RESEARCH ARTICLE Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation Sameena Parveen 1 , Amrita Khamari 1 , Jyothikamala Raju 2 , Marc G. Coppolino 3 and Sunando Datta 1, * ABSTRACT Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia- associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper. KEY WORDS: STX7, VAMP2, VAMP3, STX4, MT1-MMP, Tks5, Cortactin, Invadopodia, SNAREs INTRODUCTION Tumor cells acquire invasive capability upon sensing their extracellular milieu. Invasion involves alteration of cell-to-cell and cell–extracellular matrix (ECM) contact and remodeling of the surrounding matrix to form micro-tracks that can be used by other cells during metastasis (Friedl et al., 1997; Wolf et al., 2007; Kawauchi, 2012). ECM remodeling is achieved through the secretion of various matrix-degrading proteases, such as soluble matrix metalloproteinases (MMPs) and membrane-type MMPs. These proteases help cleave the surrounding matrix proteins, enabling pathways for the trailing cells (Stetler-Stevenson, 2001; Chun et al., 2004; Even-Ram and Yamada, 2005; Wolf et al., 2007). Proteolytically active proteases with ECM degradation capability are accumulated and secreted from a specialized protrusive structures, formed at the ventral surface of the cells, termed invadopodia (Artym et al., 2006; Steffen et al., 2008; Poincloux et al., 2009; Kessenbrock et al., 2010; Frittoli et al., 2011; Itoh, 2015; Jacob and Prekeris, 2015; Linder, 2015; Castro-Castro et al., 2016; Cepeda et al., 2016; Jablońska-Trypuć et al., 2016; Qiang et al., 2019). Invadopodium formation is a tightly regulated process involving initiation, assembly and maturation (Murphy and Courtneidge, 2011). Localized delivery of proteases to the invadopodia requires various intracellular trafficking machinery (Steffen et al., 2008; Frittoli et al., 2014; Kajiho et al., 2016; Sharma et al., 2020). Vesicles of endomembrane origin exploit soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to target and fuse to the plasma membrane (PM) (Bonifacino and Glick, 2004; Hong, 2005; Jahn and Scheller, 2006; Cai et al., 2007; Südhof and Rothman, 2009). Several studies have shown that SNAREs are involved in transport of MT1-MMP (also known as MMP14) to the cell surface (Miyata et al., 2004; Steffen et al., 2008; Kean et al., 2009; Williams et al., 2014; Clancy et al., 2015; Sneeggen et al., 2019; West et al., 2021) although the role of SNAREs in regulating the invadopodia number in cancer cells is yet to be investigated. Combining a reverse genetics approach with microscopic and biochemical studies, here we aimed at identifying SNAREs of the endocytic and recycling pathways with potential roles in invadopodia-mediated breast cancer cell invasion. The current study demonstrates that STX7 is crucial for the formation of invadopodia and the associated gelatin degradation and invasion activity. STX7 interacts with multiple SNARE proteins, forming multiple SNARE complexes. Depletion of these interacting partners phenocopies the effects of STX7 depletion. RESULTS STX7 contributes to efficient ECM degradation and Matrigel invasion To investigate the role of SNAREs in matrix remodeling and invasion, 13 SNAREs of the endocytic and recycling pathway were screened using a SMART pool siRNA-based reverse genetics approach. Depletion of six of 13 SNAREs (Fig. S1A) led to significant reduction in gelatin degradation activity in MDA-MB-231 cells (Fig. 1A; Fig. S1B). Subsequently, an Matrigel invasion assay showed a significant reduction in invasive potential upon depletion of STX2, STX4, STX7, VAMP3 and VAMP7 (Fig. 1B; Fig. S1C). Previously, STX2 was found to be involved in invasion and metastasis by activating MMP-9 expression in hepatocellular carcinoma (Jia et al., 2011). Similarly, STX4, VAMP3 and VAMP7 are known to regulate MT1-MMP trafficking during invasion (Steffen et al., 2008; Kean et al., 2009; Williams et al., 2014; Brasher et al., 2017). Although the role of STX7 in endosome maturation (Mullock et al., 2000; Nakamura et al., 2000; Ward et al., 2000) is well known, Handling Editor: Andrew Ewald Received 12 November 2021; Accepted 12 May 2022 1 Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Bhopal 462066, India. 2 Thazhathemalayil House, Thodupuzha East PO, Keerikode, Kerala 685585, India. 3 Department of Molecular and Cellular Biology, University of Guelph, Ontario N1G 2W1, Canada. *Author for correspondence (sunando@iiserb.ac.in) S.P., 0000-0002-9181-8283; A.K., 0000-0003-3454-2759; J.R., 0000-0001- 9444-6211; M.G.C., 0000-0002-4538-7929; S.D., 0000-0002-1417-0276 1 © 2022. Published by The Company of Biologists Ltd | Journal of Cell Science (2022) 135, jcs259576. doi:10.1242/jcs.259576 Journal of Cell Science