34 research outputs found
Heterogeneity of estrogen receptor α and progesterone receptor distribution in lesions of deep infiltrating endometriosis of untreated women or during exposure to various hormonal treatments
<p>Deep infiltrating endometriosis (DIE) responds variably to hormonal therapy. Mutations in cancer driver genes have been identified in a fraction of the ectopic endometrial epithelial cells, suggesting a functional heterogeneity of these lesions. To evaluate the phenotype heterogeneity of cells in DIE, we measured the expression of estrogen receptor α (ERα) and of progesterone receptor (PR) in DIE of untreated women or under various treatments. We analyzed the luminal epithelial height (LEH), immunoreactive epithelial staining (IRS) and stromal staining intensity (SSI) of ERα and PR. We observed a high variability in the same gland, among distinct glands in the same sample and among distinct patients receiving the same treatment. LEH variability was primarily due to epithelial cells heterogeneity in a gland, secondarily to the glands randomly evaluated on the same section, and tertiary to the patient category. Variability in IRS and SSI scores was primarily the consequence of their heterogeneity in the same woman and to a lesser extent to variability among patients. LEH and SSI were not modified according to treatment. IRS for PR was lower in treated patients. This heterogeneity of ERα and PR distribution could explain why endocrine treatments are unable to cure this condition.</p
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<sup>PLUS</sup>. Data are means ± SEM (n = 3). <sup>##</sup> p<0.01, <sup>###</sup> 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
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
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). <sup>#</sup> p<0.05, <sup>##</sup> p<0.01, <sup>###</sup> 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
Influence of Src Family Kinase inhibitors on 3D COL1-embedded MCF-7 and ZR-75–1 cells.
<p>MCF-7 and ZR-75–1 cells were cultured for 48h on 2D plastic (Plastic) or within 3D COL1 (COL3D) in the presence of PP2 (5 μM), saracatinib (1 μM) or vehicle (DMSO 0.1%). (<b>A</b>) 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>B</b>) Apoptosis was quantified by Cell Death Detection ELISA<sup>PLUS</sup>. Data are means ± SEM (n = 3). <sup>#</sup> p<0.01 Col3D <i>versus</i> Plastic; * p<0.05, *** p<0.001 treatment <i>versus</i> vehicle (one-way ANOVA analysis with Boneferroni post test). (<b>C</b>) Representative maximum intensity projection of serial confocal optical sections through MCF-7 cells embedded in 3D COL1 gels and treated with saracatinib (1 μM) or vehicle for 48h. F-actin (red) and DRAQ5 (blue). <i>Arrows</i>: actin-rich vesicles; <i>Arrowheads</i>: un-retracted cytoplasmic protrusions.</p
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
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
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
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