Mechanisms Controlling Luminal Identity of Breast Tumours

Breast cancer is a heterogeneous disease comprising three major molecular subtypes that can be identified by gene expression profiling: luminal, HER2-positive, and basal-like. These subtypes are thought to arise from a block at different stages of breast epithelial cell differentiation. However, the mechanisms underlying subtype specification remain largely unexplored. Luminal tumours, which account for two-thirds of breast tumours, overexpress estrogen receptor alpha (ERα), a ligand-inducible transcription factor (TF). These tumours additionally express high levels of FOXA1 and GATA3, two luminal-lineage TFs, that have been reported to regulate both expression and activity of the receptor. In the clinic, the proliferative actions of the receptor can be blocked with antiestrogens, but unfortunately ~50% of patients will relapse due to resistance, highlighting the need for additional therapies. Histone deacetylase inhibitors (HDACis) are a class of anticancer agents with anti-proliferative activity in both solid and haematological tumours. In breast cancer, these drugs were shown to suppress ERα expression/signalling, and induce differentiation and apoptosis. These properties have led to the combined use of HDACis and antiestrogens in clinical trials for treatment of ERα-positive breast tumours. In the present work, we have utilized gene correlation analysis in large breast tumour transcriptome datasets and characterized a cluster of highly correlated luminal genes containing six luminal-lineage TFs: ESR1 (gene encoding ERα), FOXA1, GATA3, SPDEF, XBP1 and AR. We show using siRNA-mediated depletion that FOXA1 dominantly positively regulates expression of the cluster despite its enrichment in ChIP-Seq binding sites of other luminal TFs. Interestingly, this cluster partitions into two subclusters, one centered around FOXA1, and the other, around ESR1 and GATA3, suggesting the presence of tumours that differentially express either subcluster. Indeed, expression of ESR1, FOXA1 and GATA3 can readily segregate luminal tumour samples (high expression) from basal-like ones (low expression). Furthermore, we found that molecular apocrine tumours, previously described as frequently HER2-positive, and androgen receptor signalling-dependent, are FOXA1high ESR1low GATA3low. Ectopic expression of ERα in molecular apocrine SK-BR-3 cells could (1) recapitulate part of the estrogen response observed in luminal cells and (2) also result in chromatin opening and transcriptional regulation of target genes weakly pre-bound by FOXA1. This is complementary to previous studies describing FOXA1 as a factor that pioneers ERα binding at its target genes. Notably, lack of ERα expression in SK-BR-3 cells, despite high expression of its upstream regulatory factor FOXA1, coincided with weak FOXA1 binding and reduced recruitment of active H3K9/K14 marks at upstream ESR1 regulatory regions.  In the second part of our study, we further show that HDACis transcriptionally reprogram breast cancer cells resulting in downregulation of luminal-lineage TFs FOXA1 and GATA3, as well as many chromatin remodelling proteins, while inducing expression of lactogenic differentiation genes. Shutdown of luminal TFs occurred through both transcriptional and post-transcriptional mechanisms that involved modulated acetylation of protein substrates to be identified. Altogether, identification of transcriptional networks that demarcate breast tumour subtypes and molecular switches controlling their function/expression will help determine whether their perturbation can rewire breast cancer cells towards a less aggressive phenotype.

Keyphrases: Breast cancer, ERα, FOXA1, GATA3, Luminal TFs, HDAC, HDAC inhibitors, Transcriptional reprogramming, Acetylation