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MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells Xiuwen Yan a, 1, Nengqi Cao b, 1, Yeh Chen c, 1, Hsin-Yi Lan d, Jong-Ho Cha e, Wen-Hao Yang a, f, *, Muh-Hwa Yang d, g, h, ** a
Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 910095, Guangdong, China Department of Surgery, Nanjing Lishui People’s Hospital, Nanjing, 211200, Jiangsu, China Institute of New Drug Development and Center for Tumor Medical Science, China Medical University, Taichung, 404, Taiwan d Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan e Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, 22212, South Korea f Graduate Institute of Biomedical Sciences and Centers for Molecular Medicine and Tumor Medical Science, China Medical University, Taichung, 40402, Taiwan g Cancer Progression Research Center, National Yang-Ming University, Taipei, 11221, Taiwan h Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, 11217, Taiwan b c
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Article history: Received 18 November 2019 Accepted 2 December 2019 Available online xxx
Hypoxia-inducible factor-1a (HIF-1a) induces cancer metastasis. We previously demonstrated that HIF1a-induced membrane-type 4 matrix metalloproteinase (MT4-MMP) is involved in hypoxia-mediated metastasis in head and neck squamous cell carcinoma (HNSCC). However, the functions and detailed mechanisms of MT4-MMP in cancer metastasis are not well understood. In this study, we investigated whether MT4-MMP regulates invadopodia formation or individual cell movementdboth critical to cancer migration and invasiondin three-dimensional (3D) environments. By expressing MT4-MMP in the HNSCC cell line FaDu, we demonstrated that MT4-MMP increases invadopodia formation and gelatin degradation. Furthermore, the amoeboid-like cell movement on collagen gel was increased by MT4-MMP expression in FaDu cells. Mechanistically, MT4-MMP may induce invadopodia formation by binding with Tks5 and PDGFRa to result in Src activation and promote amoeboid-like movement by stimulating the small GTPases Rho and Cdc42. Altogether, our data indicate that MT4-MMP induces two crucial mechanisms of cancer dissemination, invadopodia formation and amoeboid movement, and elucidate the prometastatic role of MT4-MMP in hypoxia-mediated cancer metastasis. © 2019 Elsevier Inc. All rights reserved.
Keywords: MT4-MMP Invadopodia Cell motility Amoeboid movement FaDu HNSCC
1. Introduction Matrix metalloproteinases (MMPs) are classified as a family of zinc-binding endopeptidases that degrade extracellular matrix (ECM) components and reshape the pericellular microenvironment, playing a crucial role in cancer metastasis [1,2]. This protease
Abbreviations: HIF-1a, hypoxia-inducible factor-1a; HNSCC, head and neck squamous cell carcinoma; MMP, matrix metalloproteinases; 3D, three-dimensional. * Corresponding author. Graduate Institute of Biomedical Sciences, China Medical University, No. 91, Hsueh-Shih Rd., North District, Taichung, 40402, Taiwan. ** Corresponding author. Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan. E-mail addresses: [email protected] (W.-H. Yang), [email protected] (M.-H. Yang). 1 These authors contributed equally to this study.
family includes both secreted and membrane-type MMPs (MTMMPs) which have different functions in mediating cancer cell invasiveness [3]. Membrane-type 4 MMP (MT4-MMP, also known as MMP17) is a glycosylphosphatidylinositol (GPI)-anchored MTMMP that is functionally and structurally distinct from other MTMMPs [4,5]. Studies have demonstrated that MT4-MMP is expressed in several types of human cancer, including head and neck squamous cell carcinoma (HNSCC), gastric cancer, colon cancer, and breast cancer, and promotes metastatic dissemination [6]. In comparison with other MT-MMPs, the mechanisms and functions of MT4-MMP in cancer are less understood. We previously demonstrated the role and underlying mechanism of MT4-MMP in HNSCC metastasis. Hypoxia-inducible factor-1a (HIF-1a)-induced MT4-MMP expression is critical in hypoxia-mediated cancer metastasis and a crucial prognostic indicator of HNSCC [7]. Further
https://doi.org/10.1016/j.bbrc.2019.12.009 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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in-depth investigation of MT4-MMP-mediated cancer progression may assist in development of novel therapeutic strategies for cancer. Invadopodia are actin-based membrane protrusions that degrade ECM through the localization of proteases, suggesting their critical role in tumor invasion in cancer cells [8]. As actin-based structures, the clustering and enzymatic components of invadopodia contain mainly a branched F-actin core and SH3-domain-rich adaptor proteins Tks4 and Tks5 [9e11]. Numerous proteases, including MT1-MMP, have been associated with matrix degradation by invadopodia [12,13]. Tyrosine phosphorylation of several invadopodia components including cortactin and Tks5 by Src kinases are essential for invadopodia formation [9,11]. In addition, Src activation and phosphorylation at Y416 by PDGFRa accelerate invadopodia formation [14,15]. Although the importance of invadopodia in cancer invasion and metastasis is known, whether other MT-MMPs are involved in the formation of invadopodia and the details of the interplay between invadopodia and other major metastasis mechanisms, such as individual cell movements, are unclear. Epithelial cells migrate through collective cell movement or individual cell movements, mesenchymal- or amoeboid-mode movement [16,17]. The mesenchymal mode is characterized by cells assuming an elongated shape with protruding pseudopodia that degrade ECM. By contrast, the amoeboid mode is characterized by a rounded morphology with membranous blebs as well as migration without ECM proteolysis [18]. The migration of individual cells can be clearly visualized in three-dimensional (3D) matrices [19]. Small GTPases, including Rac, Rho, and Cdc42, play
critical roles in controlling individual cell movements. Rac activated by GTP binding (GTP-Rac) promotes mesenchymal movement; by contrast, activated GTP-bound Rho (GTP-Rho) and Cdc42 (GTPCdc42) augment amoeboid movement through Rho-associated kinase (ROCK) and MLC2 phosphorylation [20e22]. Although many studies highlight the importance of individual cell movements in cancer metastasis, the mechanistic links between individual cell movement and other major mechanisms during the metastatic process remain largely unknown. This study reveals that MT4-MMP expression induces not only the formation of invadopodia but also amoeboid movement, implying that cancer cells have amoeboid movement with invadopodia and invasive capacity. 2. Materials and methods 2.1. Cell culture and plasmids The HNSCC cell lines (FaDu, OECM-1, CAL-27 and SAS) and HEK293T were collected from the Bioresource Collection and Research Center of Taiwan. All cell lines used in this research were routinely checked by STR DNA fingerprinting at Yang-Ming University and tested for mycoplasma contamination. OECM-1 and FaDu were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), and HEK-293T, SAS and CAL-27 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS. The pCDH-MT4-MMP expression plasmid was generated by inserting the full-length cDNA of MT4MMP (NM_016155) into the EcoRI/BamHI sites of pCDH-CMVMCS-EF1-puro vector. The plasmid expressing HA-tagged MT-
Fig. 1. Membrane-type 4 matrix metalloproteinase (MT4-MMP) expression increases invadopodia number and gelatin degradation in FaDu cells. (A) The expression level of MT4-MMP was tested in four head and neck cancer cell lines through immunoblotting. b-Actin served as a loading control. (B) Top: immunofluorescence staining of F-actin (red), cortactin (green), nuclei (blue), and merged image. Arrows indicate invadopodia locations. Scale bar ¼ 10 mm. Bottom: quantification of invadopodia in FaDu empty vector (EV) versus FaDu-MT4-MMP (n ¼ 6). Data are presented as mean ± standard deviation (SD). *P < 0.05 in Student t-test. (C) Gelatin degradation assays of FaDu-EV and FaDu-MT4-MMP. Top: representative images. Oregon Green 488 conjugate gelatin (green), F-actin (red), nuclei (blue), and merged image. Scale bar ¼ 10 mm. Bottom: quantification of gelatindegraded area (n ¼ 10). Data are presented as mean ± SD. *P < 0.05 in Student t-test. (D) Vertical axis (Z-axis) illustrates invadopodia in FaDu-EV versus those in FaDu-MT4MMP. Arrows indicate protrusions into gelatin. Scale bar ¼ 5 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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(1:10,000; Sigma-Aldrich #A2228) antibodies. Chemiluminescent detection reagents (Bio-Rad #170e5061 or ThermoFisher #34075) and ImageQuant LAS 4010 (GE Healthcare) were applied for detecting the signals of immunoblotting. ImageJ software (NIH) was used for analyzing the densities of the bands. 2.4. Immunofluorescence and image reconstruction by confocal microscopy For immunofluorescent staining, cells were dispensed and allowed to attach on coverslips coated with 0.2% gelatin (ThermoFisher #13186) for 16 h. After incubation, the cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 for 15 min. After blocking, cells were stained with anti-cortactin (Merck #05e180) or anti-MT4-MMP (Abcam #ab51075) antibodies overnight at 4 C and then with secondary antibodies and/or rhodamine coupled to phalloidin (ThermoFisher #R415) at RT for 2 h, followed by a nuclear staining with DAPI. Images were collected by laser confocal microscopy (Olympus FV1000, Olympus Corporation). For 3D images of the invasive cells, sequential Z sections were captured and reconstructed through Olympus FV10ASW 1.7 software. Fig. 2. MT4-MMP activates Src signaling and binds with PDGFRa and Tks5. (A) Western blot analysis of 293T cells transfected with pcDNA3.1 (empty vector, EV) or HA-tagged MT4-MMP (HA-MT4-MMP). (B) Immunoprecipitation with anti-HA antibody and Western blot analysis reveal the interaction between HA-MT4-MMP and other proteins (PDGFRa and Tks5) in HA-MT4-MMP-expressing 293T cells. Immunoblotting was analyzed using anti-HA, anti-PDGFRa, and anti-Tks5 antibodies. Immunoglobulin G (IgG) was the control for immunoprecipitation. (C) Western blot of MT4MMP and total and Y416-phsopholated Src (pY416-Src) in FaDu-EV versus FaDu-MT4MMP. b-Actin was used as a loading control. (D) Relative expression level of pY416-Src normalized by total Src in panel C.
4MMP (pcDNA3-HA-MT4-MMP) was constructed by inserting the sequence of HA-tagged MT4-MMP in pcDNA3.1 by HindIII and BamHI restriction enzyme digestion. 2.2. Generation of MT4-MMP stable cells Using the pCDH-MT4-MMP expression construct, we generated FaDu stable cell line expressing MT4-MMP (FaDu-MT4-MMP). To package lentivirus, the pCMVDR8.7, pDVsVg and pCDH-MT4-MMP were co-transfected into HEK-293T cells. The medium was replaced at 24 h after transfection. After 48 h, packaged viruses were harvested through centrifugation and filtered with a 0.45-mm filter. FaDu cells were cultured in lentivirus-containing medium with 10 mg/ml polybrene (Sigma-Aldrich Corporation) for 48 h. After virus infection, 1 mg/ml puromycin (InvivoGen) were used for selection. 2.3. Western blotting Cell lysates were collected by the lysis buffer (1.25 M urea and 2.5% SDS) after PBS wash. After centrifugation, Pierce BCA Protein Assay (ThermoFisher #PI-23227) was used for determining the protein concentration. The primary antibodies applied in this study were anti-MT4-MMP (1:1000; Abcam #ab51075), anti-HA-tag (1:3000; Cell Signaling Technology #3724S), anti-PDGFRa (1:1000; Cell Signaling Technology #3174), anti-Tks5 (1:1000; LifeSpan # LS-C169066), anti-Src (1:1000; Cell Signaling Technology #2110), anti-Src (1:1000; Cell Signaling Technology #2110), anti-pY416-Src (1:1000; Cell Signaling Technology #6943), antiMLC2 (1:1000; Cell Signaling Technology #3672), anti-p-MLC2 (1:1000; Cell Signaling Technology #3674) and anti-b-actin
2.5. Gelatin degradation assay The detail of these experiments was described previously [15,23]. Briefly, 12 mm coverslips were treated with 20% nitric acid for 2 h, following incubation with 50 mg/ml poly-L-lysine in PBS for 15 min. After PBS washes, coverslips were treated with 0.15% gluteraldehyde for 10 min. Following removal of the glutaraldehyde, coverslips were coated with a mixture at 1:9 ratio of 0.1% Oregon Green™ 488 Conjugated-gelatin (ThermoFisher #13186): 0.2% porcine gelatin for 10 min. After the preparation of gelatin-coated coverslips, cells were adhered on coverslips for 10 h, and subjected to immunofluorescence. Ten fields per sample were collected for quantification of gelatin degradation. The percentage of degraded area normalized by cell number in each field was determined by ImageJ software. 2.6. Co-immunoprecipitation assay Cells were collected and lysed by the lysis buffer (20 mM TrisHCl, pH 7.5, 1 mM EGTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM Na2EDTA, 1% Triton, 1 mM betaglycerophosphate, 1 mg/ml leupeptin, 1 mM Na3VO4) including one-fold protease inhibitor cocktail (Roche # 4693116001). Cell lysates (500 mg) were mixed with anti-HA magnetic beads (ThermoFisher #88837) at 4 C for 6 h. The magnetic beads bound with target proteins were washed with the lysis buffer three times and eluted with Blue Loading Buffer (Cell Signaling Technology # 7722S). 2.7. Time-lapse microscopy and quantification of the speed of cell motility The analysis of cell motility on collagen gel were carried out as described [24]. Briefly, the cells attached on collagen gel for 16 h, then observed in a humidified, CO2-equilibrated chamber with a Leica DM IRBE microscope (Leica Microsystems Inc) equipped with a motorized stage. MetaMorph® software (Molecular Devices, Inc., CA) was used to track the moving pathways of individual cells for 24 h in a random field. The cell speed was measured and displayed as microns per minute.
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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Fig. 3. MT4-MMP expression in FaDu cells augments the motility through amoeboid-like movement. (A) Confocal microscopy demonstrates the cellular morphology and actin organization of FaDu-EV versus FaDu-MT4-MMP on collagen gel. Green, F-actin; blue, nuclei. Scale bar ¼ 10 mm. Top: the images of single section. Bottom: the images of 3D reconstruction. Arrows indicate membrane blebs and protrusions. (B) Time-lapse microscopy tracking cell movement in FaDu-EV versus FaDu-MT4-MMP. Top: representative trajectories. Bottom: quantification of motility (n ¼ 10). Data represent sample maximum (upper end of whisker), upper quartile (top of box), median (band in the box), lower quartile (bottom of box), and sample minimum (lower end of whisker). *P < 0.01 in Student t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2.8. Small GTPase pull-down assays The active Rho/Cdc42 pull-down and detection kits (ThermoFisher) were applied to detect the active forms of Rho and Cdc42. The assays were performed according to the manufacturer’s protocol. Briefly, Glutathione S-transferase (GST)-conjugated human Rhotekin-Rho-binding domain (RBD) or Pak1-protein-binding domain (PBD) were added to cell lysates to pull down the active form of Rho or Cdc42, respectively. The active forms of Rho/Cdc42 were collected and followed by Western blot analysis. The levels of total Rho/Cdc42 were used as the controls. 2.9. Statistical analysis Graphs show the percentage and number compared to control groups with a standard deviation of three independent experiments. Statistical significance was analyzed by SPSS (Ver. 20, SPSS). The two-tailed, unpaired Student t-test was used to compare continuous variables between two groups. All experiments were performed at least twice. Results with p-values less than 0.05 were considered statistically significant. 3. Results 3.1. MT4-MMP promotes invasive ability of FaDu cells through the formation of invadopodia We previously demonstrated that MT4-MMP mediates hypoxiainduced local metastasis of HNSCC. Notably, MT4-MMP promotes cell invasiveness but does not affect cell migration in vitro, indicating its complicated role in cancer metastasis [7]. To understand the function of MT4-MMP in cancer metastasis, we investigated the effects of MT4-MMP on invadopodia formation and cell motility in a 3D environment. First, we examined the protein levels of MT4MMP in four HNSCC cell lines: CAL-27, SAS, FaDu, and OECM-1. FaDu cells exhibited the lowest MT4-MMP expression among the four cell lines (Fig. 1A). To test whether MT4-MMP is involved in invadopodia formation, FaDu cells were used for ectopic expression of MT4-MMP and subjected to immunofluorescence assays to detect colocalization of F-actin with the actin-bundling protein cortactin; this colocalization is used to detect invadopodia [25]. The result indicated that Invadopodia incidence is higher in FaDu cells expressing MT4-MMP (FaDu-MT4-MMP) than in FaDu control cells
(FaDu-EV) (Fig. 1B). Consistently, we also found that MT4-MMP promotes invadopodia-mediated gelatin degradation and is localized at invasive protrusions in FaDu cells (Fig. 1C and D). These results suggest that in FaDu cells, MT4-MMP induces invadopodia formation and focal matrix degradation.
3.2. MT4-MMP interacts with PDGFRa and Tks5 and increases phosphorylation of Src at Y416 To determine how MT4-MMP is involved in invadopodia formation, we tested whether MT4-MMP can bind with invadopodiarelated proteins to activate the major signaling pathway for invadopodia formation. To this end, HA-tagged MT4-MMP was expressed in 293T cells (Fig. 2A), then subjected to immunoprecipitation assays. Notably, PDGFRa and Tks5 could be pulled down with HA-tagged MT4-MMP by an anti-HA antibody in 293T cells (Fig. 2B). In addition, quantification data from Western blotting indicated that ectopic expression of MT4-MMP in FaDu cells increased Src phosphorylation at Y416 approximately twofold, resulting in Src activation (Fig. 2C and D). Because PDGFRa induces Src phosphorylation, which is required for invadopodia formation, and Tks5 is a critical component of invadopodia [15], MT4-MMP likely accelerates invadopodia formation through PDGFRa-mediated Src activation and is a functional component of invadopodia.
3.3. MT4-MMP increases cell motility through amoeboid-like movement Because MT4-MMP expression in HNSCC cells promotes tumor metastasis without altering cell migration in two-dimensional (2D) culture systems, we tested whether MT4-MMP contributes to tumor metastasis by influencing individual cell movement in 3D environments. For this purpose, we seeded FaDu-MT4-MMP and FaDu-EV cells on collagen gel to investigate whether MT4-MMP affects individual cell movement in 3D environments. Through confocal and time-lapse microscopy, we discovered that ectopic MT4-MMP in FaDu cells induced a rounded morphology with membrane blebs and protrusions (Fig. 3A), and increased the cellular speed on the collagen gel (Fig. 3B). Collectively, these data suggest that MT4-MMP promotes an amoeboid-like movement in FaDu cells.
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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Fig. 4. Small GTPases Rho and Cdc42, which induce amoeboid movement, are activated in MT4-MMP-expressing FaDu cells. (A) Western blot of MT4-MMP, Rho, and Cdc42 in FaDu-EV versus FaDu-MT4-MMP. Samples for detecting GTP-Rho and GTP-Cdc42 were obtained from small GTPase pull-down assays. b-Actin served as a loading control. (B) Relative levels of GTP-Rho and GTP-Cdc42 were normalized by total Rho or Cdc42 in panel A. (C) Western blot of total and phosphorylated MLC2 in FaDu-EV versus FaDu-MT4-MMP. (D) Relative level of phosphorylated MLC2 normalized by total MLC2 in panel C. (E) Proposed model of MT4-MMP-induced cancer metastasis in HNSCC. MT4-MMP may induce amoeboid movement and invadopodia formation by activating small GTPases Rho and Cdc42 and binding with PDGFRa and Tks5 in HNSCC cells.
3.4. MT4-MMP expression activates Rho and Cdc42 and induces MLC2 phosphorylation in FaDu cells Because the activation of small GTPases Rho and Cdc42 induces amoeboid movement [22], we examined whether MT4-MMP can alter Rho or Cdc42 activity. The quantitative data from small GTPase pull-down and Western blot assays revealed that ectopic MT4MMP increased GTP-bound Rho and Cdc42 (Fig. 4A and B) and upregulated the phosphorylation of MLC2 (Fig. 4C and D), a substrate phosphorylated by ROCK when Rho is activated [26]. These data imply that in HNSCC, MT4-MMP induces amoeboid movement in 3D environments possibly through activating the small GTPases Rho and Cdc42. Therefore, MT4-MMP participates in HNSCC local invasion through coordinating invadopodia formation and amoeboid movement (Fig. 4E). 4. Discussion Our results reveal a possible mechanism underlying the crosstalk of invadopodia and amoeboid movement, which is induced by MT4-MMP, contributing to local invasion and tumor metastasis in HNSCC. Although the detailed molecular mechanisms remain unclear and these biological characteristics should be further explored in more different cancer cells, the current study suggests that MT4MMP in HNSCC triggers protrusion-dependent amoeboid movement and that hypoxia/HIF-1a-induced MT4-MMP is a mediator responsible for invadopodia formation and the motility of individual cells.
Rho and Cdc42 promote MT1-MMP delivery to invadopodia, thus increasing local matrix degradation [27]. We demonstrated that MT4-MMP expression in FaDu cells not only activates Rho and Cdc42 (Fig. 4A), but also participates in invadopodia formation (Fig. 1), implying an interplay between these two membranebound MMPs in invadopodia regulation. Notably, MT4-MMP also induced amoeboid-like migration of FaDu cells in 3D environments (Fig. 3). However, studies have demonstrated that amoeboid movement of invasive cells is independent of pericellular matrix proteolysis [21,22]. Therefore, the regulation of invadopodia and single cell movement by MT4-MMP in FaDu cells indicates the possibility of amoeboid movement with the ability to degrade ECM. Hypoxic conditions promote tumor metastasis by the transcription factor HIF-1a in many cancer types. Furthermore, HIF-1aupregulated Slug, a key regulator of epithelial-mesenchymal transition (EMT), promotes MT4-MMP expression in HNSCC [7]. Here, we observed that MT4-MMP mediates invadopodia formation, highlighting its prometastatic role in hypoxic tumor microenvironments. Therefore, deciphering the network in which MT4-MMP participates in HNSCC is crucial for further understanding the biology underlying HNSCC progression and for future development of anti-invasion therapy in HNSCC. Author contributions X.Y., N.C. and Y.C. designed and carried out the experiments, and interpreted data; H.-Y.L. carried out the immunofluorescence and gelatin degradation assays; and J.-H.C. provided scientific ideas and
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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material; W.-H.Y. and M.-H.Y. supervised the entire project and prepared the manuscript.
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Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgments This research was supported by Guangzhou key medical discipline construction project fund, National Natural Science Foundation of China (81872138) and YingTsai Young Scholar Award (CMU108-YTY-04). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.009.
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MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells Xiuwen Yan a, 1, Nengqi Cao b, 1, Yeh Chen c, 1, Hsin-Yi Lan d, Jong-Ho Cha e, Wen-Hao Yang a, f, *, Muh-Hwa Yang d, g, h, ** a
Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou, 910095, Guangdong, China Department of Surgery, Nanjing Lishui People’s Hospital, Nanjing, 211200, Jiangsu, China Institute of New Drug Development and Center for Tumor Medical Science, China Medical University, Taichung, 404, Taiwan d Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan e Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, 22212, South Korea f Graduate Institute of Biomedical Sciences and Centers for Molecular Medicine and Tumor Medical Science, China Medical University, Taichung, 40402, Taiwan g Cancer Progression Research Center, National Yang-Ming University, Taipei, 11221, Taiwan h Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, 11217, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2019 Accepted 2 December 2019 Available online xxx
Hypoxia-inducible factor-1a (HIF-1a) induces cancer metastasis. We previously demonstrated that HIF1a-induced membrane-type 4 matrix metalloproteinase (MT4-MMP) is involved in hypoxia-mediated metastasis in head and neck squamous cell carcinoma (HNSCC). However, the functions and detailed mechanisms of MT4-MMP in cancer metastasis are not well understood. In this study, we investigated whether MT4-MMP regulates invadopodia formation or individual cell movementdboth critical to cancer migration and invasiondin three-dimensional (3D) environments. By expressing MT4-MMP in the HNSCC cell line FaDu, we demonstrated that MT4-MMP increases invadopodia formation and gelatin degradation. Furthermore, the amoeboid-like cell movement on collagen gel was increased by MT4-MMP expression in FaDu cells. Mechanistically, MT4-MMP may induce invadopodia formation by binding with Tks5 and PDGFRa to result in Src activation and promote amoeboid-like movement by stimulating the small GTPases Rho and Cdc42. Altogether, our data indicate that MT4-MMP induces two crucial mechanisms of cancer dissemination, invadopodia formation and amoeboid movement, and elucidate the prometastatic role of MT4-MMP in hypoxia-mediated cancer metastasis. © 2019 Elsevier Inc. All rights reserved.
Keywords: MT4-MMP Invadopodia Cell motility Amoeboid movement FaDu HNSCC
1. Introduction Matrix metalloproteinases (MMPs) are classified as a family of zinc-binding endopeptidases that degrade extracellular matrix (ECM) components and reshape the pericellular microenvironment, playing a crucial role in cancer metastasis [1,2]. This protease
Abbreviations: HIF-1a, hypoxia-inducible factor-1a; HNSCC, head and neck squamous cell carcinoma; MMP, matrix metalloproteinases; 3D, three-dimensional. * Corresponding author. Graduate Institute of Biomedical Sciences, China Medical University, No. 91, Hsueh-Shih Rd., North District, Taichung, 40402, Taiwan. ** Corresponding author. Institute of Clinical Medicine, National Yang-Ming University, Taipei, 11221, Taiwan. E-mail addresses: [email protected] (W.-H. Yang), [email protected] (M.-H. Yang). 1 These authors contributed equally to this study.
family includes both secreted and membrane-type MMPs (MTMMPs) which have different functions in mediating cancer cell invasiveness [3]. Membrane-type 4 MMP (MT4-MMP, also known as MMP17) is a glycosylphosphatidylinositol (GPI)-anchored MTMMP that is functionally and structurally distinct from other MTMMPs [4,5]. Studies have demonstrated that MT4-MMP is expressed in several types of human cancer, including head and neck squamous cell carcinoma (HNSCC), gastric cancer, colon cancer, and breast cancer, and promotes metastatic dissemination [6]. In comparison with other MT-MMPs, the mechanisms and functions of MT4-MMP in cancer are less understood. We previously demonstrated the role and underlying mechanism of MT4-MMP in HNSCC metastasis. Hypoxia-inducible factor-1a (HIF-1a)-induced MT4-MMP expression is critical in hypoxia-mediated cancer metastasis and a crucial prognostic indicator of HNSCC [7]. Further
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Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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in-depth investigation of MT4-MMP-mediated cancer progression may assist in development of novel therapeutic strategies for cancer. Invadopodia are actin-based membrane protrusions that degrade ECM through the localization of proteases, suggesting their critical role in tumor invasion in cancer cells [8]. As actin-based structures, the clustering and enzymatic components of invadopodia contain mainly a branched F-actin core and SH3-domain-rich adaptor proteins Tks4 and Tks5 [9e11]. Numerous proteases, including MT1-MMP, have been associated with matrix degradation by invadopodia [12,13]. Tyrosine phosphorylation of several invadopodia components including cortactin and Tks5 by Src kinases are essential for invadopodia formation [9,11]. In addition, Src activation and phosphorylation at Y416 by PDGFRa accelerate invadopodia formation [14,15]. Although the importance of invadopodia in cancer invasion and metastasis is known, whether other MT-MMPs are involved in the formation of invadopodia and the details of the interplay between invadopodia and other major metastasis mechanisms, such as individual cell movements, are unclear. Epithelial cells migrate through collective cell movement or individual cell movements, mesenchymal- or amoeboid-mode movement [16,17]. The mesenchymal mode is characterized by cells assuming an elongated shape with protruding pseudopodia that degrade ECM. By contrast, the amoeboid mode is characterized by a rounded morphology with membranous blebs as well as migration without ECM proteolysis [18]. The migration of individual cells can be clearly visualized in three-dimensional (3D) matrices [19]. Small GTPases, including Rac, Rho, and Cdc42, play
critical roles in controlling individual cell movements. Rac activated by GTP binding (GTP-Rac) promotes mesenchymal movement; by contrast, activated GTP-bound Rho (GTP-Rho) and Cdc42 (GTPCdc42) augment amoeboid movement through Rho-associated kinase (ROCK) and MLC2 phosphorylation [20e22]. Although many studies highlight the importance of individual cell movements in cancer metastasis, the mechanistic links between individual cell movement and other major mechanisms during the metastatic process remain largely unknown. This study reveals that MT4-MMP expression induces not only the formation of invadopodia but also amoeboid movement, implying that cancer cells have amoeboid movement with invadopodia and invasive capacity. 2. Materials and methods 2.1. Cell culture and plasmids The HNSCC cell lines (FaDu, OECM-1, CAL-27 and SAS) and HEK293T were collected from the Bioresource Collection and Research Center of Taiwan. All cell lines used in this research were routinely checked by STR DNA fingerprinting at Yang-Ming University and tested for mycoplasma contamination. OECM-1 and FaDu were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% heat-inactivated fetal bovine serum (FBS), and HEK-293T, SAS and CAL-27 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS. The pCDH-MT4-MMP expression plasmid was generated by inserting the full-length cDNA of MT4MMP (NM_016155) into the EcoRI/BamHI sites of pCDH-CMVMCS-EF1-puro vector. The plasmid expressing HA-tagged MT-
Fig. 1. Membrane-type 4 matrix metalloproteinase (MT4-MMP) expression increases invadopodia number and gelatin degradation in FaDu cells. (A) The expression level of MT4-MMP was tested in four head and neck cancer cell lines through immunoblotting. b-Actin served as a loading control. (B) Top: immunofluorescence staining of F-actin (red), cortactin (green), nuclei (blue), and merged image. Arrows indicate invadopodia locations. Scale bar ¼ 10 mm. Bottom: quantification of invadopodia in FaDu empty vector (EV) versus FaDu-MT4-MMP (n ¼ 6). Data are presented as mean ± standard deviation (SD). *P < 0.05 in Student t-test. (C) Gelatin degradation assays of FaDu-EV and FaDu-MT4-MMP. Top: representative images. Oregon Green 488 conjugate gelatin (green), F-actin (red), nuclei (blue), and merged image. Scale bar ¼ 10 mm. Bottom: quantification of gelatindegraded area (n ¼ 10). Data are presented as mean ± SD. *P < 0.05 in Student t-test. (D) Vertical axis (Z-axis) illustrates invadopodia in FaDu-EV versus those in FaDu-MT4MMP. Arrows indicate protrusions into gelatin. Scale bar ¼ 5 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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(1:10,000; Sigma-Aldrich #A2228) antibodies. Chemiluminescent detection reagents (Bio-Rad #170e5061 or ThermoFisher #34075) and ImageQuant LAS 4010 (GE Healthcare) were applied for detecting the signals of immunoblotting. ImageJ software (NIH) was used for analyzing the densities of the bands. 2.4. Immunofluorescence and image reconstruction by confocal microscopy For immunofluorescent staining, cells were dispensed and allowed to attach on coverslips coated with 0.2% gelatin (ThermoFisher #13186) for 16 h. After incubation, the cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 for 15 min. After blocking, cells were stained with anti-cortactin (Merck #05e180) or anti-MT4-MMP (Abcam #ab51075) antibodies overnight at 4 C and then with secondary antibodies and/or rhodamine coupled to phalloidin (ThermoFisher #R415) at RT for 2 h, followed by a nuclear staining with DAPI. Images were collected by laser confocal microscopy (Olympus FV1000, Olympus Corporation). For 3D images of the invasive cells, sequential Z sections were captured and reconstructed through Olympus FV10ASW 1.7 software. Fig. 2. MT4-MMP activates Src signaling and binds with PDGFRa and Tks5. (A) Western blot analysis of 293T cells transfected with pcDNA3.1 (empty vector, EV) or HA-tagged MT4-MMP (HA-MT4-MMP). (B) Immunoprecipitation with anti-HA antibody and Western blot analysis reveal the interaction between HA-MT4-MMP and other proteins (PDGFRa and Tks5) in HA-MT4-MMP-expressing 293T cells. Immunoblotting was analyzed using anti-HA, anti-PDGFRa, and anti-Tks5 antibodies. Immunoglobulin G (IgG) was the control for immunoprecipitation. (C) Western blot of MT4MMP and total and Y416-phsopholated Src (pY416-Src) in FaDu-EV versus FaDu-MT4MMP. b-Actin was used as a loading control. (D) Relative expression level of pY416-Src normalized by total Src in panel C.
4MMP (pcDNA3-HA-MT4-MMP) was constructed by inserting the sequence of HA-tagged MT4-MMP in pcDNA3.1 by HindIII and BamHI restriction enzyme digestion. 2.2. Generation of MT4-MMP stable cells Using the pCDH-MT4-MMP expression construct, we generated FaDu stable cell line expressing MT4-MMP (FaDu-MT4-MMP). To package lentivirus, the pCMVDR8.7, pDVsVg and pCDH-MT4-MMP were co-transfected into HEK-293T cells. The medium was replaced at 24 h after transfection. After 48 h, packaged viruses were harvested through centrifugation and filtered with a 0.45-mm filter. FaDu cells were cultured in lentivirus-containing medium with 10 mg/ml polybrene (Sigma-Aldrich Corporation) for 48 h. After virus infection, 1 mg/ml puromycin (InvivoGen) were used for selection. 2.3. Western blotting Cell lysates were collected by the lysis buffer (1.25 M urea and 2.5% SDS) after PBS wash. After centrifugation, Pierce BCA Protein Assay (ThermoFisher #PI-23227) was used for determining the protein concentration. The primary antibodies applied in this study were anti-MT4-MMP (1:1000; Abcam #ab51075), anti-HA-tag (1:3000; Cell Signaling Technology #3724S), anti-PDGFRa (1:1000; Cell Signaling Technology #3174), anti-Tks5 (1:1000; LifeSpan # LS-C169066), anti-Src (1:1000; Cell Signaling Technology #2110), anti-Src (1:1000; Cell Signaling Technology #2110), anti-pY416-Src (1:1000; Cell Signaling Technology #6943), antiMLC2 (1:1000; Cell Signaling Technology #3672), anti-p-MLC2 (1:1000; Cell Signaling Technology #3674) and anti-b-actin
2.5. Gelatin degradation assay The detail of these experiments was described previously [15,23]. Briefly, 12 mm coverslips were treated with 20% nitric acid for 2 h, following incubation with 50 mg/ml poly-L-lysine in PBS for 15 min. After PBS washes, coverslips were treated with 0.15% gluteraldehyde for 10 min. Following removal of the glutaraldehyde, coverslips were coated with a mixture at 1:9 ratio of 0.1% Oregon Green™ 488 Conjugated-gelatin (ThermoFisher #13186): 0.2% porcine gelatin for 10 min. After the preparation of gelatin-coated coverslips, cells were adhered on coverslips for 10 h, and subjected to immunofluorescence. Ten fields per sample were collected for quantification of gelatin degradation. The percentage of degraded area normalized by cell number in each field was determined by ImageJ software. 2.6. Co-immunoprecipitation assay Cells were collected and lysed by the lysis buffer (20 mM TrisHCl, pH 7.5, 1 mM EGTA, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM Na2EDTA, 1% Triton, 1 mM betaglycerophosphate, 1 mg/ml leupeptin, 1 mM Na3VO4) including one-fold protease inhibitor cocktail (Roche # 4693116001). Cell lysates (500 mg) were mixed with anti-HA magnetic beads (ThermoFisher #88837) at 4 C for 6 h. The magnetic beads bound with target proteins were washed with the lysis buffer three times and eluted with Blue Loading Buffer (Cell Signaling Technology # 7722S). 2.7. Time-lapse microscopy and quantification of the speed of cell motility The analysis of cell motility on collagen gel were carried out as described [24]. Briefly, the cells attached on collagen gel for 16 h, then observed in a humidified, CO2-equilibrated chamber with a Leica DM IRBE microscope (Leica Microsystems Inc) equipped with a motorized stage. MetaMorph® software (Molecular Devices, Inc., CA) was used to track the moving pathways of individual cells for 24 h in a random field. The cell speed was measured and displayed as microns per minute.
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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Fig. 3. MT4-MMP expression in FaDu cells augments the motility through amoeboid-like movement. (A) Confocal microscopy demonstrates the cellular morphology and actin organization of FaDu-EV versus FaDu-MT4-MMP on collagen gel. Green, F-actin; blue, nuclei. Scale bar ¼ 10 mm. Top: the images of single section. Bottom: the images of 3D reconstruction. Arrows indicate membrane blebs and protrusions. (B) Time-lapse microscopy tracking cell movement in FaDu-EV versus FaDu-MT4-MMP. Top: representative trajectories. Bottom: quantification of motility (n ¼ 10). Data represent sample maximum (upper end of whisker), upper quartile (top of box), median (band in the box), lower quartile (bottom of box), and sample minimum (lower end of whisker). *P < 0.01 in Student t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2.8. Small GTPase pull-down assays The active Rho/Cdc42 pull-down and detection kits (ThermoFisher) were applied to detect the active forms of Rho and Cdc42. The assays were performed according to the manufacturer’s protocol. Briefly, Glutathione S-transferase (GST)-conjugated human Rhotekin-Rho-binding domain (RBD) or Pak1-protein-binding domain (PBD) were added to cell lysates to pull down the active form of Rho or Cdc42, respectively. The active forms of Rho/Cdc42 were collected and followed by Western blot analysis. The levels of total Rho/Cdc42 were used as the controls. 2.9. Statistical analysis Graphs show the percentage and number compared to control groups with a standard deviation of three independent experiments. Statistical significance was analyzed by SPSS (Ver. 20, SPSS). The two-tailed, unpaired Student t-test was used to compare continuous variables between two groups. All experiments were performed at least twice. Results with p-values less than 0.05 were considered statistically significant. 3. Results 3.1. MT4-MMP promotes invasive ability of FaDu cells through the formation of invadopodia We previously demonstrated that MT4-MMP mediates hypoxiainduced local metastasis of HNSCC. Notably, MT4-MMP promotes cell invasiveness but does not affect cell migration in vitro, indicating its complicated role in cancer metastasis [7]. To understand the function of MT4-MMP in cancer metastasis, we investigated the effects of MT4-MMP on invadopodia formation and cell motility in a 3D environment. First, we examined the protein levels of MT4MMP in four HNSCC cell lines: CAL-27, SAS, FaDu, and OECM-1. FaDu cells exhibited the lowest MT4-MMP expression among the four cell lines (Fig. 1A). To test whether MT4-MMP is involved in invadopodia formation, FaDu cells were used for ectopic expression of MT4-MMP and subjected to immunofluorescence assays to detect colocalization of F-actin with the actin-bundling protein cortactin; this colocalization is used to detect invadopodia [25]. The result indicated that Invadopodia incidence is higher in FaDu cells expressing MT4-MMP (FaDu-MT4-MMP) than in FaDu control cells
(FaDu-EV) (Fig. 1B). Consistently, we also found that MT4-MMP promotes invadopodia-mediated gelatin degradation and is localized at invasive protrusions in FaDu cells (Fig. 1C and D). These results suggest that in FaDu cells, MT4-MMP induces invadopodia formation and focal matrix degradation.
3.2. MT4-MMP interacts with PDGFRa and Tks5 and increases phosphorylation of Src at Y416 To determine how MT4-MMP is involved in invadopodia formation, we tested whether MT4-MMP can bind with invadopodiarelated proteins to activate the major signaling pathway for invadopodia formation. To this end, HA-tagged MT4-MMP was expressed in 293T cells (Fig. 2A), then subjected to immunoprecipitation assays. Notably, PDGFRa and Tks5 could be pulled down with HA-tagged MT4-MMP by an anti-HA antibody in 293T cells (Fig. 2B). In addition, quantification data from Western blotting indicated that ectopic expression of MT4-MMP in FaDu cells increased Src phosphorylation at Y416 approximately twofold, resulting in Src activation (Fig. 2C and D). Because PDGFRa induces Src phosphorylation, which is required for invadopodia formation, and Tks5 is a critical component of invadopodia [15], MT4-MMP likely accelerates invadopodia formation through PDGFRa-mediated Src activation and is a functional component of invadopodia.
3.3. MT4-MMP increases cell motility through amoeboid-like movement Because MT4-MMP expression in HNSCC cells promotes tumor metastasis without altering cell migration in two-dimensional (2D) culture systems, we tested whether MT4-MMP contributes to tumor metastasis by influencing individual cell movement in 3D environments. For this purpose, we seeded FaDu-MT4-MMP and FaDu-EV cells on collagen gel to investigate whether MT4-MMP affects individual cell movement in 3D environments. Through confocal and time-lapse microscopy, we discovered that ectopic MT4-MMP in FaDu cells induced a rounded morphology with membrane blebs and protrusions (Fig. 3A), and increased the cellular speed on the collagen gel (Fig. 3B). Collectively, these data suggest that MT4-MMP promotes an amoeboid-like movement in FaDu cells.
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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Fig. 4. Small GTPases Rho and Cdc42, which induce amoeboid movement, are activated in MT4-MMP-expressing FaDu cells. (A) Western blot of MT4-MMP, Rho, and Cdc42 in FaDu-EV versus FaDu-MT4-MMP. Samples for detecting GTP-Rho and GTP-Cdc42 were obtained from small GTPase pull-down assays. b-Actin served as a loading control. (B) Relative levels of GTP-Rho and GTP-Cdc42 were normalized by total Rho or Cdc42 in panel A. (C) Western blot of total and phosphorylated MLC2 in FaDu-EV versus FaDu-MT4-MMP. (D) Relative level of phosphorylated MLC2 normalized by total MLC2 in panel C. (E) Proposed model of MT4-MMP-induced cancer metastasis in HNSCC. MT4-MMP may induce amoeboid movement and invadopodia formation by activating small GTPases Rho and Cdc42 and binding with PDGFRa and Tks5 in HNSCC cells.
3.4. MT4-MMP expression activates Rho and Cdc42 and induces MLC2 phosphorylation in FaDu cells Because the activation of small GTPases Rho and Cdc42 induces amoeboid movement [22], we examined whether MT4-MMP can alter Rho or Cdc42 activity. The quantitative data from small GTPase pull-down and Western blot assays revealed that ectopic MT4MMP increased GTP-bound Rho and Cdc42 (Fig. 4A and B) and upregulated the phosphorylation of MLC2 (Fig. 4C and D), a substrate phosphorylated by ROCK when Rho is activated [26]. These data imply that in HNSCC, MT4-MMP induces amoeboid movement in 3D environments possibly through activating the small GTPases Rho and Cdc42. Therefore, MT4-MMP participates in HNSCC local invasion through coordinating invadopodia formation and amoeboid movement (Fig. 4E). 4. Discussion Our results reveal a possible mechanism underlying the crosstalk of invadopodia and amoeboid movement, which is induced by MT4-MMP, contributing to local invasion and tumor metastasis in HNSCC. Although the detailed molecular mechanisms remain unclear and these biological characteristics should be further explored in more different cancer cells, the current study suggests that MT4MMP in HNSCC triggers protrusion-dependent amoeboid movement and that hypoxia/HIF-1a-induced MT4-MMP is a mediator responsible for invadopodia formation and the motility of individual cells.
Rho and Cdc42 promote MT1-MMP delivery to invadopodia, thus increasing local matrix degradation [27]. We demonstrated that MT4-MMP expression in FaDu cells not only activates Rho and Cdc42 (Fig. 4A), but also participates in invadopodia formation (Fig. 1), implying an interplay between these two membranebound MMPs in invadopodia regulation. Notably, MT4-MMP also induced amoeboid-like migration of FaDu cells in 3D environments (Fig. 3). However, studies have demonstrated that amoeboid movement of invasive cells is independent of pericellular matrix proteolysis [21,22]. Therefore, the regulation of invadopodia and single cell movement by MT4-MMP in FaDu cells indicates the possibility of amoeboid movement with the ability to degrade ECM. Hypoxic conditions promote tumor metastasis by the transcription factor HIF-1a in many cancer types. Furthermore, HIF-1aupregulated Slug, a key regulator of epithelial-mesenchymal transition (EMT), promotes MT4-MMP expression in HNSCC [7]. Here, we observed that MT4-MMP mediates invadopodia formation, highlighting its prometastatic role in hypoxic tumor microenvironments. Therefore, deciphering the network in which MT4-MMP participates in HNSCC is crucial for further understanding the biology underlying HNSCC progression and for future development of anti-invasion therapy in HNSCC. Author contributions X.Y., N.C. and Y.C. designed and carried out the experiments, and interpreted data; H.-Y.L. carried out the immunofluorescence and gelatin degradation assays; and J.-H.C. provided scientific ideas and
Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009
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material; W.-H.Y. and M.-H.Y. supervised the entire project and prepared the manuscript.
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Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgments This research was supported by Guangzhou key medical discipline construction project fund, National Natural Science Foundation of China (81872138) and YingTsai Young Scholar Award (CMU108-YTY-04). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.009.
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Please cite this article as: X. Yan et al., MT4-MMP promotes invadopodia formation and cell motility in FaDu head and neck cancer cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.009