Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology

Molecular and functional genomics analyses of estrogen- related receptor alpha (ERRα) function in breast cancer

The estrogen-related receptor α (ERRα) is an orphan nuclear receptor whose expression in breast tumors is inversely correlated to that of the ERα but positively correlates with the poor prognosis receptor tyrosine kinase (RTK) HER2. Using binding sites location analyses in breast cancer cells we show that ERRα displays strict binding site specificity and ERα-independent transcriptional programs. Ablation of ERRα in a mouse model of Neu-initiated mammary tumorigenesis significantly delays HER2-induced tumor development. ERRα regulates the expression of ERBB2 and co-amplified transcripts in a process confering resistance to tamoxifen treatment in breast cancer cells. We further use genome-wide profiling of ERRα by ChIP-Sequencing, to show that β-1-heregulin (HRG) and epidermal growth factor (EGF) lead to AP1-dependant reprogramming of ERRα recruitment to chromatin in breast cancer cells. We next show that the dual EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib induces the degradation of ERRα protein. The expression of ERRα is recovered in lapatinib-resistant breast cancer cells in an mTOR-dependant manner despite sustained inhibition of EGFR/HER2. Finally, we show that ERRα signalling in lapatinib-resistant breast cancer cells provides a suitable metabolic context to sustain growth under therapeutic pressure. Together this work has uncovered important aspects of ERRα transcriptional profiles in breast cancer cells and has established ERRα as a significant player in the development, progression and therapeutic susceptibility of breast cancer cells.L’expression du récepteur associé au récepteur des estrogènes-alpha (ERRα) est inversement corrélée a celle de ERα mais est associée a un pronostic défavorable et à l’expression de HER2. Nous avons observé que ERRα démontre une spécificité stricte d’association à son site de liaison et que ses programmes transcriptionnels sont majoritairement indépendants de ERα. Nous avons par la suite utilisé un modèle de souris pour démontrer que l’absence de ERRα retarde le développement des tumeurs mammaires initiées par HER2. Nous avons démontré que ERRα régule l’expression du gène ERBB2 et des transcripts co-amplifiés, contribuant à conférer la résistance au tamoxifen dans les cellules de cancer du sein. Nous observons que β-1-heregulin (HRG) et le facteur de croissance epidermal (EGF) engendrent une reprogrammation du recrutement de ERRα à la chromatine. Nous démontrons que l’inhibiteur de EGFR/HER2 lapatinib induit la dégradation de ERRα alors que l’expression de ERRα est préservée dans les cellules résistantes au lapatinib, d’une façon indépendante de EGFRéHER2 mais qui dépend de l’activité de mTOR. Finalement nous démontrons que ERRα contribue à conférer la résistance au lapatinib dans les cellules de cancer du sein. En résumé, nos résultats ont contribué à identifier des aspects importants des profils de transcription de ERRα et à établir ERRα comme étant un joueur important dans le développement, la progression et la réponse thérapeutique du cancer du sein

Effect of the new coactivator steroid RNA Activator on the transcriptional activity of the estrogen receptor

Nuclear receptor coactivators are factors that enhance the transcriptional activity of the receptor. Most coactivators characterized to date work in a ligand-dependent manner by interacting with the activation function-2 (AF-2) of the receptor. A newly characterized coactivator termed steroid RNA activator (SRA) was cloned as an AF-1-dependent and ligand-independent coactivator. SRA was shown to enhance the transcriptional activity of the steroid receptors by interacting with their AF-1. Here, we describe the effect of SRA on the transcriptional activity of the estrogen receptor alpha (ERalpha) and beta (ERbeta). SRA coactivates ERalpha and ERbeta in a ligand-dependent manner and the transcriptional activity of ERalpha can be enhanced by SRA in a ligand-independent manner through the AF-1 domain of the receptor. The integrity of the serine residue at position 118 (S118) in ERalpha AF-1 is required for the complete coactivation of ERalpha by SRA. Activation of MAPK induces ligand-independent activation of ERalpha by SRA, a mechanism independent of the AF-2 activity of the receptor. Mutation of S118 to an alanine residue abolishes the MAPK-induced activation of ERalpha, suggesting that phosphorylation of S118 by MAPK may be important for the ligand-independent effect of SRA on ERalpha AF-1. Thus SRA may play an important role in the regulation of ER transcriptional activity

p66ShcA potentiates the cytotoxic response of triple-negative breast cancers to PARP inhibitors

Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin

The Transcriptional Repressor Polycomb Group Factor 6, PCGF6, Negatively Regulates Dendritic Cell Activation and Promotes Quiescence

Pro-inflammatory signals provided by the microenvironment are critical to activate dendritic cells (DCs), components of the innate immune system that shape both innate and adaptive immunity. However, to prevent inappropriate immune activation, mechanisms must be in place to restrain DC activation to ensure DCs are activated only once sufficient stimuli have been received. Here, we report that DC activation and immunogenicity are regulated by the transcriptional repressor Polycomb group factor 6 (PCGF6). Pcgf6 is rapidly downregulated upon stimulation, and this downregulation is necessary to permit full DC activation. Silencing PCGF6 expression enhanced both spontaneous and stimulated DC activation. We show that PCGF6 associates with the H3K4me3 demethylase JARID1c, and together, they negatively regulate H3K4me3 levels in DCs. Our results identify two key regulators, PCGF6 and JARID1c that temper DC activation and implicate active transcriptional silencing via histone demethylation as a previously unappreciated mechanism for regulating DC activation and quiescence

A chemical toolbox for the study of bromodomains and epigenetic signaling

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation

GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer

Triple negative breast cancer is a deadly form of breast cancer with limited therapeutic options. Here the authors show the efficacy of GLUT1 pharmacological inhibition against a subset of tumors expressing RB1, thereby identifying RB1 protein level as a biomarker of sensitivity to anti-GLUT1 therapy