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A membrane-type-1 matrix metalloproteinase (MT1-MMP)-discoidin domain receptor 1 axis regulates collagen-induced apoptosis in breast cancer cells.

Assent D, Bourgot I, Hennuy B, Geurts P, Noël A, Foidart JM, Maquoi E - PLoS ONE (2015)

Bottom Line: Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling.By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis.Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a drastic remodelling of the transcriptome of MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumour and Developmental Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué (GIGA), Unit of Cancer, University of Liège, Liège, Belgium.

ABSTRACT
During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and pro-apoptotic properties. To develop metastatic capabilities, tumour cells must acquire the capacity to cope with this novel microenvironment. How cells interact with and respond to their microenvironment during cancer dissemination remains poorly understood. To address the impact of type I collagen on the fate of tumour cells, human breast carcinoma MCF-7 cells were cultured within three-dimensional type I collagen gels (3D COL1). Using this experimental model, we have previously demonstrated that membrane type-1 matrix metalloproteinase (MT1-MMP), a proteinase overexpressed in many aggressive tumours, promotes tumour progression by circumventing the collagen-induced up-regulation of BIK, a pro-apoptotic tumour suppressor, and hence apoptosis. Here we performed a transcriptomic analysis to decipher the molecular mechanisms regulating 3D COL1-induced apoptosis in human breast cancer cells. Control and MT1-MMP expressing MCF-7 cells were cultured on two-dimensional plastic plates or within 3D COL1 and a global transcriptional time-course analysis was performed. Shifting the cells from plastic plates to 3D COL1 activated a complex reprogramming of genes implicated in various biological processes. Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling. By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis. Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a drastic remodelling of the transcriptome of MCF-7 cells.

No MeSH data available.


Related in: MedlinePlus

Inhibition of DDR1 tyrosine kinase activity prevents the 3D COL1-mediated induction of BIK.MCF-7 (A) and ZR-75–1 (B) cells were cultured for 48h on 2D plastic (Plastic) or within 3D COL1 (COL3D) in the presence of DDR1-IN-1 (1 μM) or vehicle (DMSO 0.1%). RNA was extracted from each sample and BIK mRNA levels were quantified by semi-quantitative RT-PCR. Relative expression levels were obtained after normalization for the 28S rRNA levels. (C) MCF-7 cells were pre-treated with EGFP or DDR1 esiRNAs for 48h and cultured within 3D COL1 during 24h. esiRNA efficacy was analysed by western blotting. Blots were probed with an antibodies directed against the cytosolic juxtamembrane domain of DDR1 or β-actin (D), as a loading control. Relative abundances of DDR1 were quantified and normalized with respect to β-actin expression. Lysates of 3T3 cells transfected with human DDR1b cDNA [101], which contains both 120-kDa full-length and 62-kDa C-terminal DDR1 species were included as a positive control. Apoptosis was quantified by Cell Death Detection ELISAPLUS. Data are means ± SEM (n = 3). ## p<0.01, ### p<0.001 Col3D versus Plastic; ** p<0.01, *** p<0.001 treatment versus vehicle (one-way ANOVA analysis with Boneferroni post test).
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pone.0116006.g011: Inhibition of DDR1 tyrosine kinase activity prevents the 3D COL1-mediated induction of BIK.MCF-7 (A) and ZR-75–1 (B) cells were cultured for 48h on 2D plastic (Plastic) or within 3D COL1 (COL3D) in the presence of DDR1-IN-1 (1 μM) or vehicle (DMSO 0.1%). RNA was extracted from each sample and BIK mRNA levels were quantified by semi-quantitative RT-PCR. Relative expression levels were obtained after normalization for the 28S rRNA levels. (C) MCF-7 cells were pre-treated with EGFP or DDR1 esiRNAs for 48h and cultured within 3D COL1 during 24h. esiRNA efficacy was analysed by western blotting. Blots were probed with an antibodies directed against the cytosolic juxtamembrane domain of DDR1 or β-actin (D), as a loading control. Relative abundances of DDR1 were quantified and normalized with respect to β-actin expression. Lysates of 3T3 cells transfected with human DDR1b cDNA [101], which contains both 120-kDa full-length and 62-kDa C-terminal DDR1 species were included as a positive control. Apoptosis was quantified by Cell Death Detection ELISAPLUS. Data are means ± SEM (n = 3). ## p<0.01, ### p<0.001 Col3D versus Plastic; ** p<0.01, *** p<0.001 treatment versus vehicle (one-way ANOVA analysis with Boneferroni post test).

Mentions: To investigate the potential implication of DDR1 kinase activity in collagen-mediated BIK induction, 3D COL1-embedded MCF-7 cells were treated with DDR1-IN-1, a potent and selective DDR1 tyrosine kinase inhibitor devoid of inhibitory activity against SFKs [39]. DDR1-IN-1 (1μM) efficiently decreased DDR1 phosphorylation in 3D COL1-embedded CTRL cells (Fig. 10A, lanes 4 and 6 and Fig. 10E). A similar decrease of DDR1 activation was observed in MT1 cells, however, due to their lower initial level of phosphorylated DDR1, the inhibitory effect of DDR1-IN-1 failed to reach statistical significance (Fig. 10A, lanes 9 and 11 and Fig. 10E). Furthermore, DDR1-IN-1 inhibited the collagen-mediated BIK induction at both mRNA and protein levels, thereby protecting CTRL cells from apoptosis (Fig. 11A and Fig. 10C, lanes 4 and 6, respectively). A similar inhibitory effect of DDR1-IN-1 on DDR1 phosphorylation, BIK induction and apoptosis was also observed in 3D COL1-embedded ZR-75–1 cells (Fig. 11B and S16 Fig.).


A membrane-type-1 matrix metalloproteinase (MT1-MMP)-discoidin domain receptor 1 axis regulates collagen-induced apoptosis in breast cancer cells.

Assent D, Bourgot I, Hennuy B, Geurts P, Noël A, Foidart JM, Maquoi E - PLoS ONE (2015)

Inhibition of DDR1 tyrosine kinase activity prevents the 3D COL1-mediated induction of BIK.MCF-7 (A) and ZR-75–1 (B) cells were cultured for 48h on 2D plastic (Plastic) or within 3D COL1 (COL3D) in the presence of DDR1-IN-1 (1 μM) or vehicle (DMSO 0.1%). RNA was extracted from each sample and BIK mRNA levels were quantified by semi-quantitative RT-PCR. Relative expression levels were obtained after normalization for the 28S rRNA levels. (C) MCF-7 cells were pre-treated with EGFP or DDR1 esiRNAs for 48h and cultured within 3D COL1 during 24h. esiRNA efficacy was analysed by western blotting. Blots were probed with an antibodies directed against the cytosolic juxtamembrane domain of DDR1 or β-actin (D), as a loading control. Relative abundances of DDR1 were quantified and normalized with respect to β-actin expression. Lysates of 3T3 cells transfected with human DDR1b cDNA [101], which contains both 120-kDa full-length and 62-kDa C-terminal DDR1 species were included as a positive control. Apoptosis was quantified by Cell Death Detection ELISAPLUS. Data are means ± SEM (n = 3). ## p<0.01, ### p<0.001 Col3D versus Plastic; ** p<0.01, *** p<0.001 treatment versus vehicle (one-way ANOVA analysis with Boneferroni post test).
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Related In: Results  -  Collection

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pone.0116006.g011: Inhibition of DDR1 tyrosine kinase activity prevents the 3D COL1-mediated induction of BIK.MCF-7 (A) and ZR-75–1 (B) cells were cultured for 48h on 2D plastic (Plastic) or within 3D COL1 (COL3D) in the presence of DDR1-IN-1 (1 μM) or vehicle (DMSO 0.1%). RNA was extracted from each sample and BIK mRNA levels were quantified by semi-quantitative RT-PCR. Relative expression levels were obtained after normalization for the 28S rRNA levels. (C) MCF-7 cells were pre-treated with EGFP or DDR1 esiRNAs for 48h and cultured within 3D COL1 during 24h. esiRNA efficacy was analysed by western blotting. Blots were probed with an antibodies directed against the cytosolic juxtamembrane domain of DDR1 or β-actin (D), as a loading control. Relative abundances of DDR1 were quantified and normalized with respect to β-actin expression. Lysates of 3T3 cells transfected with human DDR1b cDNA [101], which contains both 120-kDa full-length and 62-kDa C-terminal DDR1 species were included as a positive control. Apoptosis was quantified by Cell Death Detection ELISAPLUS. Data are means ± SEM (n = 3). ## p<0.01, ### p<0.001 Col3D versus Plastic; ** p<0.01, *** p<0.001 treatment versus vehicle (one-way ANOVA analysis with Boneferroni post test).
Mentions: To investigate the potential implication of DDR1 kinase activity in collagen-mediated BIK induction, 3D COL1-embedded MCF-7 cells were treated with DDR1-IN-1, a potent and selective DDR1 tyrosine kinase inhibitor devoid of inhibitory activity against SFKs [39]. DDR1-IN-1 (1μM) efficiently decreased DDR1 phosphorylation in 3D COL1-embedded CTRL cells (Fig. 10A, lanes 4 and 6 and Fig. 10E). A similar decrease of DDR1 activation was observed in MT1 cells, however, due to their lower initial level of phosphorylated DDR1, the inhibitory effect of DDR1-IN-1 failed to reach statistical significance (Fig. 10A, lanes 9 and 11 and Fig. 10E). Furthermore, DDR1-IN-1 inhibited the collagen-mediated BIK induction at both mRNA and protein levels, thereby protecting CTRL cells from apoptosis (Fig. 11A and Fig. 10C, lanes 4 and 6, respectively). A similar inhibitory effect of DDR1-IN-1 on DDR1 phosphorylation, BIK induction and apoptosis was also observed in 3D COL1-embedded ZR-75–1 cells (Fig. 11B and S16 Fig.).

Bottom Line: Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling.By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis.Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a drastic remodelling of the transcriptome of MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tumour and Developmental Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué (GIGA), Unit of Cancer, University of Liège, Liège, Belgium.

ABSTRACT
During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and pro-apoptotic properties. To develop metastatic capabilities, tumour cells must acquire the capacity to cope with this novel microenvironment. How cells interact with and respond to their microenvironment during cancer dissemination remains poorly understood. To address the impact of type I collagen on the fate of tumour cells, human breast carcinoma MCF-7 cells were cultured within three-dimensional type I collagen gels (3D COL1). Using this experimental model, we have previously demonstrated that membrane type-1 matrix metalloproteinase (MT1-MMP), a proteinase overexpressed in many aggressive tumours, promotes tumour progression by circumventing the collagen-induced up-regulation of BIK, a pro-apoptotic tumour suppressor, and hence apoptosis. Here we performed a transcriptomic analysis to decipher the molecular mechanisms regulating 3D COL1-induced apoptosis in human breast cancer cells. Control and MT1-MMP expressing MCF-7 cells were cultured on two-dimensional plastic plates or within 3D COL1 and a global transcriptional time-course analysis was performed. Shifting the cells from plastic plates to 3D COL1 activated a complex reprogramming of genes implicated in various biological processes. Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling. By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis. Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a drastic remodelling of the transcriptome of MCF-7 cells.

No MeSH data available.


Related in: MedlinePlus