Abstract 854: Inhibition of PRMT5 results in radiosensitization in lung cancer cell lines
Authors: Sharma Smitha , Wu X , Smith P , Denko N , Li C , Lai H , Yan F , Shilo K , Chakravarti A , Sif S , Baiocchi R , Otterson G , Xu-Welliver Meng 
Published: May 2015

Background: Protein arginine methylation is a post translational modification that influences signal transduction, mRNA splicing, gene transcription and DNA repair. Among the PRMT family members, PRMT5 is a type II enzyme that symmetrically methylates histone H4 at Arginine 3 and histone H3 at Arginine 8. Studies have recently linked this modification to carcinogenesis and metastasis. The function of PRMT5 in carcinogenesis is related to cell proliferation through modulation of E2F1, p53, EGFR, and CRAF. It is known to accelerate progression through the G1 phase of cell cycle by influencing proteins like CDK4 and CDK6. Previous work on human lung cancer specimens has demonstrated an overexpression of PRMT5 in cancerous tissue when compared to normal lung parenchyma. Suppression of PRMT5 significantly inhibits cell proliferation in lung cancer cell lines A549 and H1299. We hypothesized inhibition of PRMT5 can lead to increased radiosensitivity in lung cancer cells.

Method: Several lung cancer cell lines were used in the experiments, including A549, H1299 and H23. SiRNA (Dharmacon) and lentiviral shRNA (Sigma) were used to knock down (KD) PRMT5 levels transiently or stably in A549 cell line in which p53 is present in its wild type form. Forty eight hours after transient transfection, cells were plated for clonogenic survival assay and subsequently exposed to ionizing radiation at 0, 2, and 8 Gy. Cellular PRMT5 protein levels were estimated by western blotting analysis for PRMT5 KD and scramble control cell lines. The scramble control and siRNA knockdown cells were subjected to cell cycle analysis by flow cytometry. We also tested specific PRMT5 inhibitors with and without radiation therapy in the lung cancer cell lines to see if PRMT5 inhibitors could lead to increased radiosensitivity.

Results: We observed a &gt;90% PRMT5 KD in transiently transfected cells at 48 h and 72 h post transfection as verified by western blot analysis. This transient KD lead to a small but significant decrease in colony survival after radiation. This radiosensitization was not observed in cells selected for stable KD of PRMT5 protein by lentiviral RNA transfection. There is an increase of cell population in G1 arrest in PRMT5 transient KD cells but not in stable KD cells. Additionally, cells treated with PRMT5 specific inhibitors (“cpd5” or “cpd65”) demonstrated increased radiosensitivity in A549 cells but not in H1299 suggesting that this effect may be p53-dependent.

Conclusion: PRMT5 inhibition by siRNA or its specific inhibitors lead to radiosensitivity in A549 lung cancer cell line. This effect may be partially dependent on p53-dependent cell cycle arrest. Further work to inhibit PRMT5 in other lung cancer cell lines with different p53 activities will be investigated.

Citation Format: Smitha Sharma, X Wu, P Smith, N Denko, C Li, H Lai, F Yan, K Shilo, A Chakravarti, S Sif, R Baiocchi, G Otterson, Meng Xu-Welliver. Inhibition of PRMT5 results in radiosensitization in lung cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 854. doi:10.1158/1538-7445.AM2014-854