Cell differentiation and dominant signaling pathway signatures in the molecular classification of human breast cancer cell lines

Background

Differentiation markers characteristic of multiple cell types in the mammary gland have emerged as a dominant feature in gene expression profiles that segregate primary human breast cancers. Immunohistochemical and mRNA expression profiling studies of large breast cancer cohorts have reproducibly identified a subset of tumors (~15%) that express markers characteristic of the basal layer of the mammary gland. This is in contrast to the many human breast cancers that uniformly express luminal markers such as the simple cytokeratins (K8/K18) and appear to originate from transformed luminal epithelial cells. A logical next step is to determine the dominant signaling pathways and genetic defects that drive tumor initiation and progression, and to understand how they are related to cell lineage in each breast cancer subtype. A large number of breast cancer cell lines have been isolated and individually characterized over the past few decades. We and others have begun to comprehensively align these cell lines with primary tumors based on gene expression profiles and other parameters in order to improve the relevance of data obtained from these experimental models for understanding human disease.

Methods

We have characterized a panel of 51 breast cell lines for a large number of properties including in vitro and in vivo growth rates, morphology on plastic and in three-dimensional matrices, and sensitivity to estrogens/anti-estrogens. Baseline microarray profiles were generated using Agilent 60-mer oligonucleotide arrays for each cell line. In addition, we have generated 'signature profiles' for selected cell lines in response to exogenous stimulation such as estrogen and TGF-β. A constraint-based analysis of microarray profiles generated from primary tumors and breast cancer cell lines in combination with response signatures was used to identify candidate genes and pathways that may play dominant roles in the breast cancer subgroups.

Results

The 51 breast cell line panel was segregated into two roughly equal sized groups comprised of those with dominant luminal features and those with progenitor or non-luminal properties. The top ~600 genes that distinguish luminal versus non-luminal cell lines were identified. All of the luminal cell lines express ESR1, HER-2 (ERBB2) or both. The non-luminal cell lines express many components of the wnt signaling pathway, including ligands, frizzled receptors and secreted inhibitory proteins. Interestingly, the majority of the non-luminal cell lines express significantly higher levels of the ROR1 receptor tyrosine kinase relative to luminal cells. The non-luminal cell lines further segregate into those with predominantly basal features versus those with mesenchymal features. A dominant feature of the mesenchymal cell lines is evidence of elevated, autocrine TGF-β signaling.

Conclusion

Based on our characterizations and array data for a large panel of breast cancer cell lines, primary tumors and response signatures, we propose an integrated model for the molecular classification of breast cancer. This model connects dominant signal transduction pathways with cell-type origin, and further resolves the biological and clinical significance of the well-established markers ER and HER-2. The proposed stratification is likely to help explain the well-known diversity in response of breast cancers to standard therapeutic regimens and, more importantly, may identify appropriate breast cancer subtypes amenable to targeted therapeutics.