The MAPK-activated protein kinase 2 mediates gemcitabine sensitivity in pancreatic cancer cells

Abstract

Pancreatic carcinoma is the major clinical entity where the nucleoside analog gemcitabine is used for first-line therapy. Overcoming cellular resistance toward gemcitabine remains a major challenge in this context. This raises the need to identify factors that determine gemcitabine sensitivity in pancreatic carcinoma cells. We previously found the MAPK-activated protein kinase 2 (MK2), part of the p38/MK2 stress response pathway, to be required for DNA replication fork stalling when osteosarcoma-derived cells were treated with gemcitabine. As a consequence, inhibition or depletion of MK2 protects these cells from gemcitabine-induced death (Köpper, et al. Proc Natl Acad Sci USA 2013; 110:16856-61). Here, we addressed whether MK2 also determines the sensitivity of pancreatic cancer cells toward gemcitabine. We found that MK2 inhibition reduced the intensity of the DNA damage response and enhanced survival of the pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and Panc-1, which display a moderate to strong sensitivity to gemcitabine. In contrast, MK2 inhibition only weakly attenuated the DNA damage response intensity and did not enhance long-term survival in the gemcitabine-resistant cell line PaTu 8902. Importantly, in BxPC-3 and MIA PaCa-2 cells, inhibition of MK2 also rescued increased H2AX phosphorylation caused by inhibition of the checkpoint kinase Chk1 in the presence of gemcitabine. These results indicate that MK2 mediates gemcitabine efficacy in pancreatic cancer cells that respond to the drug, suggesting that the p38/MK2 pathway represents a determinant of the efficacy by that gemcitabine counteracts pancreatic cancer.

Keywords: Chk1; DNA damage; MAPKAPK2; MK2; chemotherapy; gemcitabine; pancreatic carcinoma.

Figure 1. Effect of MK2 inhibition and depletion on gemcitabine-induced H2AX phosphorylation in pancreatic cancer cell lines. BxPC-3 (A), MIA PaCa-2 (B), Panc-1 (C), and PaTu 8902 cells (D) were treated with 100 nM gemcitabine and MK2 inhibitor or DMSO for 24 h. H2AX phosphorylation was analyzed by immunoblot. (E) Panc-1 cells were depleted of MK2 by siRNA-mediated knockdown. Forty-eight hours later, the cells were treated with 300 nM gemcitabine for 22 h or left untreated. The cells were fixed and stained for immunofluorescence analysis, and γH2AX fluorescence intensity was quantified. Mean ± SD from 3 technical replicates. (**P = 0.009).

Figure 2. Proliferation of pancreatic cancer cell lines upon treatment with gemcitabine and/or MK2 inhibitor. BxPC-3 (A), MIA PaCa-2 (B), Panc-1 (C), and PaTu 8902 (D) cells were treated with 100 nM gemcitabine and MK2 inhibitor or DMSO for 24 h on day 1. Then the drugs were washed out, and cell confluence was quantified by light microscopy and digital image analysis until day 18.

Figure 3. Gemcitabine-induced H2AX phosphorylation in dependence of MK2 and Chk1 inhibition in pancreatic cancer cell lines. BxPC-3 (A), MIA PaCa-2 (B), and PaTu 8902 (C) cells were treated with 100 nM gemcitabine and MK2 inhibitor, Chk1 inhibitor or both for 24 h. Then, H2AX phosphorylation was analyzed by immunoblot. “Relative γH2AX” indicates relative γH2AX intensities normalized to Hsc70 intensities. See Table S1 for raw data.