SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts

Abstract

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[Braf^V600E] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-β-galactosidase, p21^waf1 and p16^ink4a, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.