Abstract
Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3–SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.
Highlights
Cell migration and invasion are highly orchestrated, overlapping processes with invasion representing a degradative form of migration resulting from increased proteinase secretion and proteolysis of extracellular matrix (ECM) proteins within the stroma
We previously demonstrated that neural precursor cell expressed developmentally downregulated 9 (NEDD9; human enhancer of filamentation 1, HEF-1; Crk-associated substrate in Lymphocytes, CasL) is an important regulatory protein involved in head and neck squamous cell carcinoma (HNSCC) cell signaling, leading to migration and invasion [11]
We previously demonstrated that Neural precursor cell expressed developmentally downregulated 9 (NEDD9) silencing reduces migration, invasion, matrix metalloproteinase (MMP) secretion and invadopodia formation in HNSCC cells derived from the tongue (SCC9 cells; [11])
Summary
Cell migration and invasion are highly orchestrated, overlapping processes with invasion representing a degradative form of migration resulting from increased proteinase secretion and proteolysis of extracellular matrix (ECM) proteins within the stroma. Invasive cancer cells must be capable of assembling invadosome structures termed invadopodia that represent sites of focal matrix metalloproteinase (MMP) secretion [1]. MMPs, invasive cancer cells generate traction force at the rear, with the protrusive process of invadopodia formation occurring at the front, enabling cells to propel through the degraded ECM/stromal barrier. In addition to propulsive force generation, this process requires coordinated substratum attachment and detachment of cells coordinated via focal adhesions [2]
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