Integrated microRNA and mRNA signatures associated with survival in triple negative breast cancer

Abstract

Triple negative breast cancer (TNBC) is a heterogeneous disease at the molecular, pathologic and clinical levels. To stratify TNBCs, we determined microRNA (miRNA) expression profiles, as well as expression profiles of a cancer-focused mRNA panel, in tumor, adjacent non-tumor (normal) and lymph node metastatic lesion (mets) tissues, from 173 women with TNBCs; we linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify clinically and genetically different TNBC subclasses. We also assessed miRNA signatures as potential regulators of TNBC subclass-specific gene expression networks defined by expression of canonical signal pathways.Tissue specific miRNAs and mRNAs were identified for normal vs tumor vs mets comparisons. miRNA signatures correlated with prognosis were identified and predicted anti-correlated targets within the mRNA profile were defined. Two miRNA signatures (miR-16, 155, 125b, 374a and miR-16, 125b, 374a, 374b, 421, 655, 497) predictive of overall survival (P = 0.05) and distant-disease free survival (P = 0.009), respectively, were identified for patients 50 yrs of age or younger. By multivariate analysis the risk signatures were independent predictors for overall survival and distant-disease free survival. mRNA expression profiling, using the cancer-focused mRNA panel, resulted in clustering of TNBCs into 4 molecular subclasses with different expression signatures anti-correlated with the prognostic miRNAs. Our findings suggest that miRNAs play a key role in triple negative breast cancer through their ability to regulate fundamental pathways such as: cellular growth and proliferation, cellular movement and migration, Extra Cellular Matrix degradation. The results define miRNA expression signatures that characterize and contribute to the phenotypic diversity of TNBC and its metastasis.

Figure 1. Venn diagram describing miRNA expression patterns in the three classes of breast tissue.

Venn diagram (A) representing differentially expressed miRNAs observed in the comparisons among the three classes (normal, primary tumor, metastatic lesion), up or down facing arrow indicate the expression level change. Tables show: the 13 miRNAs identified by the comparison between Normal vs Tumor (B); 1 miR differentially expressed only in Tumor vs Mets (C); 1 miR differentially expressed only in Normal vs Mets (D); 4 miRNAs commonly deregulated in the Tumor vs Mets and Normal vs Mets comparisons (E).

Figure 2. Overall Survival miRNA signature and Distant Disease-Free Survival (DDFS) signature.

Overall survival (OS) of TNBC patients of 50 yrs and younger patients due to differentially expressed miRNAs in the three classes. (A) Heat map representing miRNA profiles of 75 tumor samples using average linkage clustering and Spearman Rank method as distance metrics. Bar above the dendrogram identifies 39 high risk samples shown in orange and 36 low risk cases in yellow. Samples are shown in columns, miRNAs in rows. Heat map represents relative miRNA expression as indicated in the blue to red key bar at the top. (B) Hazard ratios of protective and risky miRNAs. (C) Overall Survival miRNAs signature. Distant Disease-Free Survival (DDFS) miRNA signature of 50 yrs and younger. (D) Heat map representing miRNA profiles of 75 tumor samples using average linkage clustering and Spearman Rank method as distance metrics. Bar above the dendrogram identifies 37 high risk samples shown in orange and 38 low risk cases in yellow. Samples are shown in columns, miRNAs in rows. Heat map represent relative miRNA expression as indicated in the key bar at the top. (E) miRNAs predicting protection from or susceptibility to early recurrence. (F) Kaplan-Meier estimates of DDFS according to the seven-miRNA signature.

Figure 3. Comparison of mRNA expression profiles of normal vs tumor RNAs.

The heat maps represent hierarchical clustering of differentially expressed genes in normal and tumor-derived RNAs. mRNA profiles are clustered in 4 different subgroups (orange, blue, yellow, pink) defined by the mRNA expression patterns. Overlapping Gene Ontology terms for top canonical pathways represented by the differentially expressed genes in each subgroup, as determined by IPA-ingenuity software, are shown on the right for each of the normal/tumor comparison-defined subgroups.

Figure 4. Venn diagram and associated table describing mRNA expression profile patterns in the three classes.

Venn diagram (A) representing differentially expressed mRNAs observed in the comparison among the three classes (normal, primary tumor, metastatic lesion) for patients of 50 yrs and younger, up or down facing arrow indicate the expression level change. Tables show: the 7 mRNAs identified by the comparison between Normal vs Tumor (B); 1 mRNA differentially expressed only in Tumor vs Mets (C); 4 mRNAs differentially expressed in Normal vs Mets (D); 2 mRNAs commonly deregulated in the comparisons Tumor vs Mets and Normal vs Mets (E).