Ste20-related proline-alanine-rich kinase (SPAK) plays a role in regulating many biological activities and interacts with the K-Cl cotransporter (KCC) 3; however, the importance of SPAK for KCC3 function has not been demonstrated. Here, we investigated the role of SPAK in KCC3-regulated cell invasiveness and tumor formation. We showed that the induction of KCC3 expression triggers the activation of the NFκB and SPAK signaling cascades, leading to the activation of p38 MAPK and matrix metalloproteinase-2 (MMP2). Small interference RNA-mediated reduction in SPAK protein levels suppressed the invasive ability and oncogenic potential of cervical cancer cells and decreased tumor formation in mouse xenografts. A combination of experimental approaches, including RT-PCR and real-time RT-PCR, gelatin zymography, NFκB luciferase activity and chromatin immunoprecipitation (ChIP) assays, showed that the induction of KCC3 overexpression increased MMP2 expression and augmented binding of NFκB to its putative SPAK promoter binding site, suggesting that the SPAK-MMP2 axis is up-regulated by NFκB. The pharmacological inhibition of NFκB or MMP2 abrogated KCC3-triggered, SPAK-dependent cell invasiveness. Furthermore, p38 MAPK was identified as the upstream regulator of KCC3-dependent MMP2 activation. We concluded that SPAK may promote the KCC3-mediated tumor aggressiveness via the NFκB/p38 MAPK/MMP2 axis. This article is protected by copyright. All rights reserved.

KCC3 physically interacts with SPAK by anti bait coimmunoprecipitation (View interaction).