Basal-Type Breast Cancer Stem Cells Over-Express Chromosomal Passenger Complex Proteins

Abstract

(1) Aim: In the present paper we analyzed the transcriptome of CSCs (Cancer Stem Cells), in order to find defining molecular processes of breast cancer. (2) Methods: We performed RNA-Seq from CSCs isolated from the basal cell line MDA-MB-468. Enriched processes and networks were studied using the IPA (Ingenuity Pathway Analysis) tool. Validation was performed with qRT-PCR and the analysis of relevant genes was evaluated by overexpression, flow cytometry and in vivo zebrafish studies. Finally, the clinical relevance of these results was assessed using reported cohorts. (3) Results: We found that CSCs presented marked differences from the non-CSCs, including enrichment in transduction cascades related to stemness, cellular growth, proliferation and apoptosis. Interestingly, CSCs overexpressed a module of co-regulated Chromosomal Passenger Proteins including BIRC5 (survivin), INCENP and AURKB. Overexpression of BIRC5 increased the number of CSCs, as assessed by in vitro and in vivo zebrafish xenotransplant analyses. Analysis of previously published cohorts showed that this co-regulated module was not only overexpressed in basal breast tumors but also associated with relapse-free and overall survival in these patients. (4) Conclusions: These results underline the importance of Cancer Stem Cells in breast cancer progression and point toward the possible use of chromosomal passenger proteins as prognostic factors.

Keywords: breast cancer; networks; stem cells.

Figure 1

Isolation of Cancer Stem Cells. (A) Left panels: Flow cytometry analysis of MDA-MB-468 cells previous to sorting. The upper panel shows a dot plot with the fluorescence intensity of CD44 versus CD24 and the selected fields for sorting (CD44 high and CD24 low). The lower panel shows the third used marker (EpCam). Right panels: These plots display the purified population obtained after sorting. (B) Tumorigenicity assays using a zebrafish xenotransplant model. Upper panel: Tumor developed by MDA-MB-468 non-Cancer Stem Cells (CSCs; CD44⁺CD24⁺EpCAM^-) and CSCs (CD44⁺CD24^−/lowEpCAM⁺) at 2, 3 and 4 DPI (Days Post Injection). For each experiment, 30 embryos were used and only informative (e.g., surviving) embryos were assessed. #: The number of cells transplanted to the embryos. Lower panel: Representative image taken at 2 DPI, showing abdominal tumors (arrows) in zebrafish embryos transplanted with MDA-MB-468 non-CSCs and MDA-MB-468 CSCs. (C) Volcano plot showing DE significant genes in red (posterior probability of being differentially expressed > 95%). The line shows the cutoff for significance. (D) GSEA analysis of the DE genes found. Left panel: Enrichment against a mammary stem cells signature (derived from [17]). Right panel: Enrichment against a tissue stem cell signature (derived from [18]). (E) Top 20 DE genes with a False Discovery Rate < 0.05. (F) Absolute fold change of representative genes in CD44⁺CD24^−/lowEpCAM⁺ (Stem) cells versus CD44⁺CD24⁺EpCAM^- (Non-stem) cells analyzed by qRT-PCR. Data derived from three independent FACS isolation experiments; * p < 0.05; *** p < 0.001.

Figure 2

Network analysis of DE genes in CSCs from MDA-MB-468 cells. (A) Significant signaling cascades in the studied set. Bars length depicts the -log (p value) of each cascade, the color of the bar represents the z-score and the orange line points the ratio of genes in the pathway (e.g., the proportion of the studied DE genes present in each signaling cascade). (B) Enriched cellular and molecular functions table. The main functions and scores are shown. (C) Upstream regulators. The table shows the upstream regulator gene, the predicted activation (based on an absolute score > 2) and the p value of the gene overlap. (D) Main network in CSCs, showing two important modules in squares (Cytokeratin module and Chromosomal Passenger module). Red represents increased expression of the molecule in CSCs and green decreased expression.

Figure 3

Participation of the Chromosomal Passenger Proteins Module (CPPM) in the stem cell phenotype of MDA-MB-468 cells. (A) Gene co-expression. Dot plots show the correlation between the expression of BIRC5 and AURKB (left panel), INCENP (middle panel) and H2AFZ (right panel). (B) MDA-MB-468 cells were transiently transfected with a vector containing a BIRC5 open reading frame and subjected to qRT-PCR for the genes shown. Log fold change refers to the logarithmic ratio between BIRC5-overexpressing cells versus empty vector-expressing cells [27], using the ∆∆Ct method, with SDHA as a housekeeping gene. Each dot represents an independent transfection. BIRC5 p = 0.0012, ALDH1A1 p = 0.1547, Nanog p = 0.1137 and Oct4 p = 0.3128 (** p < 0.01, n.s. = non-significant). (C) Xenotransplant dilution assays using a zebrafish model. Table showing tumor frequencies 4 DPI. # cells: number of cells injected. For each experiment, 30 embryos were used and only informative (e.g., surviving) embryos were assessed. Percentages and number of fishes with or without tumors are shown. MDA-468-BIRC5 cells overexpress BIRC5 and MDA-468-GFP overexpress a GFP transgene (control cells). (D) Limiting dilution analysis obtained with the Extreme Limiting Dilution Assays (ELDA) software. The plot shows the percentage of the embryos with abdominal tumors injected with MDA-MB-468 cells overexpressing BIRC5 or GFP-expressing (control) cells at 4 DPI. The difference between the analyzed groups was significant (**p = 0.015; estimated stem cell frequency 1/369 vs. 1/685 in BIRC5- and GFP-expressing cells, respectively; n.s. non-significant). (E) Examples of tumors formed in these assays (arrows).

Figure 4

Clinical relevance of the CPPM in breast cancer. (A) Boxplot showing the normalized expression of BIRC5, AURKB, INCENP and H2AFZ in a group of 1881 breast cancer patients by breast cancer subtypes. The number of patients is shown above the plot. p < 0.00001 for all genes (ANOVA). (B) Boxplot showing the normalized expression of BIRC5, AURKB, INCENP and H2AFZ in a group of 1881 breast cancer patients by breast cancer ER status. The number of patients is shown above the plot. p < 0.00001 for all genes (ANOVA). (C) Boxplot showing normalized expression of the CPPM in a group of 1881 breast cancer patients. The number of patients is shown above the plot, p < 0.00001 (ANOVA). (D) Kaplan–Meier survival plot for relapse-free survival in patients with basal breast tumors derived from a published set of 249 triple-negative breast cancer patients [14], classified with the CPPM. High expression was defined as a mean expression of the module genes higher than 2093 logrank p = 0.0052, hazard ratio 1.45. (E) Kaplan–Meier survival plot for relapse-free survival in breast cancer patients derived from a published set of 249 triple-negative breast cancer patients [14], classified with the CPPM. High expression was defined as a mean expression of the module genes higher than 1223. logrank p = 1.10⁻¹⁶, hazard ratio 1.94. (F) Kaplan–Meier survival plot for overall survival in breast cancer patients derived from a published set of 249 triple-negative breast cancer patients [14], classified with the CPPM. High expression was defined as a mean expression of the module genes higher than 1454. logrank p = 1.10⁻⁰⁷, hazard ratio 1.89.