Abstract
BackgroundBreast cancer subtypes identified in genomic studies have different underlying genetic defects. Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors. Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function).MethodsThe p53-dependent gene expression signatures of four cell lines (MCF-7, ZR-75-1, and two immortalized human mammary epithelial cell lines) were identified by comparing p53-RNAi transduced cell lines to their parent cell lines. Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states. The cell line signatures were compared with p53-mutation associated genes in breast tumors.ResultsEach cell line displayed distinct patterns of p53-dependent gene expression, but cell type specific (basal vs. luminal) commonalities were evident. Further, a common gene expression signature associated with p53 loss across all four cell lines was identified. This signature showed overlap with the signature of p53 loss/mutation status in primary breast tumors. Moreover, the common cell-line tumor signature excluded genes that were breast cancer subtype-associated, but not downstream of p53. To validate the biological relevance of the common signature, we demonstrated that this gene set predicted relapse-free, disease-specific, and overall survival in independent test data.ConclusionIn the presence of breast cancer heterogeneity, experimental and biologically-based methods for assessing gene expression in relation to p53 status provide prognostic and biologically-relevant gene lists. Our biologically-based refinements excluded genes that were associated with subtype but not downstream of p53 signaling, and identified a signature for p53 loss that is shared across breast cancer subtypes.
Highlights
Breast cancer subtypes identified in genomic studies have different underlying genetic defects
Gene expression and phenotypic analysis of cell lines expressing p53 RNA interference (RNAi) To study the effects of p53 loss in vitro, an RNAi construct specific for p53 [6] was stably expressed in MCF-7, ZR-751, ME16C and Human Mammary Epithelial (HME)-CC cells
The expression of p53 was examined by Western blot analysis of extracts from MCF-7, ZR-75-1, HME-CC, and ME16C cell line parents and the same cell lines stably transduced with p53-targeted RNAi vector or p53 mismatch (MM) RNAi vector
Summary
Breast cancer subtypes identified in genomic studies have different underlying genetic defects. P53 mutations are found more frequently in aggressive estrogen receptor (ER)-negative breast cancers [1], and have been shown to correlate with breast cancer subtype in gene expression studies [2] and in a population-based study [3]. Genetic abnormalities such as amplified HER2/ ERBB2 [1] and aneuploidy [4] are frequently associated with p53 mutation status. The biological relevance of this common p53 signature was assessed by using this gene list to predict outcomes on independent test data sets of breast cancer patients
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