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

Abstract

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.

Figure 1.

Association of Stx4 1 to 15 with Munc18c inhibits Stx4–SNAP23 interaction. A, Parental MDA-MB-231 cells, and cell lines stably expressing GFP, GFP-Stx4-1-15, or GFP-Stx4-15-29 were lysed, and GFP was immunoprecipitated. Immunoprecipitates (IP) were probed for Munc18c. B, Parental MDA-MB-231 cells, and cells stably expressing Stx4-FL. Stx4–N-term, Stx4-1-15, or Stx4-15-29 were lysed, and SNAP23 was immunoprecipitated. IPs were probed for Stx4 and SNAP23. C, Densitometry of the amount of Stx4 coimmunoprecipitated relative to SNAP23 as shown in B (*, P < 0.05). Data represent three or more biological replicates with at least three technical replicates. B+α, beads plus antibody; B+L, beads plus GFP lysate. IB, immunoblot.

Figure 2.

Expression of Stx4 1 to 15 impairs invadopodium formation and gelatin degradation. A, Invadopodium-based degradation of gelatin by cells expressing GFP (control), Stx4-FL, Stx4–N-term, Stx4-1–15, or Stx4-15–29. Cells were seeded onto fluorescent gelatin for 4 hours, and then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. Scale bars = 10 μm. B, Quantification of invadopodium formation. Cells were seeded onto fluorescent gelatin and processed as in A. Cells with F-actin puncta overlying dark spots of gelatin degradation were counted as cells forming invadopodia. Percentages of cells forming invadopodia, normalized to parental cells, were determined by counting 50 cells/sample. C, Invadopodium-based degradation of gelatin (as in B) by parental MDA-MB-468 cells, or those transiently expressing GFP, GFP-Stx4-FL, or GFP-Stx4-N-term. D, Cells, as in A, were seeded onto fluorescent gelatin for 24 hours, and then fixed. Dark areas representing degradation of gelatin were analyzed and scored as described under “Materials and Methods”. All data are presented as percent of control ± SD. Asterisks denote values significantly different from control (*, P < 0.05). All data represent three or more biological replicates with at least three technical replicates.

Figure 3.

Expression of Stx4-1–15 decreases cell surface levels of MT1-MMP and EGFR, as well as cell migration and invasion. A, Parental MDA-MB-231 cells and stable cell lines expressing Stx4-FL, Stx4-N-term, Stx4-1-15, and Stx4-15-29 were plated onto gelatin for 4 hours, exposed to biotin, and then lysed and analyzed by precipitation with streptavidin beads. Cell surface levels of β₁ integrin, MT1-MMP, and EGFR were assessed by Western blotting. GAPDH was used as a loading control. B, Densitometric analysis of the amounts of β₁ integrin, EGFR, MT1-MMP in streptavidin precipitates, as in A, relative to control. Cells, as in A, were serum-starved for 24 hours, and seeded onto uncoated membranes of transwell chambers, and allowed to migrate for 20 hours (C), or seeded onto Matrigel-coated membranes and allowed to invade for 24 hours (D and E). D, Representative images of DAPI stained cells that invaded through the Matrigel-coated membrane. Quantification of invasion in MDA-MB-231 (E) and MDA-MB-468 (F) cells. Parental cells, or those expressing GFP, GFP-Stx4-FL, or Stx4-N-term were serum-starved for 24 hours and seeded onto Matrigel-coated membranes and allowed to invade for 24 ours. In C, E, and F, percentages of cells are shown from experiments in which at least 10 fields of view were counted per treatment. All data represent percent of control ± SD, from three or more biological replicates, with at least three technical replicates; asterisks denote values significantly different from controls (*, P < 0.05). IB, immunoblot.

Figure 4.

Invadopodium formation and gelatin degradation in MDA-MB-231 cells is MT1-MMP–dependent. A, Parental MDA-MB-231 cells and stable cells expressing Stx4-FL, Stx4-N-term, Stx4-1–15, and Stx4-15–29 were treated with either DMSO (control) or MAB3329 and NSC405020, plated onto gelatin for 4 hours, then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. B, Cells, as in A, were plated onto gelatin for 24 hours, then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. Invadopodia (A) and gelatin degradation (B) were quantified as described in “Materials and Methods”. Means ± SD are presented from three or more biological replicates, with at least three technical replicates; asterisks denoting values significantly different from controls (*, P < 0.05).

Figure 5.

Expression of MT1-MMP-T567E increases invadopodium formation and gelatin degradation in cells expressing Stx4-N-term or Stx4 1 to 15. A, Parental MDA-MB-231 cells and stable cells expressing Stx4-FL, Stx4-N-term, Stx4-1–15, and Stx4-15–29 were transfected with either MT1-MMP-WT-3xFLAG or MT1-MMP-T567E-3xFLAG for 20 hours, plated onto gelatin for 4 hours, then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. Cells, as in A, were plated onto gelatin for 4 hours (B), or 24 hours (C), then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. As described in “Materials and Methods”, invadopodia (B) and gelatin degradation (C) were quantified. All data are presented as percent of control ± SD from three or more biological replicates with at least three technical replicates. Asterisks denote values significantly different from control (*, P < 0.05).

Figure 6.

Expression of truncated Stx4 constructs reduces spontaneous breast cancer metastasis. A, Tumor growth of orthotopically implanted MDA-MB-231 stable cells expressing Stx4-FL, Stx4-N-term, and Stx4-Cyto. B, Kaplan–Meier curves plotting surrogate survival of mice injected with cells in A. N = 10 mice/group. C, Wet lung mass (g) for individual mice for the indicated groups. D, Histochemical (H&E) and IHC (vimentin) evaluation of whole lung sections. N = 5 lungs/group; bar = 2,000 μm. E, Quantification of metastatic burden for the indicated groups. Symbols indicate total burden for individual mice. Asterisks denote significant differences. (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Figure 7.

Expression of truncated Stx4 constructs reduces invadopodia formation, gelatin degradation, and breast cancer metastasis. A, 4T1^luc cells stably expressing GFP (control), Stx4-FL, Stx4 Cyto, or Stx4–N-term were plated onto fluorescent gelatin for 4 hours (A) or 24 hours (B), then fixed and permeabilized. As described in “Materials and Methods”, invadopodia (A) and gelatin degradation (B) were quantified. Percentages of cells forming invadopodia, normalized to GFP, were determined by counting 50 cells/sample. All data are presented as percent of control ± SD from three or more biological replicates with at least three technical replicates. Asterisks denote values significantly different from control (*, P < 0.05). C, BALB/c mice were inoculated intravenously via the lateral tail vein with 5 × 10⁵ 4T1-luciferase cells stably expressing the indicated Stx4 construct, and lung metastases were imaged 3 weeks postinjection via IVIS. D, Bioluminescent emission from the thoracic region was calculated and quantified. Data are shown for individual mice. Statistical significance was calculated via one-way ANOVA, (**, P < 0.01).