Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells

Abstract

The ability of cancer cells to degrade the extracellular matrix and invade interstitial tissues contributes to their metastatic potential. We recently showed that overexpression of sorting nexin 9 (SNX9) leads to increased cell invasion and metastasis in animal models, which correlates with increased SNX9 protein expression in metastases from human mammary cancers. Here, we report that SNX9 expression is reduced relative to neighboring normal tissues in primary breast tumors, and progressively reduced in more aggressive stages of non-small-cell lung cancers. We show that SNX9 is localized at invadopodia where it directly binds the invadopodia marker TKS5 and negatively regulates invadopodia formation and function. SNX9 depletion increases invadopodia number and the local recruitment of MT1-MMP by decreasing its internalization. Together, these effects result in increased localized matrix degradation. We further identify SNX9 as a Src kinase substrate and show that this phosphorylation is important for SNX9 activity in regulating cell invasion, but is dispensable for its function in regulating invadopodia. The diversified changes associated with SNX9 expression in cancer highlight its importance as a central regulator of cancer cell behavior.

Endocytosis, Metastasis and Beyond: Multiple Facets of SNX9

“…Consistent with this, more aggressive, late-stage primary lung cancer tumors express less SNX9 protein than their early stage counterparts [52]; whereas SNX9 expression levels were higher in breast cancer metastases than in their primary tumors [20]. It is likely that the scaffolding activity of SNX9 and its diverse sets of interactions function to spatially and temporally coordinate endocytic membrane trafficking, signaling and actin dynamics to differentially affect cell motility and invasion.…”

“…Specific adaptor proteins such as TKS5 are recruited to invadopodia [51] and are necessary for their formation. SNX9 binds TKS5 most likely via its LC domain, is localized to invadopodia (Figure 1B) and negatively regulates both their formation and function [52] (. Thus, SNX9 depletion increased invadopodia number and their matrix-degrading activity, in part by decreasing internalization of MT1-MMPs and increasing their surface expression at invadopodia [52].…”

“…Indeed, these two activities of SNX9 are independently regulated through phosphorylation by the tyrosine kinase, Src. Five Src-dependent phosphorylation sites were identified in SNX9 with Y239 being the major phosphorylated residue (Figure 3) [52,53]. The biological consequences of the single phosphorylation sites have not been evaluated, although it was suggested that phosphorylation of Y287 within the PX domain might interfere with SNX9’s membrane binding abilities [53].…”

“…Other studies have suggested that tyrosine phosphorylation within the SH3 domain of SNX9 decreases its binding affinity to N-WASP [22]. Functionally, the non-phosphorylatable mutant (in which all identified Src tyrosine phosphorylation sites were mutated to phenylalanine) was as efficient as WT-SNX9 in rescuing MT1-MMP internalization and matrix degradation following the depletion of endogenous SNX9, whereas the same non-phosphorylatable mutant had dominant negative effects on cell migration through collagen [52]. …”

“…In contrast, only SNX18 has been shown to promote autophagosome formation in mammalian cells [59,60] and to localize to a subset of AP1 and PACS1-positive endosomes [11]. SNX9 and SNX18 have opposite roles in the regulation of matrix degradation: SNX9 knockdown increases, whereas SNX18 knockdown decreases matrix degradation at invadopodia [52]. …”