• 2019-10
  • 2019-11
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  • br Finally we analyzed the protein


    Finally, we analyzed the protein expression of cell surface luminal markers and intracellular basal markers using flow cytometry (Fig. 6) and these results were in line with the gene expression data. The lu-minal markers CD24 and MUC-1 were overexpressed in both knock-down cell lines, KRT8 expression showed a tendency towards an in-crease, and the expression of EpCAM was increased in shSRC-3
    Fig. 5. SRC-2 and SRC-3 depletions promote luminal and inhibit the basal gene program. Gene expression of lineage-specific and EMT genes in untreated shMCF-7 cells was analyzed by RT-qPCR. Target gene mRNA expression is relative to the average of the expression of three reference genes (TBP, RPLP0 and PUM1) and is presented as mean of eight independent parallels ± SD. Statistical significance was calculated using Student t-test (*p ≤.05; **p ≤ .01; ***p ≤.001).
    Fig. 6. Expression of luminal markers is increased, while the expression of basal markers is decreased in shSRC-2 and shSRC-3 cells. Expression of luminal and basal markers was analyzed by flow cytometry. Median fluorecence intenisities (MFIs) for individual markers were extracted using FlowJo software. (A) Luminal markers.
    (B) Basal markers. Shown statistical significance refers to MFI values of three parallels in each cell line. Results are representative of two separate experiments. Statistical significance was calculated using One-way ANOVA corrected for multiple comparrisons by the Turkey’s range test (*p ≤.05; **p ≤.01; ***p ≤ .001).
    compared to shSRC-2 cells (Fig. 6A). As expected, the expression of basal markers p63 and KRT5 was reduced in the knockdown cell lines. Collectively, the results suggest that SRC-2 and SRC-3 depletions sti-mulate the development into the luminal lineage by increasing the expression of luminal genes and decreasing the expression of basal genes.
    3.5. SRC-2 correlates with the luminal, GSK126 and cell morphogenesis gene expression in tumor tissue from breast cancer patients
    In order to examine the role of SRC-2 in clinical breast cancer, we examined samples which are comparable to the MCF-7 cell model. More accurately, we examined 61 ER-positive, HER2-negative tumor biopsies out of 94 lymph-node negative breast cancer patient samples previously analyzed by microarray [24]. We compared the gene expression of SRC-2 and SRC-3 to the genes originally identified in MCF-7 cells as their potential targets by performing a correlation analysis. Out of 49 in-vestigated genes the gene expression of 17 genes was found to correlate to either the SRC-2 or the SRC-3 expression (Table 2). Similarly to MCF-
    Table 2
    Correlation between SRC-2 or SRC-3 and their target genes in an ER-positive HER2-negative breast cancer patient population (N = 61).
    Genea Correlation coefficient ¤ Phenotype
    7 cells, the expression of SRC-2 was negatively correlated to the luminal genes in breast cancer patients. Furthermore, SRC-2 was negatively correlated to the cell cycle, cell morphogenesis and TNBC-related genes examined. Interestingly, in this group of luminal patients we found no association between SRC-3 and cell morphogenesis genes. On the other hand we did observe a positive association between SRC-3 and the cell cycle genes CCNE1 and MKI67. It is worth noting that in the chosen group of ER-positive, HER2-negative patients most of the gene ex-pression changes correlated with SRC-2 expression, rather than SRC-3. We also found SRC-3 to be significantly upregulated (T test, p = 0.009) in the luminal HER2-positive group (while the TNBC group in this pa-tient cohort was too small to be analyzed (data not shown). Finally, we tested if SRC-2 or SRC-3 correlated with disease relapse and, once ad-justed for tumor size and treatment, found no correlation. This is most likely due to a low number (five) of relapse events in the given dataset. In conclusion, the expression of SRC-2 was negatively correlated with the expression of MCF-7-related luminal, cell cycle and cellular mor-phogenesis genes in tumor tissue from ER-positive, HER2-negative breast cancer patients.