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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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Fibroblast MMP expression profile and collagenolytic activity. (A, top) RT-PCR analysis of MMP expression of wild-type and  fibroblasts cultured atop collagen gels in 10% serum for 3 d with PDGF. (bottom) Gelatin zymography of serum-free supernatants recovered from the wild-type, MMP-2−/−, TIMP-2−/−, or MT1-MMP−/− fibroblasts cultured with PDGF alone (Control) or with either TIMP-1 or TIMP-2. Wild-type fibroblasts express pro-MMP-2 (black arrowhead) and generate mature MMP-2 (white arrowhead) via a TIMP-2-sensitive process. No MMP-2 was detected in MMP-2−/− cultures, whereas mature MMP-2 was not generated in the TIMP-2−/− cultures. The identity of the high Mr gelatinolytic species in the supernatant of TIMP-2−/− fibroblasts is unknown. (B and C) Collagenolytic activity of fibroblasts seeded on a type I collagen film as assessed by confocal laser microscopy (B) or hydroxyproline release (C). Fibroblasts were seeded atop a 100 μg/2.2 cm2 film of rhodamine-labeled collagen and stimulated with PDGF without or with BB-94 in 10% autologous mouse serum or cultured under serum-free conditions with PDGF in the presence of 20 μg/ml of plasminogen for 5 d. (B) Wild-type or MT1-MMP−/− fibroblasts were labeled with calcein-AM (green) and DAPI (blue) in the merged images (first, ninth, and last two images in the series). Bar, 100 μm. (C) Hydroxyproline release was monitored in 10% serum without or with PDGF in the absence or presence of TIMP-2. Results are expressed as the mean ± 1 SEM of three or more experiments.
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fig2: Fibroblast MMP expression profile and collagenolytic activity. (A, top) RT-PCR analysis of MMP expression of wild-type and fibroblasts cultured atop collagen gels in 10% serum for 3 d with PDGF. (bottom) Gelatin zymography of serum-free supernatants recovered from the wild-type, MMP-2−/−, TIMP-2−/−, or MT1-MMP−/− fibroblasts cultured with PDGF alone (Control) or with either TIMP-1 or TIMP-2. Wild-type fibroblasts express pro-MMP-2 (black arrowhead) and generate mature MMP-2 (white arrowhead) via a TIMP-2-sensitive process. No MMP-2 was detected in MMP-2−/− cultures, whereas mature MMP-2 was not generated in the TIMP-2−/− cultures. The identity of the high Mr gelatinolytic species in the supernatant of TIMP-2−/− fibroblasts is unknown. (B and C) Collagenolytic activity of fibroblasts seeded on a type I collagen film as assessed by confocal laser microscopy (B) or hydroxyproline release (C). Fibroblasts were seeded atop a 100 μg/2.2 cm2 film of rhodamine-labeled collagen and stimulated with PDGF without or with BB-94 in 10% autologous mouse serum or cultured under serum-free conditions with PDGF in the presence of 20 μg/ml of plasminogen for 5 d. (B) Wild-type or MT1-MMP−/− fibroblasts were labeled with calcein-AM (green) and DAPI (blue) in the merged images (first, ninth, and last two images in the series). Bar, 100 μm. (C) Hydroxyproline release was monitored in 10% serum without or with PDGF in the absence or presence of TIMP-2. Results are expressed as the mean ± 1 SEM of three or more experiments.

Mentions: As fibroblasts and tumor cells both express collagenolytic activity during invasion, efforts were initiated to identify the proteolytic systems that mediate pericellular degradation. During matrix remodeling events in vivo, fibroblasts express MMP zymogens that can undergo processing to active forms (Brinckerhoff and Matrisian, 2002). Similar to the repertoire of MMPs expressed by fibroblasts activated in vivo (Okada et al., 1997; Bullard et al., 1999; Wu et al., 2002), fibroblasts stimulated with PDGF atop a three-dimensional collagen substratum in vitro express MMP-13, MMP-8, MMP-2, MT1-MMP, MT3-MMP, MMP-3, and MMP-9 as well as the MMP inhibitor TIMP-2 (Fig. 2 A). Further, wild-type cells process proMMP-2 to an active form as observed in vivo (Wang et al., 2000; Fig. 2 A).


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)

Fibroblast MMP expression profile and collagenolytic activity. (A, top) RT-PCR analysis of MMP expression of wild-type and  fibroblasts cultured atop collagen gels in 10% serum for 3 d with PDGF. (bottom) Gelatin zymography of serum-free supernatants recovered from the wild-type, MMP-2−/−, TIMP-2−/−, or MT1-MMP−/− fibroblasts cultured with PDGF alone (Control) or with either TIMP-1 or TIMP-2. Wild-type fibroblasts express pro-MMP-2 (black arrowhead) and generate mature MMP-2 (white arrowhead) via a TIMP-2-sensitive process. No MMP-2 was detected in MMP-2−/− cultures, whereas mature MMP-2 was not generated in the TIMP-2−/− cultures. The identity of the high Mr gelatinolytic species in the supernatant of TIMP-2−/− fibroblasts is unknown. (B and C) Collagenolytic activity of fibroblasts seeded on a type I collagen film as assessed by confocal laser microscopy (B) or hydroxyproline release (C). Fibroblasts were seeded atop a 100 μg/2.2 cm2 film of rhodamine-labeled collagen and stimulated with PDGF without or with BB-94 in 10% autologous mouse serum or cultured under serum-free conditions with PDGF in the presence of 20 μg/ml of plasminogen for 5 d. (B) Wild-type or MT1-MMP−/− fibroblasts were labeled with calcein-AM (green) and DAPI (blue) in the merged images (first, ninth, and last two images in the series). Bar, 100 μm. (C) Hydroxyproline release was monitored in 10% serum without or with PDGF in the absence or presence of TIMP-2. Results are expressed as the mean ± 1 SEM of three or more experiments.
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Related In: Results  -  Collection

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fig2: Fibroblast MMP expression profile and collagenolytic activity. (A, top) RT-PCR analysis of MMP expression of wild-type and fibroblasts cultured atop collagen gels in 10% serum for 3 d with PDGF. (bottom) Gelatin zymography of serum-free supernatants recovered from the wild-type, MMP-2−/−, TIMP-2−/−, or MT1-MMP−/− fibroblasts cultured with PDGF alone (Control) or with either TIMP-1 or TIMP-2. Wild-type fibroblasts express pro-MMP-2 (black arrowhead) and generate mature MMP-2 (white arrowhead) via a TIMP-2-sensitive process. No MMP-2 was detected in MMP-2−/− cultures, whereas mature MMP-2 was not generated in the TIMP-2−/− cultures. The identity of the high Mr gelatinolytic species in the supernatant of TIMP-2−/− fibroblasts is unknown. (B and C) Collagenolytic activity of fibroblasts seeded on a type I collagen film as assessed by confocal laser microscopy (B) or hydroxyproline release (C). Fibroblasts were seeded atop a 100 μg/2.2 cm2 film of rhodamine-labeled collagen and stimulated with PDGF without or with BB-94 in 10% autologous mouse serum or cultured under serum-free conditions with PDGF in the presence of 20 μg/ml of plasminogen for 5 d. (B) Wild-type or MT1-MMP−/− fibroblasts were labeled with calcein-AM (green) and DAPI (blue) in the merged images (first, ninth, and last two images in the series). Bar, 100 μm. (C) Hydroxyproline release was monitored in 10% serum without or with PDGF in the absence or presence of TIMP-2. Results are expressed as the mean ± 1 SEM of three or more experiments.
Mentions: As fibroblasts and tumor cells both express collagenolytic activity during invasion, efforts were initiated to identify the proteolytic systems that mediate pericellular degradation. During matrix remodeling events in vivo, fibroblasts express MMP zymogens that can undergo processing to active forms (Brinckerhoff and Matrisian, 2002). Similar to the repertoire of MMPs expressed by fibroblasts activated in vivo (Okada et al., 1997; Bullard et al., 1999; Wu et al., 2002), fibroblasts stimulated with PDGF atop a three-dimensional collagen substratum in vitro express MMP-13, MMP-8, MMP-2, MT1-MMP, MT3-MMP, MMP-3, and MMP-9 as well as the MMP inhibitor TIMP-2 (Fig. 2 A). Further, wild-type cells process proMMP-2 to an active form as observed in vivo (Wang et al., 2000; Fig. 2 A).

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