Changes in EMT markers levels are associated with CD146 modulation.

<p>The CD146-pCMV6-XL4 vector was transfected in MCF-7 and SKBR3 cells, CD146 expression was down-modulated (KD) in CAL51 and MDA-MB-231 cells with siRNAs. mRNAs from CD146 or siRNAs transfected cells were analyzed against mRNAs from mock transfected cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> are expressed as the mean of the fold change (± standard error of the mean) for at least six different mRNA preparations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon matched pairs test and considered as significant when p<0.05 (indicated with S for significant).</p

Functional properties of breast cancer cells depend on the level of CD146 expression.

<p>A: Anchorage-dependent growth was estimated by the cell number after four days of culture. B: Anchorage-independent growth was assessed by soft agar colony-forming assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s4" target="_blank">Materials and Methods</a>. C, Chemotactic migration evaluated after 36 hours (CAL51 cells) or 96 hours (MCF-7 and SKBR3 cells) using uncoated Boyden chambers and 10% FCS as chemo-attractant. Migrating cells were stained with Crystal Violet solution, lysed and absorbance measured at 550 nm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> represented as the mean ± standard error of seven independent experiments were analyzed with the Wilcoxon signed rank test. White bars: CD146^low cells, black bars: CD146^high cells.</p

IC50 values for doxorubicin and docetaxel depending on the level of CD146 expression.

<p>The specific MFI represents the ratio of the mean fluorescence intensity for the CD146 antibody over the mean fluorescence intensity of the isotypic control (mean ± standard error of the mean of at least six independent experiments). IC50 is expressed as the mean (± standard error of the mean) for six different experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon matched pairs test and considered as significant when p<0.05.</p

CD146 Expression in Human Breast Cancer Cell Lines Induces Phenotypic and Functional Changes Observed in Epithelial to Mesenchymal Transition

<div><h3>Background</h3><p>Metastasis is an important step in tumor progression leading to a disseminated and often incurable disease. First steps of metastasis include down-regulation of cell adhesion molecules, alteration of cell polarity and reorganization of cytoskeleton, modifications associated with enhanced migratory properties and resistance of tumor cells to anoikis. Such modifications resemble Epithelial to Mesenchymal Transition (EMT). In breast cancer CD146 expression is associated with poor prognosis and enhanced motility.</p> <h3>Methodology/Principal Findings</h3><p>On 4 different human breast cancer cell lines, we modified CD146 expression either with shRNA technology in CD146 positive cells or with stable transfection of CD146 in negative cells. Modifications in morphology, growth and migration were evaluated. Using Q-RT-PCR, we analyzed the expression of different EMT markers. We demonstrate that high levels of CD146 are associated with loss of cell-cell contacts, expression of EMT markers, increased cell motility and increased resistance to doxorubicin or docetaxel. Experimental modulation of CD146 expression induces changes consistent with the above described characteristics: morphology, motility, growth in anchorage independent conditions and Slug mRNA variations are strictly correlated with CD146 expression. These changes are associated with modifications of ER (estrogen receptor) and Erb receptors and are enhanced by simultaneous and opposite modulation of JAM-A, or exposure to heregulin, an erb-B4 ligand.</p> <h3>Conclusions</h3><p>CD146 expression is associated with an EMT phenotype. Several molecules are affected by CD146 expression: direct or indirect signaling contributes to EMT by increasing Slug expression. CD146 may also interact with Erb signaling by modifying cell surface expression of ErbB3 and ErbB4 and increased resistance to chemotherapy. Antagonistic effects of JAM-A, a tight junction-associated protein, on CD146 promigratory effects underline the complexity of the adhesion molecules network in tumor cell migration and metastasis.</p> </div

JAM-A expression and migration abilities of breast cancer cells.

<p>A: Mean Fluorescence Intensities (MFI) of CD146 and JAM-A expression on indicated human breast cancer cell lines are shown. Values indicate the specific mean fluorescence intensity (sMFI) ± the standard error of the mean of at least seven experiments. The sMFI was defined as the ratio of the MFI for the considered antibody over the MFI obtained with the appropriate isotypic control. B: down-regulation of JAM-A expression by over-expression of CD146 in MCF-7 cells. JAM-A expression measured by flow cytometry in CD146⁺ MCF-7 cells, one representative experiment. C, migration of mock-transfected or CD146⁺ MCF-7 cells after JAM-A knockdown. JAM-A was inhibited with a siRNA in mock transfected and CD146⁺ MCF-7 cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> represent the mean ± standard error of the mean of three independent experiments.</p

Morphologic changes associated with the modulation of CD146 expression.

<p>A: Morphology of MCF-7 cells stably transfected with CD146 and of CAL51 cells stably transfected with a shRNA against CD146 compared to mock transfected cells. The diminution in cell-cell contacts is associated with CD146 expression (magnification×40). B: Proportion of scattered colonies in mocked-transfected and CD146-modulated MCF-7 and CAL51 cells. Cells expressing high levels of CD146 produce a higher number of scattered colonies than their CD146 low or negative counterparts. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> expressed as the mean ± standard error of the mean of at least six independent experiments were analyzed with the Wilcoxon signed rank test.</p

CD146 modulation reveals a link with ER, PR and Erb receptor expression and function.

<p>A: analysis of ER, PR and Erb receptors in CD146 transfected MCF-7 cells versus mock transfected cells. Q-RT-PCR was performed on six different RNA preparations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> expressed as the mean ± standard error to the mean were analyzed with the one sample t-test assuming a theoretical mean equal to 1 (** = p<0.01). B: CD146 increases the response to heregulin in MCF-7 cells. Q-RT-PCR was performed on six different RNA preparations isolated from mock transfected cells (CD146 negative cells) or CD146 transfected cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> are expressed as fold change in expression comparing HRG-treated cells with untreated cells for mock transfected cells (CD146 negative) as well as CD146 transfected cells. Mean ± standard error to the mean is shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043752#s2" target="_blank">Results</a> were analyzed with the Wilcoxon signed rank test.</p

Characteristics of the breast cancer cell lines used in this study.

<p>The specific MFI represents the ratio of the mean fluorescence intensity for the CD146 antibody over the mean fluorescence intensity of the isotypic control (mean ± standard error of the mean of at least six independent experiments).</p