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Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.

Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S, Allen E, Chung D, Weiss SJ - J. Cell Biol. (2004)

Bottom Line: As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity.Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM.Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)-dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

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MT1-MMP drives cancer cell invasion and intravasation. (A and B) HT-1080 or SCC-1 cells were incubated alone with TIMP-1, TIMP-2, a 21-bp MT1-MMP siRNA, or a 21-bp control siRNA and cultured atop a rhodamine-labeled type I collagen gel (2.2 mg/ml) in 10% serum with 50 ng/ml HGF for 3 d. Tunneling behavior of HT-1080 and SCC-1 cells (A) was monitored by confocal laser microscopy (bottom, merged images of phalloidin/DAPI-labeled SCC-1 cells and the surrounding rhodamine-labeled collagen are shown). Invasive foci and hydroxyproline release for HT-1080 and SCC-1 cells were quantified as described in B. Where indicated, MT1-MMP siRNA-treated cells were transfected with a mouse MT1-MMP (mMT1-MMP) expression vector (MT1-siRNA/mMT1). (C) CAM invasion by fluorescent nanobead-labeled HT-1080 or SCC-1 cells incubated alone, electroporated with MT1-siRNA alone, or coelectroporated with MT1-siRNA and a mMT1-MMP expression vector. Tumor cell invasion was visualized by fluorescent microscopy of CAM cross sections after a 3-d incubation period. The CAM surface is marked by arrows. Percent invasion for control-siRNA–, MT1-siRNA–, and mMT1-MMP–transfected MT1-siRNA–treated HT-1080 cells was 24 ± 7%, 1 ± 1%, and 25 ± 3%, respectively, and for SCC-1 cells was 15.8 ± 1%, 1 ± 1%, and 16 ± 4%, respectively (mean ± 1 SD; n = 3). (D) Invasion of human dermal explants by HT-1080 cells after electroporation with a control siRNA or MT1-siRNA and cultured atop the CAM for 3 d. The percentage of invading cells and invasion depth for control siRNA- and MT1 siRNA-treated HT-1080 cells, respectively, were 16.9 ± 5.9% and 144.1 ± 47.6 μm and 2.9 ± 0.2% and 22.9 ± 10.8 μm. (E) Tumor cell intravasation/extravasation was detected as Alu-sequences by PCR on DNA extracted from the lower CAM for either HT-1080 or SCC-1 cells after a 3-d incubation period. Chick GAPDH (chGAPDH) serves as the loading control. Results are representative of four experiments performed. Bars, 100 μm.
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fig6: MT1-MMP drives cancer cell invasion and intravasation. (A and B) HT-1080 or SCC-1 cells were incubated alone with TIMP-1, TIMP-2, a 21-bp MT1-MMP siRNA, or a 21-bp control siRNA and cultured atop a rhodamine-labeled type I collagen gel (2.2 mg/ml) in 10% serum with 50 ng/ml HGF for 3 d. Tunneling behavior of HT-1080 and SCC-1 cells (A) was monitored by confocal laser microscopy (bottom, merged images of phalloidin/DAPI-labeled SCC-1 cells and the surrounding rhodamine-labeled collagen are shown). Invasive foci and hydroxyproline release for HT-1080 and SCC-1 cells were quantified as described in B. Where indicated, MT1-MMP siRNA-treated cells were transfected with a mouse MT1-MMP (mMT1-MMP) expression vector (MT1-siRNA/mMT1). (C) CAM invasion by fluorescent nanobead-labeled HT-1080 or SCC-1 cells incubated alone, electroporated with MT1-siRNA alone, or coelectroporated with MT1-siRNA and a mMT1-MMP expression vector. Tumor cell invasion was visualized by fluorescent microscopy of CAM cross sections after a 3-d incubation period. The CAM surface is marked by arrows. Percent invasion for control-siRNA–, MT1-siRNA–, and mMT1-MMP–transfected MT1-siRNA–treated HT-1080 cells was 24 ± 7%, 1 ± 1%, and 25 ± 3%, respectively, and for SCC-1 cells was 15.8 ± 1%, 1 ± 1%, and 16 ± 4%, respectively (mean ± 1 SD; n = 3). (D) Invasion of human dermal explants by HT-1080 cells after electroporation with a control siRNA or MT1-siRNA and cultured atop the CAM for 3 d. The percentage of invading cells and invasion depth for control siRNA- and MT1 siRNA-treated HT-1080 cells, respectively, were 16.9 ± 5.9% and 144.1 ± 47.6 μm and 2.9 ± 0.2% and 22.9 ± 10.8 μm. (E) Tumor cell intravasation/extravasation was detected as Alu-sequences by PCR on DNA extracted from the lower CAM for either HT-1080 or SCC-1 cells after a 3-d incubation period. Chick GAPDH (chGAPDH) serves as the loading control. Results are representative of four experiments performed. Bars, 100 μm.

Mentions: To determine if a required role for MT1-MMP in fibroblast invasion extends to tumor cells, collagen-invasive activity of the MT1-MMP siRNA-treated cancer cells was assessed in vitro and in vivo. As shown in Fig. 6, type I collagen invasion and the associated degradation of the surrounding matrix by HT-1080 or SCC-1, although unaffected by the control siRNA, are suppressed almost completely by the MT1-MMP siRNA (Fig. 6, A and B). Validated siRNA-mediated targeting of either tumor cell MMP-1, MMP-2, or MMP-9 did not affect collagen-invasive activity (unpublished data). A role for MT1-MMP in tumor cell invasion was not limited to HT-1080 or SCC-1 cells, as similar results are obtained when MT1-MMP expression is silenced in MDA-MB-231, PANC-1, or DU-145 cells where the number of invasive foci is reduced by more than 75% (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408028/DC1). MT1-MMP siRNA-treated tumor cells retain full ability to invade either pepsin-extracted collagen gels devoid of intermolecular cross-links or fibrin matrices (Fig. S3). Similarly, (though in contrast with other recent reports; Ueda et al., 2003; Iida et al., 2004) invasion through Matrigel, an extract of noncross-linked ECM macromolecules (Kleinman et al., 1982), is unaffected in MT1-MMP–targeted tumor cells (Fig. S3). However, invasion through cross-linked collagen matrices and the associated collagenolytic activity are restored fully by transfecting the MT1-MMP siRNA-treated tumor cells with the mouse MT1-MMP construct (Fig. 6, A and B).


Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.

Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, Lopez-Otin C, Shapiro S, Inada M, Krane S, Allen E, Chung D, Weiss SJ - J. Cell Biol. (2004)

MT1-MMP drives cancer cell invasion and intravasation. (A and B) HT-1080 or SCC-1 cells were incubated alone with TIMP-1, TIMP-2, a 21-bp MT1-MMP siRNA, or a 21-bp control siRNA and cultured atop a rhodamine-labeled type I collagen gel (2.2 mg/ml) in 10% serum with 50 ng/ml HGF for 3 d. Tunneling behavior of HT-1080 and SCC-1 cells (A) was monitored by confocal laser microscopy (bottom, merged images of phalloidin/DAPI-labeled SCC-1 cells and the surrounding rhodamine-labeled collagen are shown). Invasive foci and hydroxyproline release for HT-1080 and SCC-1 cells were quantified as described in B. Where indicated, MT1-MMP siRNA-treated cells were transfected with a mouse MT1-MMP (mMT1-MMP) expression vector (MT1-siRNA/mMT1). (C) CAM invasion by fluorescent nanobead-labeled HT-1080 or SCC-1 cells incubated alone, electroporated with MT1-siRNA alone, or coelectroporated with MT1-siRNA and a mMT1-MMP expression vector. Tumor cell invasion was visualized by fluorescent microscopy of CAM cross sections after a 3-d incubation period. The CAM surface is marked by arrows. Percent invasion for control-siRNA–, MT1-siRNA–, and mMT1-MMP–transfected MT1-siRNA–treated HT-1080 cells was 24 ± 7%, 1 ± 1%, and 25 ± 3%, respectively, and for SCC-1 cells was 15.8 ± 1%, 1 ± 1%, and 16 ± 4%, respectively (mean ± 1 SD; n = 3). (D) Invasion of human dermal explants by HT-1080 cells after electroporation with a control siRNA or MT1-siRNA and cultured atop the CAM for 3 d. The percentage of invading cells and invasion depth for control siRNA- and MT1 siRNA-treated HT-1080 cells, respectively, were 16.9 ± 5.9% and 144.1 ± 47.6 μm and 2.9 ± 0.2% and 22.9 ± 10.8 μm. (E) Tumor cell intravasation/extravasation was detected as Alu-sequences by PCR on DNA extracted from the lower CAM for either HT-1080 or SCC-1 cells after a 3-d incubation period. Chick GAPDH (chGAPDH) serves as the loading control. Results are representative of four experiments performed. Bars, 100 μm.
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fig6: MT1-MMP drives cancer cell invasion and intravasation. (A and B) HT-1080 or SCC-1 cells were incubated alone with TIMP-1, TIMP-2, a 21-bp MT1-MMP siRNA, or a 21-bp control siRNA and cultured atop a rhodamine-labeled type I collagen gel (2.2 mg/ml) in 10% serum with 50 ng/ml HGF for 3 d. Tunneling behavior of HT-1080 and SCC-1 cells (A) was monitored by confocal laser microscopy (bottom, merged images of phalloidin/DAPI-labeled SCC-1 cells and the surrounding rhodamine-labeled collagen are shown). Invasive foci and hydroxyproline release for HT-1080 and SCC-1 cells were quantified as described in B. Where indicated, MT1-MMP siRNA-treated cells were transfected with a mouse MT1-MMP (mMT1-MMP) expression vector (MT1-siRNA/mMT1). (C) CAM invasion by fluorescent nanobead-labeled HT-1080 or SCC-1 cells incubated alone, electroporated with MT1-siRNA alone, or coelectroporated with MT1-siRNA and a mMT1-MMP expression vector. Tumor cell invasion was visualized by fluorescent microscopy of CAM cross sections after a 3-d incubation period. The CAM surface is marked by arrows. Percent invasion for control-siRNA–, MT1-siRNA–, and mMT1-MMP–transfected MT1-siRNA–treated HT-1080 cells was 24 ± 7%, 1 ± 1%, and 25 ± 3%, respectively, and for SCC-1 cells was 15.8 ± 1%, 1 ± 1%, and 16 ± 4%, respectively (mean ± 1 SD; n = 3). (D) Invasion of human dermal explants by HT-1080 cells after electroporation with a control siRNA or MT1-siRNA and cultured atop the CAM for 3 d. The percentage of invading cells and invasion depth for control siRNA- and MT1 siRNA-treated HT-1080 cells, respectively, were 16.9 ± 5.9% and 144.1 ± 47.6 μm and 2.9 ± 0.2% and 22.9 ± 10.8 μm. (E) Tumor cell intravasation/extravasation was detected as Alu-sequences by PCR on DNA extracted from the lower CAM for either HT-1080 or SCC-1 cells after a 3-d incubation period. Chick GAPDH (chGAPDH) serves as the loading control. Results are representative of four experiments performed. Bars, 100 μm.
Mentions: To determine if a required role for MT1-MMP in fibroblast invasion extends to tumor cells, collagen-invasive activity of the MT1-MMP siRNA-treated cancer cells was assessed in vitro and in vivo. As shown in Fig. 6, type I collagen invasion and the associated degradation of the surrounding matrix by HT-1080 or SCC-1, although unaffected by the control siRNA, are suppressed almost completely by the MT1-MMP siRNA (Fig. 6, A and B). Validated siRNA-mediated targeting of either tumor cell MMP-1, MMP-2, or MMP-9 did not affect collagen-invasive activity (unpublished data). A role for MT1-MMP in tumor cell invasion was not limited to HT-1080 or SCC-1 cells, as similar results are obtained when MT1-MMP expression is silenced in MDA-MB-231, PANC-1, or DU-145 cells where the number of invasive foci is reduced by more than 75% (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408028/DC1). MT1-MMP siRNA-treated tumor cells retain full ability to invade either pepsin-extracted collagen gels devoid of intermolecular cross-links or fibrin matrices (Fig. S3). Similarly, (though in contrast with other recent reports; Ueda et al., 2003; Iida et al., 2004) invasion through Matrigel, an extract of noncross-linked ECM macromolecules (Kleinman et al., 1982), is unaffected in MT1-MMP–targeted tumor cells (Fig. S3). However, invasion through cross-linked collagen matrices and the associated collagenolytic activity are restored fully by transfecting the MT1-MMP siRNA-treated tumor cells with the mouse MT1-MMP construct (Fig. 6, A and B).

Bottom Line: As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity.Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM.Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)-dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity.

Show MeSH
Related in: MedlinePlus