MK2 Pathway Mediates Radiation-Induced Tumor Inflammation and Is a Poor Prognostic Factor in Head and Neck Cancer

Abstract

Epithelial-to-mesenchymal transition (EMT) pathway is a known mechanism for tumor metastasis, invasion, proliferation and treatment resistance. We have identified the MAP kinase-activated protein kinase 2 (MK2) pathway as a regulator of inflammatory cytokine production and EMT pathway activation. However, no link has been established between radiotherapy (RT) and MK2 pathway, tumor inflammation, EMT and tumor repopulation in head and neck squamous cell carcinoma (HNSCC). We hypothesize that RT can activate MK2 signaling which in turn leads to increased tumor inflammation, EMT, tumor repopulation and high MK2 phosphorylation (pMK2) is a poor prognostic factor in HNSCC. A 180 patient oropharyngeal SCC (OSCC) tissue microarray, with annotated patient characteristics and survival outcomes, was stained for pMK2, p16, and HPV (ISH, RNA-seq). H-score was calculated for all samples and correlated to stage, HPV-p16 status and survival outcomes (OS and DSS) by log-rank analysis. For in vitro studies, HNSCC cell lines (SCC47, SCC90, TU167, HN11) were treated in 1 of 4 arms: control, MK2 inhibitor (MK2i), RT, or both. Intensity of pMK2 was assessed by immunoblot. Cytokine and EMT gene expression was evaluated by RT-qPCR. Cytokine production was assessed using a commercial kit. The siRNA experiments were performed to assess specificity of RT signaling through the MK2 pathway. For in vivo studies, HNSCC patient-derived xenografts (PDX) were treated and analyzed as the groups described above and survival studies performed. Patients bearing tumors with high pMK2 vs low pMK2 were associated with worse cancer-specific survival. This was more pronounced in p16-negative compared to p16-positive OSCC patients (p<0.0002). In vitro, RT increased TNFA (8-15x), IL6 (5-7x), VIM (4.5x), SNAI1 (2-5x) gene expression significantly. This effect was significantly reduced with the addition of MK2i prior to RT. MK2 siRNA successfully knocked down MK2 expression in all cell lines tested (to 7-14% of scramble). Similar to the small molecule inhibitor, MK2 siRNA could substantially reduce inflammatory cytokine production. The in vivo animal studies demonstrate that combined modality therapy was superior to monotherapy alone (p = 0.0123). Tumor PDX volumes captured at day 19 demonstrated the following average tumor sizes: control (994mm3), MK2i (440mm3), RT(479mm3), and RT+MK2i (185mm3). We observe that RT-induced inflammation, EMT and tumor regrowth appears to be regulated by MK2 pathway. OSCC patients who have p16-negative tumors with high pMK2 have a worse prognosis compared to patients with p16-positive tumors with low pMK2. We demonstrate for the first time that RT can induce MK2 phosphorylation in HNSCC leading to increased tumor inflammatory cytokine and EMT gene expression. Inhibition of the MK2 pathway may help to decrease RT resistance and improve HNSCC treatment outcomes.