Rap1B-induced p38 Promotes TTP phosphorylation and Invasion in HNSCC

Tumor-secreted inflammatory cytokines and matrix metalloproteinases (MMPs) play a crucial role in tumor invasion, metastasis and tumor recurrence in head and neck squamous cell carcinoma (HNSCC). Tristetraprolin (TTP) promotes degradation of cytokine mRNA. Although downregulation of TTP increases invasion, its role in MMP regulation has not been investigated. Additionally upstream regulators of TTP have not been elucidated in cancer. Rap1B, a member of the ras family of proteins, is a small GTPase. Objective: Our goal is to determine how rap1B modulates TTP-mediated cytokine and MMP secretion and tumor progression. Method:  Rap1B mRNA and protein expression were evaluated in HNSCC cell lines via RT-qPCR and immunoblot, respectively. Active rap1B was quantified by the ralGDS pulldown assay. To investigate the signaling mechanism, experiments were performed in two HNSCC cell lines stimulated with IL-1β. Rap1B and TTP expression were modulated by siRNA. Cytokines in conditioned medium were quantified by ELISA. mRNA decay in actinomycin D-treated cells, was quantified by RT-qPCR. Cell motility and invasion were determined by migration and Matrigel assays. MMP secretion was evaluated by zymography. Result:  Both total rap1B and active rap1B were highly expressed in multiple HNSCC compared to normal keratinocytes. IL-1β mediates rap1B activation. Suppression of rap1B expression attenuated IL-1β-induced p38 activation. siTTP increased MMP secretion and invasion. Knockdown of TTP stabilized MMP-2 and MMP-9 mRNA resulting in increased MMP-2 and MMP-9 secretion. Conclusion: Rap1B induces p38 which phosphorylates TTP. Since enhanced MMP secretion is associated with tumor invasion and metastasis, TTP may be a promising target to inhibit cancer progression and improve HNSCC treatment options. This study was supported in part by DE018512-01, DE017977-01, DE0219513-01, 1R01DE021423, DE007057-32, and 5F32-DE21305-02.