NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma
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Stéphane Grauzam1,*, Amanda M. Brock1,*, Casey O. Holmes1, Jessica A. Tiedeken1, Samantha G. Boniface1, Bailey N. Pierson1, Daniel G. Patterson1, Sonya D. Coaxum2, David M. Neskey1,2,3 and Steven A. Rosenzweig1,3
1Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA
2Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, Charleston, SC 29425, USA
3Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA
*These authors contributed equally to this work
Steven A. Rosenzweig, email: [email protected]
Keywords: invasion; metastasis; invadopodia; matrix metalloproteinases; NEDD9
Received: December 21, 2017 Accepted: April 24, 2018 Published: May 22, 2018
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 (ΔSH3NEDD9) 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 ΔCTNEDD9 which lacked the C-terminus (ΔC-terminal; ΔCT). 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.
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