CD146 expression is associated with a poor prognosis in human breast tumors and with enhanced motility in breast cancer cell lines

Abstract

Introduction: Metastasis is a complex process involving loss of adhesion, migration, invasion and proliferation of cancer cells. Cell adhesion molecules play a pivotal role in this phenomenon by regulating cell-cell and cell-matrix interactions. CD146 (MCAM) is associated with an advanced tumor stage in melanoma, prostate cancer and ovarian cancer. Studies of CD146 expression and function in breast cancer remain scarce except for a report concluding that CD146 could act as a tumor suppressor in breast carcinogenesis.

Methods: To resolve these apparent discrepancies in the role of CD146 in tumor cells, we looked at the association of CD146 expression with histoclinical features in human primary breast cancers using DNA and tissue microarrays. By flow cytometry, we characterized CD146 expression on different breast cancer cell lines. Using siRNA or shRNA technology, we studied functional consequences of CD146 downmodulation of MDA-MB-231 cells in migration assays. Wild-type, mock-transfected and downmodulated transfected cells were profiled using whole-genome DNA microarrays to identify genes whose expression was modified by CD146 downregulation.

Results: Microarray studies revealed the association of higher levels of CD146 with histoclinical features that belong to the basal cluster of human tumors. Expression of CD146 protein on epithelial cells was detected in a small subset of cancers with histoclinical features of basal tumors. CD146+ cell lines displayed a mesenchymal phenotype. Downmodulation of CD146 expression in the MDA-MB-231 cell line resulted in downmodulation of vimentin, as well as of a set of genes that include both genes associated with a poor prognosis in a variety of cancers and genes known to promote cell motility. In vitro functional assays revealed decreased migration abilities associated with decreased CD146 expression.

Conclusions: In addition to its expression in the vascular compartment, CD146 is expressed on a subset of epithelial cells in malignant breast. CD146 may directly or indirectly contribute to tumor aggressiveness by promoting malignant cell motility. Changes in molecular signatures following downmodulation of CD146 expression suggest that CD146 downmodulation is associated with the reversal of several biological characteristics associated with epithelial to mesenchymal transition, and the phenomenon associated with the metastatic process.

Figure 1

CD146 protein expression in human primary breast cancer and specific survival. Examples of CD146 staining for (a) a metaplastic carcinoma and (b) an invasive adenocarcinoma.

Figure 2

CD146 mRNA expression in human primary breast tumors. (a) Hierarchical clustering of 227 breast cancer tissue samples and 14,486 genes/Expressed Sequence Tags based on mRNA expression levels. Each row represents a gene, and each column represents a tumor. The expression level of each gene in a single tumor is relative to its median abundance across all tumors and is depicted according to a color scale shown at the bottom. Red and green, expression levels respectively above and below the median. The magnitude of deviation from the median is represented by the color saturation. The dendrogram of samples (above matrix) represents overall similarities in gene expression profiles and is magnified in (b), colored bars to the left, locations of seven gene clusters of interest. The stromal/vascular gene cluster (orange bar) includes MCAM. ER, estrogen receptor. (b) Dendrogram of breast cancer samples. (c) Expanded view of CD146/MCAM expression. Also see Additional data file 4.

Figure 3

Specific survival of patients with CD146^- and CD146⁺ tumors. CD146 expression was defined using immunohistochemistry on tissue microarrays. (a) All tumors. (b) Estrogen receptor-negative/progesterone receptor-negative/ERBB2-negative (triple-negative) tumors.

Figure 4

CD146 mRNA expression in human breast cancer cell lines. (a) Hierarchical clustering of 34 mammary cell lines and 13,976 genes/Expressed Sequence Tags based on mRNA expression levels. The legend is similar to Figure 2. Colored bars to the left, locations of four gene clusters of interest. The stromal/mesenchymal gene cluster (orange bar) includes MCAM. ER, estrogen receptor. (b) Dendrogram of cell lines. *Cell lines analyzed in the present study by flow cytometry for CD146 expression (low expression, MCF-7, ZR-75-30 and MDA-MB-453; high expression, MDA-MB-231, Hs578T and MCF-10A). (c) Expanded view of the stromal/mesenchymal gene cluster, which includes the four probe sets representing CD146/MCAM. Genes are referenced by their HUGO abbreviation as used in Entrez Gene.

Figure 5

CD146 expression in breast cancer cell lines. Values indicate the specific mean fluorescence intensity (sMFI) ± standard error of the mean in at least six independent experiments, using the P1H12 mAb. The sMFI was defined as the ratio of the mean fluorescence intensity for the considered mAb over the mean fluorescence intensity obtained with the appropriate isotypic control.

Figure 6

Downmodulation of CD146 expression and migration abilities of the MDA-MB-231 cell line. (a) CD146 mRNA expression in the MDA-MB-231 cell line 72 hours after transfection with siRNAs targeting CD146. Two different siRNAs (si78 and si79) and two controls (si78mut and siGFP) were used. CD146mRNA expression was normalized to GAPDH and expressed relatively to the native cells (arbitrarily 100%). (b) Protein expression (PE) measured by flow cytometry, one representative experiment. (c) Chemotactic migration evaluated after 24 hours, using uncoated Boyden chambers and 10% FCS as chemoattractant. (d) Transmigration through the established human endothelial HBMEC cell line. (e) and (f) Wound healing assay. (e) Wound healing repair was evaluated after 24 hours; percentage of the initial wound surface repaired after 24 hours was estimated using ImageJ software. (f) A representative experiment of wound healing: MDA-MB-231 cells were transfected with siRNAs and, 3 days after transfection, a cell-free area (wound) was created in confluent cultures (t = 0); cells were allowed to migrate for 24 hours (t = 24) before image analysis. For (c), (d) and (e) results are expressed relative to the results obtained with the mutated si78 RNA (arbitrarily 100%) and represent the mean ± standard error of the mean of four independent experiments (except for (d), which represents two experiments).