SLUG/SNAI2 and tumor necrosis factor generate breast cells with CD44+/CD24- phenotype.
BACKGROUND
Breast 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.


METHODS
MCF-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.


RESULTS
Two thousand thirty five genes were differentially expressed (p < 0.001, fold change >or= 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 TGFbeta, NF-kappaB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-kappaB 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-kappaB 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.


CONCLUSIONS
EMT-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.
Bhat-Nakshatri
Poornima
P
Department of Surgery, Indiana University School of Medicine, West Walnut Street, Indianapolis, IN 46202, USA.
Appaiah
Hitesh
H
Ballas
Christopher
C
Pick-Franke
Patricia
P
Goulet
Robert
R
Badve
Sunil
S
Srour
Edward F
EF
Nakshatri
Harikrishna
H
D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001940:Breast / Q000378:metabolism* / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051927:CD24 Antigen / Q000378:metabolism*; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D016335:Zinc Fingers
D014408:Biomarkers, Tumor; D051927:CD24 Antigen; D018960:Hyaluronan Receptors; D012333:RNA, Messenger; C000604407:SNAI1 protein, human; C000604408:SNAI2 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha