talazoparib). ΙΙ. Investigate the mechanism by which LB-100 and Talazoparib effect pancreatic cancer cells by studying effect of drugs on cell cycle and modulation of cell cycle regulatory proteins and assess the role of treatment combination in control DSB and HR repair through modulation of ATM phosphorylation. Human pancreatic cancer cell lines Panc-1, MIA-Pa-Ca-2 and BxPC-3 were obtained from European Collection of Authenticated Cell Culture (ECACC) and grown in either Dulbecco’s modified Eagle medium (DMEM) (MiaPaCa-2 and Panc-1) or RPMI medium (BxPC-3) with 10% FBS. Cell cultures were maintained in an atmosphere of 5% CO2/95% air at 37 C. PP2A activity was measured by PP2A Immunoprecipitation phosphatase assay Kit (Millipore), cell cytotoxicity were measured by MTT assay, cell cycle determined by flow cytometry. Western blot techniques were used to assess levels of proteins associated with regulation of cell cycle (Cdc2, p-Cdc2, and Cdc25c), apoptosis (caspase3) and DNA damage (γ-H2AX). Data are expressed as the mean ± (SEM) and analysed by an analysis of variance (ANOVA). The results show that LB-100 decreased PP2A activity in all pancreatic cancer cells in dose dependent manner. LB-100 significantly decreased cell viability of Panc-1, MIA-Pa-Ca-2 and BxPC-3 with IC50 (3.94 μM, 6.86 μM, and 10.87 μM) respectively. Interestingly adding 25 nm of Talazoparib further decreased IC50 in all cells (1.88 μM, 5.24 μM and 5.83 μM). Treatment combination attenuated pancreatic cancer cells growth through caspase activation and G2/M cell-cycle arrest. Combination therapy impaired cellular repair and induced DNA double-strand breaks by inducing Υ-H2AX. Our results suggest that treatment combination of LB100 and talazoparib in vitro inhibit cancer cells growth by modulation of the DNA damage response pathway and cell cycle checkpoint abrogation. The combination of PP2A inhibitor with PARP inhibitor has a synergistic effect in vitro. Further in vivo studies are needed to explore this combination as an effective option in the treatment of pancreatic cancer.