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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 regulates collagen-degradative activity in tumor cells. (A) Degradative activity of HT-1080 cells (5 × 104) cultured atop a film of rhodamine-labeled type I collagen (100 μg/2.2 cm2) in the presence of 10% serum alone (HT-1080; stained with phalloidin/DAPI), or with BB-94, TIMP-1, or TIMP-2. Zones of collagen degradation were monitored by confocal laser microscopy after 3 d. (B) Western blot analysis of MT1-MMP (top) expression in HT-1080 or SCC-1 cells before or after electroporation with a 21-bp MT1-MMP siRNA (MT1-siRNA) or after coelectroporation of MT1-siRNA with a mouse MT1-MMP plasmid (mMT1). Gelatin zymography (bottom) of serum-free supernatants recovered from control or MT1-siRNA–treated HT-1080 or SCC-1 cells after 2 d in culture. MMP-2 (72 kD) and MMP-9 (92 kD) expression are not affected by MT1-siRNA as assessed by zymography. Control HT-1080 cells generate pro-MMP-2 (black arrowhead) and mature MMP-2 (white arrowhead). (C) HT-1080 cells were either treated with a control-siRNA, MT1-siRNA, or coelectroporated with MT1-siRNA and either MMP-1RXKR, MMP-13RXKR, MMP-2RXKR, or mMT1-MMP expression vectors, cultured for 3 d atop a film of rhodamine-labeled type I collagen in 10% serum, and zones of collagen degradation monitored by confocal laser microscopy. In the top section of the split image of MT1-siRNA–treated HT-1080 cells, the phalloidin/DAPI-stained tumor cells are shown atop the collagen film, whereas the lower section displays the underlying collagen layer alone. Results are representative of four performed. Bar, 100 μm.
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fig3: MT1-MMP regulates collagen-degradative activity in tumor cells. (A) Degradative activity of HT-1080 cells (5 × 104) cultured atop a film of rhodamine-labeled type I collagen (100 μg/2.2 cm2) in the presence of 10% serum alone (HT-1080; stained with phalloidin/DAPI), or with BB-94, TIMP-1, or TIMP-2. Zones of collagen degradation were monitored by confocal laser microscopy after 3 d. (B) Western blot analysis of MT1-MMP (top) expression in HT-1080 or SCC-1 cells before or after electroporation with a 21-bp MT1-MMP siRNA (MT1-siRNA) or after coelectroporation of MT1-siRNA with a mouse MT1-MMP plasmid (mMT1). Gelatin zymography (bottom) of serum-free supernatants recovered from control or MT1-siRNA–treated HT-1080 or SCC-1 cells after 2 d in culture. MMP-2 (72 kD) and MMP-9 (92 kD) expression are not affected by MT1-siRNA as assessed by zymography. Control HT-1080 cells generate pro-MMP-2 (black arrowhead) and mature MMP-2 (white arrowhead). (C) HT-1080 cells were either treated with a control-siRNA, MT1-siRNA, or coelectroporated with MT1-siRNA and either MMP-1RXKR, MMP-13RXKR, MMP-2RXKR, or mMT1-MMP expression vectors, cultured for 3 d atop a film of rhodamine-labeled type I collagen in 10% serum, and zones of collagen degradation monitored by confocal laser microscopy. In the top section of the split image of MT1-siRNA–treated HT-1080 cells, the phalloidin/DAPI-stained tumor cells are shown atop the collagen film, whereas the lower section displays the underlying collagen layer alone. Results are representative of four performed. Bar, 100 μm.

Mentions: Tumor cells, like fibroblasts, also express secreted and membrane-anchored MMPs (Seiki et al., 2003) and efficiently degrade subjacent collagen via a BB-94–sensitive process (Fig. 3 A). Consistent with a role for MT1-MMP in pericellular collagenolysis, high dose TIMP-1 is unable to block degradation, whereas proteolysis is inhibited completely by TIMP-2 (Fig. 3 A). To determine directly the role of MT1-MMP in the tumor cell collagenolytic phenotype, the expression of the membrane-anchored enzyme was targeted with a siRNA construct in HT-1080 or SCC-1 cells. As shown in Fig. 3 B, MT1-MMP is silenced effectively without affecting MMP-2 or MMP-9 expression, though, as expected, the MT1-MMP–dependent processing of proMMP-2 to its active form is suppressed. Further, like MT1-MMP−/− fibroblasts, MT1-MMP siRNA-treated cancer cells display no defects in two-dimensional proliferation or migration across collagen-coated surfaces (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200408028/DC1). However, in the absence of endogenous MT1-MMP, the pericellular collagenolytic activity of HT-1080 cells or SCC-1 cells was lost completely (Fig. 3 C). Further, the inability of MT1-MMP siRNA-treated tumor cells to mediate subjacent collagenolysis is not reversed when the cells are transfected with chimeric constructs of human MMP-1, MMP-13, or MMP-2 that undergo constitutive processing to active forms (i.e., MMP-1RXKR, MMP-13RXKR, or MMP-2RXKR expression vectors; Hotary et al., 2003; Fig. 3 C). Although siRNAs can potentially induce nonspecific effects by mediating off-target gene regulation (Scacheri et al., 2004), collagenolytic activity is restored completely when the MT1-MMP siRNA-treated tumor cells are transfected with a mouse MT1-MMP construct that displays only limited homology through the human MT1-MMP siRNA-targeted sequence (Fig. 3 C). Thus, cancer cells, like fibroblasts, rely on MT1-MMP to express subjacent collagenolytic activity.


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 regulates collagen-degradative activity in tumor cells. (A) Degradative activity of HT-1080 cells (5 × 104) cultured atop a film of rhodamine-labeled type I collagen (100 μg/2.2 cm2) in the presence of 10% serum alone (HT-1080; stained with phalloidin/DAPI), or with BB-94, TIMP-1, or TIMP-2. Zones of collagen degradation were monitored by confocal laser microscopy after 3 d. (B) Western blot analysis of MT1-MMP (top) expression in HT-1080 or SCC-1 cells before or after electroporation with a 21-bp MT1-MMP siRNA (MT1-siRNA) or after coelectroporation of MT1-siRNA with a mouse MT1-MMP plasmid (mMT1). Gelatin zymography (bottom) of serum-free supernatants recovered from control or MT1-siRNA–treated HT-1080 or SCC-1 cells after 2 d in culture. MMP-2 (72 kD) and MMP-9 (92 kD) expression are not affected by MT1-siRNA as assessed by zymography. Control HT-1080 cells generate pro-MMP-2 (black arrowhead) and mature MMP-2 (white arrowhead). (C) HT-1080 cells were either treated with a control-siRNA, MT1-siRNA, or coelectroporated with MT1-siRNA and either MMP-1RXKR, MMP-13RXKR, MMP-2RXKR, or mMT1-MMP expression vectors, cultured for 3 d atop a film of rhodamine-labeled type I collagen in 10% serum, and zones of collagen degradation monitored by confocal laser microscopy. In the top section of the split image of MT1-siRNA–treated HT-1080 cells, the phalloidin/DAPI-stained tumor cells are shown atop the collagen film, whereas the lower section displays the underlying collagen layer alone. Results are representative of four performed. Bar, 100 μm.
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fig3: MT1-MMP regulates collagen-degradative activity in tumor cells. (A) Degradative activity of HT-1080 cells (5 × 104) cultured atop a film of rhodamine-labeled type I collagen (100 μg/2.2 cm2) in the presence of 10% serum alone (HT-1080; stained with phalloidin/DAPI), or with BB-94, TIMP-1, or TIMP-2. Zones of collagen degradation were monitored by confocal laser microscopy after 3 d. (B) Western blot analysis of MT1-MMP (top) expression in HT-1080 or SCC-1 cells before or after electroporation with a 21-bp MT1-MMP siRNA (MT1-siRNA) or after coelectroporation of MT1-siRNA with a mouse MT1-MMP plasmid (mMT1). Gelatin zymography (bottom) of serum-free supernatants recovered from control or MT1-siRNA–treated HT-1080 or SCC-1 cells after 2 d in culture. MMP-2 (72 kD) and MMP-9 (92 kD) expression are not affected by MT1-siRNA as assessed by zymography. Control HT-1080 cells generate pro-MMP-2 (black arrowhead) and mature MMP-2 (white arrowhead). (C) HT-1080 cells were either treated with a control-siRNA, MT1-siRNA, or coelectroporated with MT1-siRNA and either MMP-1RXKR, MMP-13RXKR, MMP-2RXKR, or mMT1-MMP expression vectors, cultured for 3 d atop a film of rhodamine-labeled type I collagen in 10% serum, and zones of collagen degradation monitored by confocal laser microscopy. In the top section of the split image of MT1-siRNA–treated HT-1080 cells, the phalloidin/DAPI-stained tumor cells are shown atop the collagen film, whereas the lower section displays the underlying collagen layer alone. Results are representative of four performed. Bar, 100 μm.
Mentions: Tumor cells, like fibroblasts, also express secreted and membrane-anchored MMPs (Seiki et al., 2003) and efficiently degrade subjacent collagen via a BB-94–sensitive process (Fig. 3 A). Consistent with a role for MT1-MMP in pericellular collagenolysis, high dose TIMP-1 is unable to block degradation, whereas proteolysis is inhibited completely by TIMP-2 (Fig. 3 A). To determine directly the role of MT1-MMP in the tumor cell collagenolytic phenotype, the expression of the membrane-anchored enzyme was targeted with a siRNA construct in HT-1080 or SCC-1 cells. As shown in Fig. 3 B, MT1-MMP is silenced effectively without affecting MMP-2 or MMP-9 expression, though, as expected, the MT1-MMP–dependent processing of proMMP-2 to its active form is suppressed. Further, like MT1-MMP−/− fibroblasts, MT1-MMP siRNA-treated cancer cells display no defects in two-dimensional proliferation or migration across collagen-coated surfaces (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200408028/DC1). However, in the absence of endogenous MT1-MMP, the pericellular collagenolytic activity of HT-1080 cells or SCC-1 cells was lost completely (Fig. 3 C). Further, the inability of MT1-MMP siRNA-treated tumor cells to mediate subjacent collagenolysis is not reversed when the cells are transfected with chimeric constructs of human MMP-1, MMP-13, or MMP-2 that undergo constitutive processing to active forms (i.e., MMP-1RXKR, MMP-13RXKR, or MMP-2RXKR expression vectors; Hotary et al., 2003; Fig. 3 C). Although siRNAs can potentially induce nonspecific effects by mediating off-target gene regulation (Scacheri et al., 2004), collagenolytic activity is restored completely when the MT1-MMP siRNA-treated tumor cells are transfected with a mouse MT1-MMP construct that displays only limited homology through the human MT1-MMP siRNA-targeted sequence (Fig. 3 C). Thus, cancer cells, like fibroblasts, rely on MT1-MMP to express subjacent collagenolytic activity.

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