SLUG/SNAI2 and Tumor Necrosis Factor Generate Breast Cells With CD44+/CD24- Phenotype

Abstract

BackgroundBreast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.MethodsMCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.ResultsTwo thousand thirty five genes were differentially expressed (p < 0.001, fold change ≥ 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFβ, NF-κB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-κB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-κB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.ConclusionsEMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.

Cross-regulation between protein L-isoaspartyl O-methyltransferase and ERK in epithelial mesenchymal transition of MDA-MB-231 cells

“…Therefore, it could be interesting to determine whether our EMT conditions are capable of transforming MDA-MB-231 cells into highly metastatic stem-like cells. To do this, phenotype of these cells will be determined by measuring the surface levels of CD44/CD24 [45] .Taken together, the results presented here provide in vitro support for the idea that PIMT and ERK are tightly expressed and activated in EMT conditions generated during the detachment of MDA-MB-231 cells from poly-HEMA coated dishes and by TGF-β/TNF-α stimulation, as summarized in Figure 6. These findings suggest that the development of specific inhibitors of PIMT may lead to a novel route of inhibition of the EMT and metastasis.…”

“…Therefore, it could be interesting to determine whether our EMT conditions are capable of transforming MDA-MB-231 cells into highly metastatic stem-like cells. To do this, phenotype of these cells will be determined by measuring the surface levels of CD44/CD24 [45] .…”

Epithelial-to-mesenchymal transition (EMT) confers primary resistance to trastuzumab (Herceptin)

“…To evaluate the role of EMT drivers in regulating the CD44 + CD24 -/low mesenchymal subpopulation [44][45][46] in basal/ HER2 + JIMT1 cells, we used flow cytometry to determine the amount of JIMT1 cells bearing the CD44 + CD24 -/low mesenchymal immunophenotype before and after the lentiviral-mediated small hairpin (sh) RNA knockdowns of the EMT-driving expression of both ZEB1 and ZEB2 transcripts significantly, a result that was accompanied by the slight downregulation of TWIST1 and VIM. However, we failed to detect a significant increase in the expression of CD24 in the ZEB1 KD-JIMT1 cells.…”

Nuclear receptors agonists exert opposing effects on the inflammation dependent survival of breast cancer stem cells

“…5,12 According to these findings, tumor necrosis factor-a (TNFa), a major NF-kB inducer, was shown to enhance MS formation, 8,9 by upregulating SLUG, an NF-kB controlled regulator of the breast CSC phenotype. 9,10 In hematopoietic and prostate CSCs, NF-kB upregulation has been reported. 13,14 A kind of 'NF-kB activity addiction' has been therefore proposed to likely render CSCs more susceptible to NF-kB inhibitors than their normal counterparts.…”

“…3 Cells disclosing a CSCs phenotype can be expanded in vitro as multicellular spheroids, named mammospheres (MS), obtained from breast cancer surgical specimens and cell lines. [6][7][8][9][10][11] The activity of nuclear factor-kB (NF-kB) pathway has been recognized to be of pivotal importance in MS survival. [8][9][10][11] In MS from aggressive breast cancer the over-expression of interleukin-6 (IL6), an NF-kB controlled gene, has been reported.…”