Abstract 4305: TET increases p27Kip1 protein stability and p21WAF1/Cip1 promoter activity to block deregulated cell cycle progression in pancreatic cancer cells

Introduction: Pancreatic cancer (PaCa) is the fourth 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 ineffective thus there is an urgent need for development of new therapeutic agents for its treatment. Owing to its mutational landscape, which includes mutated KRAS and p53 these cells have a deregulated G1 checkpoint of cell cycle and thus confer high proliferation rate and therapeutic resistance. Recently we observed that Tetrandrine derivative (TET), a bis-benzylisoquinoline alkaloid halts cell cycle progression in PaCa cells at G1/S boundary in vitro and in vivo. In the present study we explored potential mechanism of TET induced cell cycle arrest in two PaCa cells with either wild type or mutant KRAS, and mutant p53 by evaluating critical cell cycle regulatory proteins involved in G1/S transition. Method: PaCa cell lines: BxPC3 (PDAC) and MiaPaCa2 (Pancreatic Carcinoma) were used. Cells were treated with various concentrations of TET for different time points. Cell growth was checked using the MTT assay. Cell cycle was analyzed after PI staining via Flow cytometry. Changes in protein levels were assessed by Western Blot analysis. mRNA changes were evaluated by real time PCR. Promoter activity was checked by luciferase reporter assay. Knockdown was achieved by lentiviral transduction. Results: Treatment of PaCa cells with TET resulted in decreased levels of cyclin D1 and phospho-Rb, two critical proteins required for G1/S transition and increased levels of p21WAF1/Cip1 and p27Kip1 proteins. Real time PCR revealed that cyclin D1 mRNA was decreased and p21WAF1/Cip1 mRNA was increased while there was no significant change in p27Kip1 mRNA levels. The cycloheximide chase showed that TET treatment increases p27Kip1 protein levels by improving its protein stability (t1/2 6h vs 2h: TET treated vs control). Further studies into the mechanism revealed that treatment of PaCa cells with TET resulted in decreased protein levels of Skp2 E3 ligase which is specific for degrading p27Kip1 protein during the cell cycle. Moreover TET also increased the p21WAF1/Cip1 promoter activity accounting for increased mRNA levels of p21WAF1/Cip1. These data suggest a notable mechanism by which TET regulates expression of critical cell cycle regulators at both transcriptional and post-translational level. Such a mechanism would provide for cell cycle block in PaCa cells following TET treatment. Conclusion: TET halts deregulated cell cycle in PaCa cells in part by modulating p27Kip1 and p21WAF1/Cip1 protein levels. Moreover p27Kip1 is regulated post-translationally (via Skp2 regulation) and p21WAF1/Cip1 is regulated at transcriptional level. These results suggest that TET can not only halt deregulated cell cycle but also overcome the therapeutic resistance in PaCa cells. Citation Format: Karnika Singh, Qin Dong, Sweaty Koul, Hari K. Koul. TET increases p27Kip1 protein stability and p21WAF1/Cip1 promoter activity to block deregulated cell cycle progression in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4305.