Abstract 2367: RSK2 promotes head and neck cancer cell invasion through direct phosphorylation of Hsp27 to regulate actin filaments

Abstract

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancers and frequently metastasizes to lymph nodes. We found that the protein expression pattern of p90 ribosomal S6 kinase 2 (RSK2) correlates with the invasive ability of diverse HNSCC cell lines, and with human head and neck cancer progression. Continued RSK2 expression is vital for maintenance of the invasive and metastatic potential of HNSCC cells in vitro and in vivo, respectively. In a phospho-antibody microarray-based study, we identified pro-metastatic protein factors that are phosphorylated and activated by RSK2, including the previously identified CREB and a newly identified RSK2 substrate Hsp27, which are important for the RSK2-mediated pro-invasive ability of HNSCC cells. Hsp27 has been found to be overexpressed in many types of cancers, and its overexpression is associated aggressive tumor behavior. Here we found that RSK2 promotes stabilization of actin filaments in HNSCC cells through phosphorylation and activation of Hsp27 RNAi-mediated knockdown of Hsp27 significantly attenuated HNSCC Tu212 cell invasion conferred by exogenous expression of RSK2. In an in vitro kinase assay using purified recombinant Hsp27 WT and individual S15A, S78A or S82A mutant proteins incubated with active recombinant RSK2, we found that RSK2 directly phosphorylates Hsp27 at S78 and S82, but not S15. Moreover, stable expression of phospho-mimetic Hsp27 S78D/S82D double mutant led to further significantly enhanced cell invasion of poorly invasive HNSCC Tu212 and 686LN cells, compared to exogenous expression of Hsp27 WT. Stable expression of Hsp27 S78D/S82D mutant, but not WT or S78A/S82A mutant, rescued the cell invasion and tumor metastasis attenuated by stable knockdown of RSK2 in highly invasive HNSCC M4e cells in vitro and in vivo using xenograft mice, respectively. Hsp27 regulates actin dynamics and apoptosis. Actin immunofluorescent staining showed that RNAi-mediated stable knockdown of RSK2 resulted in disruption of actin filaments in 212LN and M4e cells. However, stable expression of the Hsp27 phospho-mimetic mutant S78D/S82D, but not WT or the phospho-deficient S78A/S82A mutant, rescued the formation of actin filaments. Our findings demonstrate that RSK2 signals through Hsp27 to regulate actin filaments and cell invasion via direct phosphorylation of Hsp27 at Ser78 and Ser82. This may represent a transcription-independent mechanism underlying RSK2-mediated pro-metastatic signaling in HNSCC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2367.