Abstract 305: TET blocks cell cycle progression in pancreatic cancer cells at G1-S boundary by decreasing cyclin D1 and increasing CDK inhibitors (CKIs), p21 (WAF1, Cip1) and p27 (Kip1)

Abstract

Abstract Introduction: Pancreatic cancer (PaCa) is the fourth leading cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PaCa are largely ineffective thus there is an urgent need for development of therapeutic agents for the treatment of PaCa. Owing to its mutational landscape, which involves mutated KRAS and p53 genes these cancer cells have a deregulated G1 checkpoint of cell cycle control and therefore confer high rate of proliferation and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression at G0/G1 boundary in vitro and in vivo. In the present study we evaluated potential mechanism of TET induced cell cycle arrest in two pancreatic cancer cells with either wild type KRAS or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G0/G1 to S transition. Methods: Pancreatic Cancer cell lines: BxPC3 (Pancreatic Ductal Adeno-Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in the study. Cells were treated with various concentrations of TET for different time points. Cell proliferation was evaluated using the MTT survival assay. Cell cycle was analyzed following PI staining via Flow cytometry. Changes in protein expression were analyzed by Western Blot analysis in cells. Changes in mRNA were evaluated by real time PCR. Results: Treatment of pancreatic cancer cells with TET resulted in decreased levels cyclin D1 and phospho-Rb, two critical proteins required for G0/G1 to S transition. Pretreatment of the cells with proteasomal inhibitor MG132 was able to increase the levels of cyclin D1 in control cells but was unable to prevent its loss upon TET treatment. Our results also show that levels of CKIs, p21WAF1/Cip1 and p27Kip1 were increased at the protein level. Gene expression analysis revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27 mRNA levels. The cycloheximide chase revealed that TET stabilizes p27Kip1 and to some extent p21WAF1/Cip1 as well, suggesting that TET increased p27Kip1 and also p21WAF1/Cip1 levels in part by increasing protein stability. These data suggest a complex mechanism by which TET regulates expression of critical cell cycle regulatory molecules at both transcriptional, translational and post-translational level. Such a mechanism would provide for cell cycle arrest in pancreatic cancer cells following TET treatment. Conclusion: These results show that TET halts cell cycle at G0/G1 boundary in PaCa cells by modulating the cell cycle regulatory proteins and stabilizing the CKIs by altering their protein stability, suggesting that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in pancreatic cancer cells. Citation Format: Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. TET blocks cell cycle progression in pancreatic cancer cells at G1-S boundary by decreasing cyclin D1 and increasing CDK inhibitors (CKIs), p21 (WAF1, Cip1) and p27 (Kip1) [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 305. doi:10.1158/1538-7445.AM2017-305