Abstract 2079: RSK2 mediates anti-anoikis signals by regulating downstream factors through both transcription -independent and -dependent manner in human cancers

Abstract

Abstract Metastasis is the most dangerous switch during tumor progression, which involves complicated steps of events including anoikis resistance. Anoikis is an anchorage-dependent cell death induced by loss of cell or extracellular matrix attachment. Metastatic tumor cells may escape from anoikis and invade other organs. However, the signaling mechanisms by which invading tumor cells become resistant to the anoikis process remains unclear. We recently reported that continued RSK2 expression contributes to the maintenance of the invasive and metastatic potential of human head and neck squamous cell carcinoma (HNSCC). Further studies revealed that RSK2 signaling is commonly important to protect metastatic human cancer cell lines including 212LN (HNSCC), A549 (lung cancer), and SKBR3 (breast cancer) from anoikis. Stable knockdown of RSK2 did not significantly affect apoptosis induced by control anti-cancer agent cycloheximide, but sensitized various cancer cell lines to detachment-induced anoikis. Phosphor-proteomics based study identified two RSK2 phosphorylation targets, including a newly identified RSK2 substrate, apoptosis signal-regulating kinase 1 (ASK1), and a known target, CREB. Further study revealed that they are involved in RSK2 dependent anti-anoikis signaling. ASK1 is a mitogen-activated protein kinase kinase kinase, and its activity is regulated in various ways, including phosphorylation. Our study showed that RSK2 directly phosphorylates ASK1 at multiple sites, which negatively contribute to ASK1 kinase and pro-apoptotic activity. Knockdown of ASK1 in cells resulted in decreased anoikis, while overexpression of ASK1 sensitized cells to anoikis which suggest that RSK2 phosphorylates and inhibits ASK1 to protect cancer cells from anoikis. CREB is a transcription factor activated by RSK2 whose signaling is implicated in promoting tumor progression and metastasis. We identify RSK2αCREB transcription targets by performing DNA microarray using metastatic HNSCC cells with stable knockdown of RSK2 or CREB. We identified 115 and 45 genes that are down-regulated or up-regulated in both knockdown cells. Among these genes, we identified a group of anti-apoptotic genes down-regulated, and pro-apoptotic genes up-regulated as novel RSK2 and CREB transcription targets. Over-expression of the anti-apoptotic targets or transient knockdown of the pro-apoptotic targets rescued the anoikis induced by RSK2 knockdown and conferred resistance to cells from undergoing anoikis. These data suggest a transcription-dependent mechanism in which RSK2 may in part signal through CREB to provide anti-anoikis signals. These data together suggest that RSK2 provides anti-anoikis signals via ASK1 and CREB in both transcription-independent and -dependent manners, respectively, which may promote cancer cell invasion and tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2079. doi:1538-7445.AM2012-2079