Androgen receptor signalling driving cancer stem cells (CSCs) state in triple-negative breast cancer

Therapeutic strategies that improve survival outcomes for advanced-stage breast cancers have proven a major clinical challenge. Here, we define an androgen receptor signalling network that governs the maintenance and de novo formation of cancer stem cells in triple-negative breast cancer. In response to chemotherapy, androgen receptor activation switches cells into a cancer stem cell state, while androgen receptor antagonism suppresses cancer stem cell formation and function. In vivo, we validate that the androgen receptor antagonist, seviteronel, significantly improves chemotherapy-mediated inhibition of primary and metastatic tumour growth. MCF7 cells (Michigan Cancer Foundation) and ZR75-1 cells (American Type Culture Collection) lines were verified through short tandem repeat profiling and tested negative for mycoplasma contamination. Parental cell lines were cultured in RPMI 1640 media (Thermo Fisher) supplemented with 10% fetal bovine serum (GE Healthcare) and 20 mM HEPES (Thermo Fisher). HMEC cells were purchased from ATCC and transformed into HMLER cells by the sequential addition of hTERT, SV40-ER and RAS as previously described21. Cells were cultured in serum-free mammary epithelial growth medium (MEGM) and MEGM Single Quots (Lonza, cat no. CC-4136: 1 ml bovine pituitary extract (BPE), 0.5 ml GA-1000 (30 mg/ml Gentamicin and 15 µg/ml Amphotericin), 0.5 ml insulin, 0.5 ml hydrocortisone and 0.5 ml hEGF). Cells were maintained at 37oC with 5% CO2. HCC38 cells were purchased from ATCC and cultured in RPMI containing 10% FBS containing penicillin/streptomycin (PS; 5.000 units penicillin and 5 mg streptomycin/ml in H2O, Sigma Aldrich, cat no. P4333). All cell lines were routinely tested to confirm the absence of mycoplasma contamination. Cells were grown in P150 plates for 48h-72h, depending on growth kinetics, till plates reached 80% confluence. At harvest time, cells were washed twice with cold PBS1x (Invitrogen), scraped and collected in eppendorfs. Cells were spun down, washed once more with cold PBS1x, removing the supernatant, followed by snap freezing in liquid nitrogen. Frozen cell pellets (1 x 10^6 cells) were used for RNA extraction of RNA using the commercial RNeasy Kit (Qiagen). Quality of RNA was assessed using a bioanalyzer Eukaryote Total RNA Nano chip. Samples that scored above RIN:9 were used to generate the mRNA library, using a TruSeq Stranded mRNA LT - Set A kit (Illumina: RS-122-2101). CD44-low versus CD44-high cell lines: MCF-7, ZR75-1, HMLER and HCC38. Samples were submitted for depth pair-end sequencing (Illumina HiSeq 2500 v4.0) at the Garvan Molecular Genetics.