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Published on: December 22, 2020
SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion
Masashi Muramatsu1, Lingqiu Gao2, Jennifer Peresie2
1Institute of Resource Development and Analysis, Kumamoto University, Kumamoto 860-0811, Japan.
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[BrafV600E] 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, p21waf1 and p16ink4a, 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.
Insights
Stromal SSeCKS/AKAP12 loss enhances peritoneal metastasis by increasing inflammatory mediators like Cxcl10. This occurs via altered PKC, PKA, and PI3K/Akt signaling in peritoneal membrane fibroblasts, promoting tumor cell chemotaxis.
Area of Science:
- Oncology
- Cell Biology
- Immunology
Background:
- SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein that suppresses metastasis by inhibiting tumor cell signaling pathways.
- SSeCKS is downregulated not only in metastatic cells but also in the tumor microenvironment, suggesting a role for stromal cells in cancer progression.
Purpose of the Study:
- To investigate the role of stromal SSeCKS/AKAP12 in controlling peritoneal metastasis.
- To elucidate the mechanisms by which SSeCKS deficiency in the stroma promotes cancer spread.
Main Methods:
- Orthotopic melanoma models in SSeCKS-null (KO) and wild-type (WT) mice.
- Adoptive transfer experiments using peritoneal fluid (PF) and peritoneal cells (PC).
- Chemotaxis assays, gene knockdown (Cxcr3), and analysis of inflammatory mediators (Cxcl9, Cxcl10).
- Senescence marker analysis (SA-β-galactosidase, p21waf1, p16ink4a) in peritoneal membrane fibroblasts (PMF).
- Investigation of signaling pathways (PKC, PKA, PI3K/Akt) using kinase inhibitors and scaffolding-site mutants.
Main Results:
- SSeCKS-null hosts exhibited significantly higher peritoneal metastasis compared to WT hosts, which was transferable via peritoneal fluid.
- Peritoneal fluid from SSeCKS-null mice enhanced melanoma cell chemotaxis, correlating with increased Cxcr3 ligand levels (Cxcl9, Cxcl10).
- Loss of SSeCKS in peritoneal membrane fibroblasts led to increased senescence and enhanced secretion of Cxcl10, driven by inflammatory mediators and regulated by PKC, PKA, and PI3K/Akt pathways.
Conclusions:
- Stromal SSeCKS/AKAP12 plays a critical role in suppressing peritoneal metastasis.
- SSeCKS deficiency in stromal cells promotes metastasis by increasing the secretion of tumor chemoattractants like Cxcl10, mediated by specific signaling pathways.
