Transcriptomic Characterization, Chemosensitivity and Regulatory Effects of Exosomes in Spontaneous EMT/MET Transitions of Breast Cancer Cells

Abstract

Background/aim: We examined the gene expression changes of breast cancer cells spontaneously undergoing epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) and the role of exosomes in these transitions.

Materials and methods: Highly invasive mesenchymal-like breast cancer cells, MDA-MB-231 (basal cells), EMT and MET variants, were characterized by microarray gene expression profiling, immunocytochemistry and chemo-sensitivity.

Results: Spontaneously disseminated cells were anoikis resistant, exhibited a dissociative, EMT-like phenotype and underwent MET when reseeded in cell-free plates. MET was inhibited by exosomes secreted by basal cells. Chemo-sensitivity to doxorubicin, vincristine and paclitaxel decreased in the order EMT<MET<basal. Phenotypic plasticity arose with differential expression of metastasis and stemness associated genes (LGR5, FZD10, DTX1, ErbB3, FTH1 and DLL4) and pathways (DNA replication and repair, ABC transporter, Hedgehog, Notch and metabolic pathways).

Conclusion: This is an appropriate model for studying EMT/MET transitions, drug targets and the role of exosomes in breast cancer dissemination.

Keywords: Breast cancer; chemoresistance; epithelial–mesenchymal transition (EMT); exosomes; mesenchymal–epithelial transition (MET); metastasis.

Figure 1. Behavioral characterization of suspension EMT cells and effect of exosomes secreted by basal cells on the reverse transition from EMT to MET. A: Time-dependent increase in the number of viable MDA-MB-231 cells spontaneously growing in suspension. Data are expressed as means±SEM of 3 independent experiments. ***p<0.001 vs. 48 h. B: Number of adherent new colonies (MET cells) formed by EMT cells in DMEM alone or in the presence of 10% and 20% exosomes isolated from the culture medium of basal cells. ***p<0.001 vs. DMEM alone.

Figure 2. Expression of the EMT hallmarks vimentin and E-cadherin. Immunocytochemical evaluation of the expression of E-cadherin (A) and vimentin (B) in adherently growing MDA-MB-231 cells (Basal). Expression of E-cadherin (C) and vimentin (D) in MDA-MB-231 growing in suspension (EMT). Expression of E-cadherin (E) and vimentin (F) in adherent new colonies (MET) obtained by seeding disseminated MDA-MB- 231 cells in cell-free wells (20x magnification; Barr 100 mm objective). Densitometric analysis of E-cadherin in Basal, EMT and MET cells (G). Densitometric analysis of vimentin in Basal, EMT and MET cells. **p<0.01 vs. Basal and MET cells (H).

Figure 2. Expression of the EMT hallmarks vimentin and E-cadherin. Immunocytochemical evaluation of the expression of E-cadherin (A) and vimentin (B) in adherently growing MDA-MB-231 cells (Basal). Expression of E-cadherin (C) and vimentin (D) in MDA-MB-231 growing in suspension (EMT). Expression of E-cadherin (E) and vimentin (F) in adherent new colonies (MET) obtained by seeding disseminated MDA-MB- 231 cells in cell-free wells (20x magnification; Barr 100 mm objective). Densitometric analysis of E-cadherin in Basal, EMT and MET cells (G). Densitometric analysis of vimentin in Basal, EMT and MET cells. **p<0.01 vs. Basal and MET cells (H).

Figure 3. Chemosensitivity assay in basal, EMT and MET cells. Data are expressed as means±SEM of 3 independent experiments. ***p<0.001 vs. adherent MDA-MB-231.

Figure 4. Gene Expression Analysis. A: Hierarchical clustering of gene expression profiles of EMT, MET and basal cells. B: Venn diagram showing the number of differentially expressed genes in the three cell clones, the degree of overlap between the differentially expressed genes in the three comparisons; non-overlapping numbers specify the genes unique to each comparison.

Figure 5. Heat maps representing the three KEGG pathways pointed out by Gene Set Enrichment Analysis (GSEA) of MDA-MB-231 cells transcriptomic data: DNA replication (hsa03030), base excision repair (hsa03410) and TCA cycle (hsa00020). Up-regulated genes are shown in dark blue, down-regulated genes in light blue color.

Figure 6. Heat maps representing KEGG pathways pointed out by Gene Set Enrichment Analysis (GSEA) of MDA-MB-231 cells transcriptomic data. Up-regulated genes are shown in dark blue, down-regulated genes in light blue color.

Figure 7. Heat maps representing the gene sets predicted to be targets of miR-30d, miR-181a and miR-134. Up-regulated genes are shown in dark blue, down-regulated genes in light blue color.