Comparative Transcriptomics of Triple Negative Breast Cancer Stem Cells and Differentiated Tumor Cells Identifies Teneurin-4 as a Potential Therapeutic Target

BACKGROUND: Triple-negative breast cancer (TNBC) is insensitive to the most effective therapies for other breast cancers, including endocrine and Her2-directed therapies, thus the lack of specific treatments prompted us to search for new TNBC-associated molecules to be used as targets for cancer therapy. As patients with TNBC usually experience a quicker relapse and metastatic progression compared to other breast cancer subtypes, we hypothesized that cancer stem cells (CSC) could play a central role in TNBC. We thus directed our focus on genes differentially expressed between CSC and differentiated cancer cells of TNBC cell lines. RESULTS: We established tumorsphere cultures from mouse and human mammary cancer cell lines to enrich the CSC population. RNA-Seq was used to identify differences in gene expression between tumorspheres and their monolayer counterparts. Seventy-four transcripts were found up-regulated in the tumorspheres, while forty-two genes were down-regulated. Enrichment analysis of biological processes showed an up-regulation in genes involved in regulation of apoptosis in tumorspheres, and a down-regulation in genes involved in lipid metabolism and cell cycle regulation. By focusing on up-regulated genes coding for cell membrane-associated proteins, we selected Teneurin-4 (TENM4) as a candidate for further studies. Meta-analysis of publicly available datasets revealed that TENM4 mRNA is up-regulated in both lobular and ductal invasive carcinoma specimens compared to normal breast, and that high expression of TENM4 in TNBC patients shows a trend of correlation with a shorter relapse-free survival. 4T1 tumorspheres treated with a siRNA specific to TENM4 showed a decrease in TENM4 mRNA and protein levels, which was reflected by a significant impairment of tumorsphere-forming ability. TENM4 silencing also led to a decrease in Focal Adhesion Kinase (FAK) phosphorylation, which has been previously linked to CSC biology, thus strengthening the possible link between TENM4 and a CSC-like phenotype. CONCLUSIONS: Overall, our results indicate that the stem-like status of TNBC cells is accompanied by altered regulation of apoptosis, cell cycle and lipid metabolism pathways. Furthermore, we identified TENM4 as a potential novel player in CSC biology, and its potential role as a novel target to improve the outcome of TNBC patients in the future. Overall design: 4T1 and HCC1806 cell lines as model for TNBC in mouse and human, respectively. Based on culture conditions previously established for these and other breast cancer cell lines, we successfully generated tumorspheres from the parental cells. For both cell lines three replicates were done for the cell grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) and Penicillin/Streptomycin solution at 1x dilution (Sigma-Aldrich) (bulk), and those grown in DMEM/Nutrient Mixture F-12 Ham (Sigma-Aldrich) supplemented with 0.4% bovine serum albumin (BSA, Sigma-Aldrich), 20 ng/mL bFGF (PeproTech EC Ltd., London, UK), 20 ng/mL EGF (Sigma-Aldrich) and 5 µg/mL insulin (Sigma-Aldrich). (spheroids)