Abstract 4438: Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic cancer
Authors: Rauth Sanchita , Karmakar Saswati , Shah Ashu , Nimmakayala Rama Krishna , Bhatia Rakesh , Muniyan Sakthivel , Kumar Sushil , Dutta Samikshan , Datta Kaustubh , Batra Surinder K. , Ponnusamy Moorthy Palanimuthu 
Published: August 2019

Background: RNA polymerase associated factor 1 (PAF1)/Pancreatic differentiation 2 (PD2) is one of the core subunit of the human PAF1 complex (PAF1C), which regulates various cellular functions such as transcriptional elongation and histone modification. We have previously demonstrated its unique role in oncogenesis and stem cell maintenance. Studies have demonstrated that PAF1/PD2 gene yields a protein of 59.9 Kda (531 amino acids), however it has been found that it always gives a band at 80 Kda. Further, previous studies suggests that the 60 Kda protein represents the precursor, which rapidly process into an 80 Kda mature protein. SUMOylation is a process of reversible posttranslational modification that adds a small ubiquitin-related modifier (SUMO)-1 protein to the target protein. SUMOylation plays various molecular biology function such as transcriptional regulation, protein-protein interaction, and DNA damage repair and in cell cycle. DNA damage happen due to several physiological processes, but can also be caused by genotoxic agents. Promyelocytic Leukemia (PML) is protein that forms nuclear bodies and may be modified by SUMO1 and act as a DNA-damage sensor.

Hypothesis: PAF1/PD2 is interacting with SUMO-1 and PML and, thus, plays an important function in providing gemcitabine resistance to pancreatic cancer cells

Methods: SW1990, F9 (mouse embryonic cells) and CD18/HPAF cells were treated with 2-D08, a potent SUMOylation inhibitor for 24 hours and with siRNA against SUMO1 for 48 hours followed by protein isolation and western blotting. Immunoprecipitation and immunofluorescence were done to show the interaction between PAF1/PD2 and SUMO1 and with PML. To study the effect of PAF1/PD2 on gemcitabine resistance, SW1990 and Capan-1 cells were treated with different concentration of gemcitabine and then the expression of PAF1/PD2 along with SUMO1 was checked through immunoblotting and confocal imaging.

Results: Results shows that inhibiting SUMOylation with both 2D08 and siRNA resulted in a decrease expression of PAF1/PD2 80 Kda protein. Immunoprecipitation and immunofluorescence analysis showed that endogenous PAF1/PD2 interacts with SUMO1. This finding was further verified using ectopically overexpressed Flag- tagged PAF1/PD2 and HA-tagged SUMO1, which showed a physical interaction between PAF1/PD2 and SUMO1. Interestingly, we observed that gemcitabine treatment significantly increased the SUMOylated status of PAF1/PD2 in pancreatic cancer cells. Further our results proved that SUMOylated PAF1/PD2 form nuclear bodies along with PML in pancreatic cancer cells.

Conclusions: Our observation suggest that PAF1/PD2 undergoes SUMOylation and covalently interacts with SUMO1. Treatment with gemcitabine results in enhanced expression of PAF1/PD2 and increased co-localization with SUMO1 and PML, indicates a role of SUMOylated PAF1/PD2 in gemcitabine resistance.

Citation Format: Sanchita Rauth, Saswati Karmakar, Ashu Shah, Rama Krishna Nimmakayala, Rakesh Bhatia, Sakthivel Muniyan, Sushil Kumar, Samikshan Dutta, Kaustubh Datta, Surinder K. Batra, Moorthy Palanimuthu Ponnusamy. Role of post translational modification of PAF1/PD2 in gemcitabine resistance of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4438.