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The biological and molecular heterogeneity of breast cancers correlate with their cancer stem cell content

ABSTRACT: Pathways that govern normal stem cell (SC) function are often subverted in cancer. Here, we report the isolation to near purity of human normal mammary SC (hNMSCs), from cultured mammospheres, based on their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. We demonstrated that PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we could prospectively isolate SCs from the normal gland and from breast tumors. Poorly-differentiated aggressive (G3) cancers displayed higher content of prospectively isolated cancer SCs, than well-differentiated less aggressive (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in cancer SCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their SC content. Epithelial cells, from reductive mammoplasties, labeled with PKH26 (Sigma, 10-7 M, 5 min) and subjected to FACS analysis using a FACS Vantage SE flow cytometer (Becton&Dickinson) to yield PKH-POS and PKH-NEG cells, were prepared for RNA extraction and hybridization on Affymetrix microarrays. Three independent pools of cells were used, and six Gene Expression Arrays were obtained. No replicates are available.

ORGANISM(S): Homo sapiens

SUBMITTER: Pece S

PROVIDER: S-ECPF-GEOD-18931 | biostudies-other | 2010 Jan

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. METHODS: We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. RESULTS: Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. CONCLUSIONS: We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to CSCs and tumor progression should consider the expression of various CD44 isoforms.

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The biological and molecular heterogeneity of breast cancers correlate with their cancer stem cell content

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