Cancer cell extravasation requires iplectin-mediated delivery of MT1-MMP at invadopodia.

BACKGROUND: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear. METHODS: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo. RESULTS: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed iplectin (i = invadopodial). iPlectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed iplectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency. CONCLUSIONS: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and iplectin. CLINICAL TRIAL REGISTRATION NUMBER: NCT04608357.

Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

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.

Syntaxin4-Munc18c Interaction Promotes Breast Tumor Invasion and Metastasis by Regulating MT1-MMP Trafficking.

Invasion of neighboring extracellular matrix (ECM) by malignant tumor cells is a hallmark of metastatic progression. This invasion can be mediated by subcellular structures known as invadopodia, the function of which depends upon soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated vesicular transport of cellular cargo. Recently, it has been shown the SNARE Syntaxin4 (Stx4) mediates trafficking of membrane type 1-matrix metalloproteinase (MT1-MMP) to invadopodia, and that Stx4 is regulated by Munc18c in this context. Here, it is observed that expression of a construct derived from the N-terminus of Stx4, which interferes with Stx4-Munc18c interaction, leads to perturbed trafficking of MT1-MMP, and reduced invadopodium-based invasion in vitro, in models of triple-negative breast cancer (TNBC). Expression of Stx4 N-terminus also led to increased survival and markedly reduced metastatic burden in multiple TNBC models in vivo. The findings are the first demonstration that disrupting Stx4-Munc18c interaction can dramatically alter metastatic progression in vivo, and suggest that this interaction warrants further investigation as a potential therapeutic target. IMPLICATIONS: Disrupting the interaction of Syntaxin4 and Munc18c may be a useful approach to perturb trafficking of MT1-MMP and reduce metastatic potential of breast cancers.

Targeting SNARE-Mediated Vesicle Transport to Block Invadopodium-Based Cancer Cell Invasion.

During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells' invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types.

Inhibition of ß1 integrin induces its association with MT1-MMP and decreases MT1-MMP internalization and cellular invasiveness.

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of ß1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of ß1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and ß1 integrin were sequestered at the cell surface when ß1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of ß1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and ß1 integrin ultimately led to a loss of invasive behaviour.

Adaptor Protein ShcD/SHC4 Interacts with Tie2 Receptor to Synergistically Promote Glioma Cell Invasion.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

ß1 integrin-mediated signaling regulates MT1-MMP phosphorylation to promote tumor cell invasion.

Malignant cancer cells can invade extracellular matrix (ECM) through the formation of F-actin-rich subcellular structures termed invadopodia. ECM degradation at invadopodia is mediated by matrix metalloproteinases (MMPs), and recent findings indicate that membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP14) has a primary role in this process. Maintenance of an invasive phenotype is dependent on internalization of MT1-MMP from the plasma membrane and its recycling to sites of ECM remodeling. Internalization of MT1-MMP is dependent on its phosphorylation, and here we examine the role of ß1 integrin-mediated signaling in this process. Activation of ß1 integrin using the antibody P4G11 induced phosphorylation and internalization of MT1-MMP and resulted in increased cellular invasiveness and invadopodium formation in vitro We also observed phosphorylation of Src and epidermal growth factor receptor (EGFR) and an increase in their association in response to ß1 integrin activation, and determined that Src and EGFR promote phosphorylation of MT1-MMP on Thr567 These results suggest that MT1-MMP phosphorylation is regulated by a ß1 integrin-Src-EGFR signaling pathway that promotes recycling of MT1-MMP to sites of invadopodia formation during cancer cell invasion.This article has an associated First Person interview with the first author of the paper.

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion.

SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) mediate trafficking of membrane type 1-matrix metalloproteinase (MT1-MMP) during invadopodium formation and tumor cell invasion.

Movement through the extracellular matrix (ECM) requires cells to degrade ECM components, primarily through the action of matrix metalloproteinases (MMPs). Membrane type 1-matrix metalloproteinase (MT1-MMP) has an essential role in matrix degradation and cell invasion and localizes to subcellular degradative structures termed invadopodia. Trafficking of MT1-MMP to invadopodia is required for the function of these structures, and here we examine the role of N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE)-mediated membrane traffic in the transport of MT1-MMP to invadopodia. During invadopodium formation in MDA-MB-231 human breast cancer cells, increased association of SNAP23, Syntaxin4, and vesicle-associated membrane protein 7 (VAMP7) is detected by coimmunoprecipitation. Blocking the function of these SNAREs perturbs invadopodium-based ECM degradation and cell invasion. Increased level of SNAP23-Syntaxin4-VAMP7 interaction correlates with decreased Syntaxin4 phosphorylation. These results reveal an important role for SNARE-regulated trafficking of MT1-MMP to invadopodia during cellular invasion of ECM.

SNARE-dependent interaction of Src, EGFR and ß1 integrin regulates invadopodia formation and tumor cell invasion.

Acquisition of an invasive phenotype is prerequisite for tumor metastasis. Degradation of the extracellular matrix (ECM), and subsequent invasion by tumor cells, is mediated, in part, through subcellular structures called invadopodia. Src-dependent cytoskeletal rearrangements are required to form invadopodia, and here we identify an association between Src, epidermal growth factor receptor (EGFR), and ß1 integrin that facilitates invadopodia formation. The association of Src, EGFR and ß1 integrin is dependent upon membrane traffic that is mediated by syntaxin13 (officially known as STX12) and SNAP23; a similar dependence on these two SNARE proteins was observed for invadopodium-based matrix degradation and cell invasion. Inhibition of SNARE function impaired the delivery of Src and EGFR to developing invadopodia, as well as the ß1-integrin-dependent activation of Src and phosphorylation of EGFR on Tyr residue 845. We also identified an association between SNAP23 and ß1 integrin, and inhibition of ß1 integrin increased this association, whereas the interaction between syntaxin13 and SNAP23 was reduced. The results suggest that SNARE-dependent trafficking is regulated, in part, by ß1 integrin and is required for the delivery of Src and EGFR to sites of invadopodia formation in order to support tumor cell invasion.

Phosphorylation of membrane type 1-matrix metalloproteinase (MT1-MMP) and its vesicle-associated membrane protein 7 (VAMP7)-dependent trafficking facilitate cell invasion and migration.

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover.

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels.

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity (MIA), secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins α(4)ß(1) and α(5)ß(1), facilitating cell detachment and promoting formation of metastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I (COPI)- and coat protein complex II (COPII)-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca(2+) ions. Interestingly, the Ca(2+)-activated K(+)-channel, subfamily N, member 4 (KCa3.1), known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant-negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

VAMP3, syntaxin-13 and SNAP23 are involved in secretion of matrix metalloproteinases, degradation of the extracellular matrix and cell invasion.

Cellular remodeling of the extracellular matrix (ECM), an essential component of many physiological and pathological processes, is dependent on the trafficking and secretion of matrix metalloproteinases (MMPs). Soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic has documented roles in cell-ECM interactions and the present study specifically examines SNARE function in the trafficking of MMPs during ECM degradation. Using the invasive human fibrosarcoma cell line HT-1080, we demonstrate that a plasma membrane SNARE, SNAP23, and an endosomal v-SNARE, VAMP3 (also known as cellubrevin), partly colocalize with MMP2 and MMP9, and that inhibition of these SNAREs using dominant-negative SNARE mutants impaired secretion of the MMPs. Inhibition of VAMP3, SNAP23 or syntaxin-13 using dominant-negative SNARES, RNA interference or tetanus toxin impaired trafficking of membrane type 1 MMP to the cell surface. Consistent with these observations, we found that blocking the function of these SNAREs reduced the ability of HT-1080 cells to degrade a gelatin substrate in situ and impaired invasion of HT-1080 cells in vitro. The results reveal the importance of VAMP3, syntaxin-13 and SNAP23 in the trafficking of MMP during degradation of ECM substrates and subsequent cellular invasion.

Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis.

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP(2) from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcgamma receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP(2) and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP(2) levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.