The Mapkapk2 Pathway Mediates Radiation-Induced Tumor Inflammation And Proliferation In Bladder Cancer

Muscle-invasive bladder cancer survival remains poor because of development of distant metastatic disease. Epithelial-to-mesenchymal transition (EMT) is a known mechanism for tumor metastasis, invasion, proliferation and treatment resistance. We have previously identified the 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. We hypothesize that MK2 pathway activation in bladder cancer following RT can lead to increased tumor inflammation, EMT, and tumor repopulation. The bladder cancer cell lines T24, HTB9, UMUC were treated with either sham-control, radiotherapy (RT), MK2 inhibitor (MK2i; PF364402, 50 mM), or both. Standard immunoblot was performed to assess protein phosphorylation and pathway activation. qRT-PCR was performed to evaluate cytokine and EMT gene expression. Cytokine production was assessed using a commercial cytokine magnetic bead kit and assayed using the Luminex 200 platform. siRNA knockdown of MK2 expression was used to evaluate specificity of RT activation of the MK2 pathway. Cell proliferation studies were performed over a 72-hour period by addition of excipient or inflammatory cytokine (IL-6, 10ng/ml) to plated cells. Following RT, we observe an increase in MK2 phosphorylation by immunoblot over non-treated bladder cancer cells, in vitro. Pre-treatment with an MK2i prior to RT led to abrogation of MK2 phosphorylation. The downstream consequence of RT-mediated MK2 pathway activation consistently demonstrated in all three bladder cancer cell lines an increase in inflammatory cytokines production: IL-1a, IL-6, and TNFa; and an increase in EMT genes: SNAI1 (all 3 cells) and SNAI2 (only in 2 cell lines). MK2i prior to RT led to significant reduction in IL-1a, IL-6, TNFa and SNAI1 and SNAI2. Bladder cancer exposure to IL-6 led to increased cell proliferation compared to sham-treated cells (253K vs 165K, p = 0.039). Furthermore, we successfully used siRNA to knockdown MK2 expression in T24 bladder cells to 7-14% of MK2 expression seen in cells treated with scramble siRNA. Like the small molecule inhibitor, MK2 siRNA could substantially reduce RT-induced MK2 phosphorylation and IL-6 production as determined by immunoblot and by gene expression analysis. We have demonstrated for the first time that RT can regulate MK2 pathway activation with subsequent activation of tumor EMT and inflammatory cytokine production. Furthermore, pharmacologic or genetic MK2 pathway blockade could abrogate this RT-induced effect and reduce tumor cell proliferation. Potential inhibition of the MK2 pathway may help to decrease RT resistance and improve bladder-organ preservation therapy.