Molecular mechanisms of type I collagen-induced apoptosis in breast carcinoma cells

Objective: As invading breast carcinoma cells breach the underlying basement membrane, they become confronted with a dense three-dimensional reactive stroma dominated by type I collagen. To develop metastatic capabilities, invading tumour cells must acquire the capacity to negotiate this hostile microenvironment. By enmeshing cells in a dense fibrillar network, type I collagen acts as a physical barrier for cell migration as well as an endogenous antigrowth signal, partly by inducing apoptosis in epithelial cells. Aberrant cell survival resulting from an acquired resistance toward apoptosis represents a prominent hallmark of cancers. However, the molecular mechanisms implicated in collagen-induced apoptosis remain poorly defined. Here, we investigate the molecular mechanisms by which type I collagen induces apoptosis in breast carcinoma cells and identify MMP-14, a membrane-anchored matrix metalloproteinase, as a key anti-apoptotic factor. Methods: To investigate the induction of apoptosis by collagen, human breast adenocarcinoma MCF-7 cells overexpressing or not MMP-14 were plated on plastic plates or embedded within three dimensional type I collagen gels (Col3D). Cell death was evaluated by measuring cytoplasmic histone-associated DNA fragments (Cell Death Detection ELISA). The percentage of cells with an apoptotic nuclear morphology was also determined. The interactions between cancer cells and Col3D were analyzed by confocal microscopy and the impact of Col3D on the transcriptome of cancer cells was investigated using Illumina HT-12 BeadArrays. Results: When cultured within Col3D gels, MCF-7 cells displayed a round morphology and a cell death characterized by a Z-VAD-FMK-dependent chromatin condensation, nuclear segmentation and oligonucleosomal DNA fragmentation was induced. Transfection of MCF-7 cells with MMP-14 cDNA promoted the interactions between cells and collagen and prevented apoptosis. A transcriptomic analysis revealed that culturing MCF-7 cells within Col3D altered the expression of about 700 genes, irrespective of MMP-14 expression. Col3D modulated the expression of several apoptosis-related genes. Interestingly, MMP-14 activity was sufficient to prevent the Col3D-dependent induction of Bcl2-Interacting Killer (BIK), a pro-apoptotic member of the Bcl-2 family. Conclusions: Our results shed light on the molecular mechanisms by which a collagen-rich microenvironment triggers apoptosis in invading breast cancer cells. Furthermore, we demonstrate that MMP-14 promotes tumour progression by circumventing apoptosis

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

During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and proapoptotic 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 demonstrate 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. A transcriptomic analysis was performed 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 drastic alterations of the transcriptome of MCF-7 cells

A Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) – Discoidin Domain Receptor 1 Axis Regulates Collagen-Induced Apoptosis in Breast Cancer Cells

<div><p>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.</p></div

Inhibition of DDR1 tyrosine kinase activity prevents the 3D COL1-mediated induction of BIK.

<p>MCF-7 (<b>A</b>) and ZR-75–1 (<b>B</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. (<b>C</b>) 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 <b>β</b>-actin (<i>D</i>), as a loading control. Relative abundances of DDR1 were quantified and normalized with respect to <b>β</b>-actin expression. Lysates of 3T3 cells transfected with human DDR1b cDNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116006#pone.0116006.ref101" target="_blank">101</a>], 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 ELISA^PLUS. Data are means ± SEM (n = 3). ^## p<0.01, ^### p<0.001 Col3D <i>versus</i> Plastic; ** p<0.01, *** p<0.001 treatment <i>versus</i> vehicle (one-way ANOVA analysis with Boneferroni post test).</p

Pharmacological inhibition of 3D COL1-induced BIK expression in MCF-7 cells.

<p>Pharmacological inhibition of 3D COL1-induced BIK expression in MCF-7 cells.</p

Gene ontology terms associated with the 3D COL1 signature.

<p>Gene ontology terms associated with the 3D COL1 signature.</p

Top 5 significant GO terms enriched in individual lists of 3D COL1-regulated genes.

<p>Top 5 significant GO terms enriched in individual lists of 3D COL1-regulated genes.</p

Transcriptional regulators of the MT1-MMP modulated genes in 3D COL1.

<p>The IPA Upstream regulator analysis identified transcription factors with direct actions on differentially expressed target genes. The different molecules are presented by cellular localization. HIF1A and EPAS1 were predicted to be activated (or to have increased activity) in MT1 cells relative to CTRL cells. Genes in red and green are up- and down-regulated in response to MT1-MMP expression, respectively. E: expression; PD: protein-DNA binding; RB: regulation of binding; T: transcription.</p

3D COL1 and MT1-MMP regulate DDR1 activation and cleavage.

<p>Control (CTRL) and MT1-MMP (MT1) expressing MCF-7 cells were cultured for 24h on 2D plastic (Plastic) or within 3D COL1 (Col3D) in the presence (+) of BB-94 (1 μM), DDR1-IN-1 (1 μM) or vehicle (DMSO 0.1%). 3D COL1 gels were mechanically disrupted and lysed in RIPA buffer. Lysates were resolved by reducing 8% SDS-PAGE followed by immunoblot analysis. Blots were probed with phospho-DDR1 (Tyr792) antibody (<i>A</i>) and then reprobed with antibodies directed against the cytosolic juxtamembrane domain of DDR1 (<i>B</i>), BIK (<i>C</i>) or <b>β</b>-actin (<i>D</i>), as a loading control. Relative abundances of phosphorylated DDR1, 120-kDa full-length, 62-kDa C-terminal forms of DDR1 and BIK were quantified and normalized with respect to <b>β</b>-actin expression. Data are means ± SEM (n = 3). ^# p<0.05, ^## p<0.01, ^### p<0.001 treatment <i>versus</i> vehicle-treated cells plated on plastic; * p<0.05, *** p<0.001 MT1 <i>versus</i> CTRL cells (one-way ANOVA analysis with Boneferroni post test) (<i>E</i>). Lysates of 3T3 cells transfected with human DDR1b cDNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116006#pone.0116006.ref101" target="_blank">101</a>], which contains both 120-kDa full-length and 62-kDa C-terminal DDR1 species were included as a positive control.</p

3D COL1 modulates the expression of small nuclear RNAs (snRNAs)-coding genes.

<p>Control (CTRL) and MT1-MMP (MT1) expressing MCF-7 cells were cultured for 24, 48 and 72h on 2D plastic (Plastic) or within 3D COL1 (Col3D). RNA was extracted from each sample and gene expression values measured using the Illumina Human HT-12 BeadChip array. Microarray data were expressed as fluorescence intensities. Dashed line represents the background fluorescence.</p