First, the authors isolated CD44+, CD24− (thought to be stem cell-like) and CD44−, CD24+ (thought to be more differentiated luminal epithelial-like) populations from breast cancer samples and normal breast tissue. They also used the cell surface receptor, PROCR, to purify a CD44+, PROCR+ population. PROCR is expressed on breast epithelial cells but not on leukocytes or myofibroblasts, unlike CD44. Reverse transcription PCR showed that CD44+ cells expressed stem cell markers, whereas CD24+ cells expressed luminal epithelial markers. Single nucleotide polymorphism analysis confirmed that CD44+ cells are genetically identical to CD24+ cells in some breast cancer samples. However, in other samples, fluorescence in situ hybridization analyses indicated that the CD24+ population can acquire additional mutations and so continue to evolve, contributing to the heterogeneity of this disease.
The authors also generated serial analysis of gene expression (SAGE) libraries for each cell population and used MetaCore data mining technology to look for evidence of signalling pathway disruption. They found that the transforming growth factor β (TGFβ) pathway is important in CD44+ breast cancer cells. These cells express TGFβ receptor 2 (
TGFBR2
), whereas CD24+ cells express much lower levels of TGFBR2 owing, the authors confirmed, to the methylation of this gene. Moreover, a TGFBR1 and TGFBR2 small-molecule inhibitor promoted CD44+ cell differentiation in vitro from a mesenchymal to epithelial-like phenotype, but had no effect on CD24+ cells.
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