Bromodomain (BRD)-containing proteins are chemically tractable multi-domain scaffolding molecules involved in acetyl lysine (Kac) signaling. BRD inhibitors have shown promise in clinical oncology, including melanomas; however, their narrow therapeutic windows and issues with resistance in pre-clinical models highlight the need to better understand the functions of and interconnection between BRD-containing proteins. Here, we use complementary interaction-mapping techniques (affinity purification and proximity-dependent biotinylation) to map the interactions of 39 of the 42 BRD-containing proteins and 110 additional proteins that physically or functionally associate with them. We uncover 3,892 novel interactions and reveal the intricate connectivity of the Kac machinery. Chemical inhibition of multiple BRD classes revealed that inhibiting BETs—but not mSWI/SNF or CREBBP/EP300 proteins—dramatically rewired the interactome. Finally, we identified MAPKAPK2 activity as a critical determinant of BET inhibitor sensitivity in melanoma through its impact on chromatin composition remodeling. In Brief Kougnassoukou Tchara et al . generate a static protein interaction map of the human acetyl lysine machinery by coupling two complementary functional proteomics approaches (FLAG affinity purification and proximity-dependent biotinylation) to mass spectrometry. They also investigate network changes upon bromodomain inhibition, and describe a novel resistance mechanism mediated by the p38 stress signaling pathway that causes significant metabolic changes. Highlights ### Competing Interest Statement The authors have declared no competing interest.

Just like the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using 2 human PCa tissue microarray cohorts, we first demonstrate that nuclear ERα expression was heterogeneous among patients, being detected in only half of the tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimicked the androgen transcriptional response and activated specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprogrammed PCa metabolism, was associated with disease progression, and could be targeted for therapeutic purposes.

Sex hormones are necessary for the development and functions of the normal prostate as well as for the initiation and progression of prostate tumors. Indeed, androgens and estrogens can activate their respective nuclear receptors to modulate the expression of multiple genes and pathways in prostate cells. Nevertheless, the androgen and estrogen responses in the normal prostate, and the transcriptomic changes occurring after carcinogenesis, remain poorly understood. Here, wildtype mice and transgenic mice that spontaneously develop prostate cancer (C57BL/6J PB-Cre4+/−;Ptenfl/fl) were castrated to ensure hormone deprivation. After three days, animals received injections of testosterone and/or estradiol. After one day, the prostates were harvested, and RNA was purified for sequencing. Sequencing data were then analyzed to study transcriptional modulations following hormonal exposures in normal and tumoral murine prostates. New analyses can be carried out with specific fold-change thresholds for gene expression, or with different pair-wise combinations between conditions (treatments and/or mouse models). Together, the data generated herein are a useful tool to study hormonal transcriptional responses in prostate and prostate cancer biology.

Prostate cancer is a hormone-dependent disease that relies on the androgen signaling, as well as on the estrogen signaling, for growth and survival. To identify the genes regulated by these sex-steroid hormones in the human prostate cancer cell line VCaP, these cells were treated for 24 h with either androgens and/or estrogens. Then, the RNA of each sample was purified for sequencing to generate bulk mRNA-seq data. After verifying raw quality, reads were pseudo-aligned on the human reference transcriptome (Gencode v27). Analysis was carried out on aligned and quantified data to determine the transcriptomic changes following each hormonal treatment. These data presented herein can be reanalyzed with specific fold-change thresholds for gene expression, or with different pair-wise combinations to compare the hormones’ transcriptional impacts on VCaP cells and better understand prostate cancer cell biology.

Winter flounder (Pseudopleuronectes americanus) is a North Atlantic flatfish that inhabits cold-water environments already affected by global warming. Flatfishes are particularly sensitive during their juvenile stage to a phenomenon known as temperature-dependent sex determination (TSD). In this study, we hypothesized that many genes involved in the steroidogenesis pathway are already expressed at the larval stage in winter flounder and that temperature conditions may influence this pathway prior to the juvenile stage, which is usually considered the TSD-sensitive period. We also hypothesized that temperature effects on the steroidogenesis pathway may carry over from the larval to the juvenile stage. We surveyed three cohorts issued from three different spawning events during spring. Rearing temperature and salinity conditions followed seasonal conditions in the St. Lawrence Estuary (Québec, Canada). As a result, cohorts were exposed to different temperature regimes during egg and larval development. Once metamorphosis occurred, all juveniles were held at a stable temperature of 10.1 ± 0.8 °C. Larvae were sampled at 20, 30, and, 40 days post-hatching and juveniles at one and two months post-metamorphosis. RNA was extracted from these individuals and then sequenced. Transcriptome analysis showed clear differentiation between larvae and juveniles. Gene expression patterns showed that the steroidogenesis pathway, including genes involved in gonadal differentiation, was influenced by temperature during larval development, which indicates that sexual determination at the cellular level is an early process in winter flounder development. Carry-over effects of larval rearing temperature conditions were present at the juvenile stage, with an increased number of genes involved in the steroidogenesis pathway being affected. Altogether, our study highlights the important role of larval development and temperature exposure on the transcriptome of winter flounder.

Prostate cancer is well known to be dependent on the androgen receptor (AR) for growth and survival. Thus, AR is the main pharmacological target to treat this disease. However, after an initially positive response to AR-targeting therapies, prostate cancer will eventually evolve to castration-resistant prostate cancer, which is often lethal. Tumour growth was initially thought to become androgen-independent following treatments; however, results from molecular studies have shown that most resistance mechanisms involve the reactivation of AR. Consequently, tumour cells become resistant to castration — the blockade of testicular androgens — and not independent of AR per se. However, confusion still remains on how to properly define preclinical models of prostate cancer, including cell lines. Most cell lines were isolated from patients for cell culture after evolution of the tumour to castration-resistant prostate cancer, but not all of these cell lines are described as castration resistant. Moreover, castration refers to the blockade of testosterone production by the testes; thus, even the concept of “castration” in vitro is questionable. To ensure maximal transfer of knowledge from scientific research to the clinic, understanding the limitations and advantages of preclinical models, as well as how these models recapitulate cancer cell androgen dependency and can be used to study castration resistance mechanisms, is essential.

Due to its sensitivity to hormonal signaling, the mammary gland is often referred to as a sentinel organ for the study of endocrine-disrupting chemicals (EDCs), environmental pollutants that can interfere with the estrogen signaling pathway and induce mammary developmental defects. If and how EDCs impact mammary epithelial cell metabolism has not yet been documented. Herein, to study how estrogens and EDCs modulate mammary gland metabolism, we performed bioenergetic flux analyses using mouse mammary epithelial organoids compared to cells grown in monolayer culture. Several EDCs were tested, including bisphenol A (BPA), its close derivative BPS, a new BPA replacement copolyester called TritanTM, and the herbicide glyphosate. We report that estrogens reprogrammed mammary epithelial cell metabolism differently when grown in two- and three-dimensional models. Specific EDCs were also demonstrated to alter bioenergetic fluxes, thus identifying a new potential adverse effect of these molecules. Notably, organoids were more sensitive to low EDC concentrations, highlighting them as a key model for screening the impact of various environmental pollutants. Mechanistically, transcriptomic analyses revealed that EDCs interfered with the regulation of estrogen target genes and the expression of metabolic genes in organoids. Furthermore, co-treatment with the anti-estrogen fulvestrant blocked these metabolic impacts of EDCs, suggesting that, at least partially, they act through modulation of the estrogen receptor activity. Finally, we demonstrate that mammary organoids can be used for long-term studies on EDC exposure to study alterations in organogenesis/morphogenesis and that past pregnancies can modulate the sensitivity of mammary epithelial organoids to specific EDCs. Overall, this study demonstrates that estrogens and EDCs modulate mammary epithelial cell metabolism in monolayer and organoid cultures. A better understanding of the metabolic impacts of EDCs will allow a better appreciation of their adverse effects on mammary gland development and function.

The estrogen signaling pathway has been reported to modulate prostate cancer (PCa) progression through the activity of estrogen receptors α and β (ERα and ERβ). Given that selective estrogen receptor modulators (SERMs) are used to treat breast cancer, ERs have been proposed as attractive therapeutic targets in PCa. However, many inconsistencies regarding the expression of ERs and the efficacy of SERMs for PCa treatment exist, notably due to the use of ERβ antibodies lacking specificity and treatments with high SERM concentrations leading to off-target effects. To end this confusion, our objective was to study the impact of estrogenic and anti-estrogenic ligands in well-studied in vitro PCa models with appropriate controls, dosages, and ER subtype-specific antibodies. When using physiologically relevant concentrations of nine estrogenic/anti-estrogenic compounds, including five SERMs, we observed no significant modulation of PCa cell proliferation. Using RNA-seq and validated antibodies, we demonstrate that these PCa models do not express ERs. In contrast, RNA-seq from PCa samples from patients have detectable expression of ERα. Overall, our study reveals that commonly used PCa models are inappropriate to study ERs and indicate that usage of alternative models is essential to properly assess the roles of the estrogen signaling pathway in PCa.

The androgen receptor (AR) is a transcription factor that drives prostate cancer (PCa) by modulating the expression of thousands of genes to promote proliferation and survival and to reprogram metabolism. However, how AR activation controls alternative splicing is mostly unknown. Our objective was to define its role in the transcriptome-wide regulation of alternative splicing. Three human PCa models-LNCaP, LAPC4, and 22Rv1 cells-were treated with and without androgens, and RNA was purified for deep-sequencing analyses (RNA-seq). Several bioinformatic tools were then used to study alternative splicing. We demonstrate that in the absence of androgens, alternative splicing complexity is similar among AR-positive PCa cells, with 48 % of all transcripts having various levels of alternative splicing. We also describe alternative splicing differences among cell lines, such as specific splicing of AR, REST, TSC2, and CTBP1. Interestingly, AR activation changed the alternative splicing of thousands of genes in all the PCa cell lines tested. Overlap between AR-sensitive alternative splicing events revealed that genes linked to cell metabolism are major targets for this specific modulation. These genes encode metabolic enzymes such as the prostate-specific membrane antigen, encoded by FOLH1, and the malate dehydrogenase 1 (MDH1). Overall, our study presents a comprehensive analysis of the PCa cell transcriptome and its modulation by AR, revealing a significant enrichment of metabolic genes in this AR-dependent regulation of alternative splicing.