The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

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.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Preclinical models of prostate cancer - modelling androgen dependency and castration resistance in vitro, ex vivo and in vivo.

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.

Impacts of endocrine-disrupting chemicals on prostate function and cancer.

Because of their historical mode of action, endocrine-disrupting chemicals (EDCs) are associated with sex-steroid receptors, namely the two estrogen receptors (ERα and ERß) and the androgen receptor (AR). Broadly, EDCs can modulate sex-steroid receptor functions. They can also indirectly impact the androgen and estrogen pathways by influencing steroidogenesis, expression of AR or ERs, and their respective activity as transcription factors. Additionally, many of these chemicals have multiple cellular targets other than sex-steroid receptors, which results in a myriad of potential effects in humans. The current article reviews the association between prostate cancer and the endocrine-disrupting functions of four prominent EDC families: bisphenols, phthalates, phytoestrogens, and mycoestrogens. Results from both in vitro and in vivo models are included and discussed to better assess the molecular mechanisms by which EDCs can modify prostate biology. To overcome the heterogeneity of results published, we established common guidelines to properly study EDCs in the context of endocrine diseases. Firstly, the expression of sex-steroid receptors in the models used must be determined before testing. Then, in parallel to EDCs, pharmacological compounds acting as positive (agonists) and negative controls (antagonists) have to be employed. Finally, EDCs need to be used in a precise range of concentrations to modulate sex-steroid receptors and avoid off-target effects. By adequately integrating molecular endocrinology aspects in EDC studies and identifying their underlying molecular mechanisms, we will truly understand their impact on prostate cancer and distinguish those that favor the progression of the disease from those that slow down tumor development.

KLF5 and NFYA factors as novel regulators of prostate cancer cell metabolism.

Prostate cancer (PCa) cells rely on the androgen receptor (AR) signaling axis to reprogram metabolism to sustain aberrant proliferation. Whether additional transcription factors participate to this reprogramming remains mostly unknown. To identify such factors, DNA motif analyses were performed in the promoter and regulatory regions of genes sensitive to androgens in PCa cells. These analyses identified two transcription factors, KLF5 and NFYA, as possibly associated with PCa cell metabolism. In clinical datasets, KLF5 and NFYA expression levels were associated with disease aggressiveness, being significantly decreased and increased, respectively, during PCa progression. Their expression was next investigated by qPCR and Western blot in human PCa cell models, revealing a positive regulation of KLF5 by androgens and a correlation between NFYA and AR protein expression status. siRNA-mediated knockdown of KLF5 increased human PCa cell proliferation rate in AR-positive cell models, suggesting a tumor suppressor function. Live-cell metabolic assays showed that knockdown of KLF5 promoted mitochondrial respiration, a key metabolic pathway associated with PCa progression. The opposite was observed for knockdown of NFYA regarding proliferation and respiration. RNA-seq analyses following the knockdown of either KLF5 and NFYA confirmed that both factors regulated distinct metabolic gene signatures, as well as other gene signatures, explaining their differential impact on PCa cell proliferation and metabolism. Overall, our findings identify KLF5 and NFYA as novel regulators of PCa cell metabolism.

Alternative splicing regulation by the androgen receptor in prostate cancer cells.

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.

A Systematic Study of the Impact of Estrogens and Selective Estrogen Receptor Modulators on Prostate Cancer Cell Proliferation.

The estrogen signaling pathway has been reported to modulate prostate cancer (PCa) progression through the activity of estrogen receptors α and ß (ERα and ERß). 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 ERß 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.