Abstract 792: Knockdown of MK2 in bladder cancer cells increases their radiosensitivity

Abstract

Abstract We have previously identified the p38 substrate MAPK-activated protein kinase-2 (MK2) as a radiation response stress pathway regulating inflammatory cytokine production that facilitates EMT pathway activation and tumor growth in head and neck cancer. Here we examine whether MK2 knockdown in bladder cancer cells alters their ability to produce immune mediators and to resist radiation.In order to study the impact of MK2 in bladder cancer (i.e., T24, HTB9), we generated both scrambled control (SCR) and MK2 shRNA using a lentiviral transduction system and selected for clones via puromycin selection. Generally, all MK2 shRNA clones demonstrated >88% reduction in MK2 protein expression by immunoblot. Increased phosphorylation of MK2 (p272) compared to control was detected 48 hours after the exposure of cells to 10 Gy radiation which was not seen in shRNA cells. Gene expression analysis of the bladder cells at 48 or 72 hours after 10 Gy radiation showed an increase in the expression of the inflammatory cytokines IL-6 and TNFα and genes that regulate epithelial to mesenchymal transition; SNAI1 and vimentin. A reduced expression of these genes was seen in the shRNA cells following radiation demonstrating MK2 as an important signaling molecule in this response. We then examined whether MK2 also impacted tumor proliferation, radiosensitivity and the cell cycle. The proliferation rates of shRNA cells compared to SCR cells showed no difference under control conditions in both cell lines. However, in response to radiation both T24 and HTB9 shRNA cells showed a greater loss of cells compared to SCR as determined by CyQUANT DNA fluorescence at 24, 48, 72 and 144 hours. Furthermore, shRNA cells showed reduced clonogenic activity after radiation compared to SCR as determined by colony-forming assays using 0-10 Gy doses in 2 Gy increments. Analysis of cell cycle changes induced by radiation at 28 hours showed a pronounced shift of HTB9 cells from 57.8 (% G1 Phase), 19.1 (% S phase) and 22.1 (% G2/M) phase to 7.6, 3.4, 86.7. This pattern was significantly different in shRNA cells that showed proportions of 46.2, 17.8, 32.0 without radiation which shifted to 20.6, 5.9 and 67.3 28 hours after irradiation. The percentage of cells in G1, and G2/M phase 28 hours after irradiation was significantly different between SCR and shRNA (p<0.001) suggesting that MK2 knockdown altered radiosensitivity by impacting cell cycle kinetics.In summary, we measured a significant increase in the sensitivity of bladder cancer cells to radiation following knockdown of MK2. The data suggests a reduction in survival of tumors cells exposed to radiation with MK2 knockdown through cell cycle changes and reduced production of survival and growth signals. Citation Format: Deri Morgan, Grace Millington, Hanna Smith, Colby Spiess, Kiersten L. Berggren, Xinglei Shen, Gregory N. Gan. Knockdown of MK2 in bladder cancer cells increases their radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 792.