Table 1.
Characteristics of the breast cancer cell lines used in this study.
Figure 1.
Morphologic changes associated with the modulation of CD146 expression.
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. Results expressed as the mean ± standard error of the mean of at least six independent experiments were analyzed with the Wilcoxon signed rank test.
Table 2.
Changes in EMT markers levels are associated with CD146 modulation.
Figure 2.
CD146 modulation reveals a link with ER, PR and Erb receptor expression and function.
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. Results 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. Results 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. Results were analyzed with the Wilcoxon signed rank test.
Figure 3.
Functional properties of breast cancer cells depend on the level of CD146 expression.
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 Materials and Methods. 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. Results represented as the mean ± standard error of seven independent experiments were analyzed with the Wilcoxon signed rank test. White bars: CD146low cells, black bars: CD146high cells.
Table 3.
IC50 values for doxorubicin and docetaxel depending on the level of CD146 expression.
Figure 4.
JAM-A expression and migration abilities of breast cancer cells.
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. Results represent the mean ± standard error of the mean of three independent experiments.