Androgen Receptor Signaling Pathway Research Articles

Octopus maya white body show sex-specific transcriptomic profiles during the reproductive phase, with high differentiation in signaling pathways.

White bodies (WB), multilobulated soft tissue that wraps the optic tracts and optic lobes, have been considered the hematopoietic organ of the cephalopods. Its glandular appearance and its lobular morphology suggest that different parts of the WB may perform different functions, but a detailed functional analysis of the octopus WB is lacking. The aim of this study is to describe the transcriptomic profile of WB to better understand its functions, with emphasis on the difference between sexes during reproductive events. Then, validation via qPCR was performed using different tissues to find out tissue-specific transcripts. High differentiation in signaling pathways was observed in the comparison of female and male transcriptomic profiles. For instance, the expression of genes involved in the androgen receptor-signaling pathway were detected only in males, whereas estrogen receptor showed higher expression in females. Highly expressed genes in males enriched oxidation-reduction and apoptotic processes, which are related to the immune response. On the other hand, expression of genes involved in replicative senescence and the response to cortisol were only detected in females. Moreover, the transcripts with higher expression in females enriched a wide variety of signaling pathways mediated by molecules like neuropeptides, integrins, MAPKs and receptors like TNF and Toll-like. In addition, these putative neuropeptide transcripts, showed higher expression in females’ WB and were not detected in other analyzed tissues. These results suggest that the differentiation in signaling pathways in white bodies of O. maya influences the physiological dimorphism between females and males during the reproductive phase.

Androgen-responsive lncRNA LINC00304 promotes cell cycle and proliferation via regulating CCNA1.

Long noncoding RNA (lncRNA) plays a vital role in the development of many diseases. The abnormal expression of lncRNA is closely related to the occurrence and development of different kinds of tumors including prostate cancer (PCa). Differentially expressed lncRNA LINC00304 was identified using a publicly available gene expression data set (GSE38241) and quantitative polymerase chain reaction validation. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict the molecular function of LINC00304. A lncRNA microarray, bioinformatic analysis, and chromatin immunoprecipitation assay were carried out to verify the upstream androgen receptor (AR) signaling pathway. Subsequently, the function of LINC00304 was observed by a series of in vitro assays. We observed higher expression of LINC00304 in PCa cells and samples compared with normal prostate cells and tissues. Functional analysis of LINC00304 showed it was related to regulating cell cycle process, cellular developmental process, and focal adhesion. Further, we identified androgen-inhibited lncRNA, LINC00304 as a direct target of AR. A series of functional studies revealed that overexpression of LINC00304 could significantly promote cell proliferation and cell cycle progression in PCa cells. We also find that LINC00304 can significantly promote CCNA1 expression in PCa cells. Our results indicate that LINC00304 may represent a new diagnostic and therapeutic biomarker for PCa.

Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer.

Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients' outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

Interactions between Germline and Somatic Mutated Genes in Aggressive Prostate Cancer.

Prostate cancer (PCa) is the most common diagnosed malignancy and the second leading cause of cancer-related deaths among men in the USA. Advances in high-throughput genotyping and next generation sequencing technologies have enabled discovery of germline genetic susceptibility variants and somatic mutations acquired during tumor formation. Emerging evidence indicates that germline variations may interact with somatic events in carcinogenesis. However, the possible oncogenic interactions and cooperation between germline and somatic variation and their role in aggressive PCa remain largely unexplored. Here we investigated the possible oncogenic interactions and cooperation between genes containing germline variation from genome-wide association studies (GWAS) and genes containing somatic mutations from tumor genomes of 305 men with aggressive tumors and 52 control samples from The Cancer Genome Atlas (TCGA). Network and pathway analysis were performed to identify molecular networks and biological pathways enriched for germline and somatic mutations. The analysis revealed 90 functionally related genes containing both germline and somatic mutations. Transcriptome analysis revealed a 61-gene signature containing both germline and somatic mutations. Network analysis revealed molecular networks of functionally related genes and biological pathways including P53, STAT3, NKX3-1, KLK3, and Androgen receptor signaling pathways enriched for germline and somatic mutations. The results show that integrative analysis is a powerful approach to uncovering the possible oncogenic interactions and cooperation between germline and somatic mutations and understanding the broader biological context in which they operate in aggressive PCa.

Combination of phospholipase Cε knockdown with GANT61 sensitizes castration‑resistant prostate cancer cells to enzalutamide by suppressing the androgen receptor signaling pathway.

Castration-resistant prostate cancer (CRPC) is a major challenge in the treatment of prostate cancer (PCa). Phospholipase Cε (PLCε), an oncogene, has been found to be involved in the carcinogenesis, tumor proliferation and migration of several types of cancer. The effects, however, of PLCε on CRPC remains unclear. In the present study, the expression of PLCε and glioma-associated homolog (Gli)-1/Gli-2 in benign prostatic hyperplasia (BPH), PCa and CRPC tissues and cells was investigated, and the correlations between PLCε and Gli-1/Gli-2 in CRPC tissues and cell lines were further explored. In addition, the effect of PLCε on cell proliferation and invasion was assessed in CRPC cell lines, and the sensitivity of EN-R and 22RV1 cells to enzalutamide following the downregulation of PLCε expression was determined using lentivirus-mediated shPLCε and/or treatment with specific Gli inhibitor GANT61. It was found that the PLCε expression was excessively upregulated in the majority of CRPC tissues, and PLCε positivity was linked to poor progression-free survival (PFS) and overall survival (OS) in patients with PCa. Furthermore, PLCε knockdown significantly suppressed CRPC cell proliferation and invasion. Of note, it was found that PLCε knockdown increased the sensitivity of CRPC cells to enzalutamide in vitro by suppressing androgen receptor (AR) activities via the non-canonical Hedgehog/Gli-2 and p-STAT3 signaling pathways. PLCε knockdown was shown to increase the sensitivity of CRPC cell xenografts to enzalutamide in vivo. Finally, the combination of PLCε knockdown with GANT61 significantly sensitized CRPC cells to enzalutamide. Collectively, the results of the present study suggest that PLCε is a potential therapeutic target for CRPC.

Research progress in chemo-resistance mechanism of castration resistant prostate cancer

Prostate cancer is one of the most common malignancies in old males. While chemotherapy remains an effective method for metastatic castration resistant prostate cancer (mCRPC), most patients develop chemo-resistance with median survival less than 1 year after six to eight cycles of systematic chemotherapy. Therefore, it is very important to elucidate the mechanisms of CRPC and chemotherapy resistance. This article is to focus on the androgen receptor signaling pathway, micro-RNA, tumor microenvironment, cancer stem cells as well as related molecular events, and to summarize the research progress. Key words: Castration resistant prostate cancer; Chemotherapy resistance; Mechanism

Novel therapeutic avenues in triple-negative breast cancer: PI3K/AKT inhibition, androgen receptor blockade, and beyond.

Multiomic analyses have shed light upon the molecular heterogeneity and complexity of triple-negative breast cancers (TNBCs). With increasing recognition that TNBC is not a single disease entity but encompasses different disease subtypes, a one-size-fits-all treatment paradigm has become obsolete. In this context, the inhibition of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and androgen receptor (AR) signaling pathways have emerged as potential therapeutic strategies against selected tumors. In this paper, we reviewed the preclinical rationale, predictive biomarkers, efficacy, and safety data from early phase trials, and the future directions for these two biomarker-directed treatment approaches in TNBC.

4-1BBL has a Possible Role in Mediating Castration-Resistant Conversion of Prostate Cancer via Up-Regulation of Androgen Receptor.

4-1BB ligand (4-1BBL) was a transmembrane glycoprotein belonging to the tumor necrosis factor family. It was expressed on activated T lymphocytes and function as a co-stimulatory molecule via cross-linking with 4-1BB (a.k.a, CD137). In addition to its role in immune regulation, 4-1BBL transmitted signals into the cells on which it was expressed (reverse signaling). 4-1BBL represented a promising target for enhancing antitumor immune responses. Recent studies indicated that 4-1BBL also expressed in non-immune cells and possessed different functions in various types of cells. Here, we reported that 4-1BBL didn't express in normal prostate tissues and benign prostatic hyperplasia tissues, but it expressed in prostate cancer (PCa) tissues at moderate level. Expression of 4-1BBL was up-regulated during the transition from PCa to castration resistant prostate cancer (CRPC). Increasing expression of 4-1BBL not only promoted expression of androgen receptor (AR), but also augmented proliferation and invasion ability of prostate cancer cells in androgen deprivation environment. These results were further verified by xenograft tumor experiments. Meanwhile, inhibiting AR signal pathway by chemical antagonist was able to significantly reduce 4-1BBL mediated proliferation and invasion of PCa cells. These novel findings indicated that 4-1BBL might mediate prostate cancer progression to castration-resistant prostate cancer via enhancing expression and function of AR.

Androgen receptor and soy isoflavones in prostate cancer.

Androgens and androgen receptor (AR) play a critical role not only in normal prostate development, but also in prostate cancer. For that reason, androgen deprivation therapy (ADT) is the primary treatment for prostate cancer. However, the majority of patients develop castration-resistant prostate cancer, which eventually leads to mortality. Novel therapeutic approaches, including dietary changes, have been explored. Soy isoflavones have become a focus of interest because of their positive health benefits on numerous diseases, particularly hormone-related cancers, including prostate and breast cancers. An important strategy for the prevention and/or treatment of prostate cancer might thus be the action of soy isoflavones on the AR signaling pathway. The current review article provides a detailed overview of the anticancer potential of soy isoflavones (genistein, daidzein and glycitein), as mediated by their effect on AR.

Discovery of Novel Androgen Receptor Ligands by Structure-based Virtual Screening and Bioassays

Androgen receptor (AR) is a ligand-activated transcription factor that plays a pivotal role in the development and progression of many severe diseases such as prostate cancer, muscle atrophy, and osteoporosis. Binding of ligands to AR triggers the conformational changes in AR that may affect the recruitment of coactivators and downstream response of AR signaling pathway. Therefore, AR ligands have great potential to treat these diseases. In this study, we searched for novel AR ligands by performing a docking-based virtual screening (VS) on the basis of the crystal structure of the AR ligand binding domain (LBD) in complex with its agonist. A total of 58 structurally diverse compounds were selected and subjected to LBD affinity assay, with five of them (HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58) exhibiting strong binding to AR-LBD. The IC50 values of HBP1-51 and HBP1-58 are 3.96 µM and 4.92 µM, respectively, which are even lower than that of enzalutamide (Enz, IC50 = 13.87 µM), a marketed second-generation AR antagonist. Further bioactivity assays suggest that HBP1-51 is an AR agonist, whereas HBP1-58 is an AR antagonist. In addition, molecular dynamics (MD) simulations and principal components analysis (PCA) were carried out to reveal the binding principle of the newly-identified AR ligands toward AR. Our modeling results indicate that the conformational changes of helix 12 induced by the bindings of antagonist and agonist are visibly different. In summary, the current study provides a highly efficient way to discover novel AR ligands, which could serve as the starting point for development of new therapeutics for AR-related diseases.

Exploration of the diagnostic value and molecular mechanism of miR‑1 in prostate cancer: A study based on meta‑analyses and bioinformatics.

Prostate cancer (PCa) remains a principal issue to be addressed in male cancer-associated mortality. Therefore, the present study aimed to examine the clinical value and associated molecular mechanism of microRNA (miR)-1 in PCa. A meta-analysis was conducted to evaluate the diagnosis of miR-1 in PCa via Gene Expression Omnibus and ArrayExpress datasets, The Cancer Genome Atlas miR-1 expression data and published literature. It was identified that expression of miR-1 was significantly downregulated in PCa. Decreased miR-1 expression possessed moderate diagnostic value, with area under the curve, sensitivity, specificity and odds ratio values at 0.73, 0.77, 0.57 and 4.60, respectively. Using bioinformatics methods, it was revealed that a number of pathways, including the ‘androgen receptor signaling pathway’, ‘androgen receptor activity’, ‘transcription factor binding’ and ‘protein processing in the endoplasmic reticulum’, were important in PCa. A total of seven hub genes, including phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccin ocarboxamide synthase (PAICS), cadherin 1 (CDH1), SRC proto-oncogene, non-receptor tyrosine kinase, twist family bHLH transcription factor 1 (TWIST1), ZW10 interacting kinetochore protein (ZWINT), PCNA clamp associated factor (KIAA0101) and androgen receptor, among which, five (PAICS, CDH1, TWIST1, ZWINT and KIAA0101) were significantly upregulated and negatively correlated with miR-1, were identified as key miR-1 target genes in PCa. Additionally, it was investigated whether miR-1 and its hub genes were associated with clinical features, including age, tumor status, residual tumor, lymph node metastasis, pathological T stage and prostate specific antigen level. Collectively the results suggest that miR-1 may be involved in the progression of PCa, and consequently be a promising diagnostic marker. The ‘androgen receptor signaling pathway’, ‘androgen receptor activity’, ‘transcription factor binding’ and ‘protein processing in the endoplasmic reticulum’ may be crucial interactive pathways in PCa. Furthermore, PAICS, CDH1, TWIST1, ZWINT and KIAA0101 may serve as crucial miR-1 target genes in PCa.

Enzalutamide-resistant castration-resistant prostate cancer: challenges and solutions.

The new-generation hormonal agent enzalutamide has been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC), in both post- and predocetaxel setting, due to the significant improvement in overall survival. More recently, enzalutamide also showed impressive results in the treatment of men with nonmetastatic CRPC. Unfortunately, not all patients with CRPC are responsive to enzalutamide, and even in responders, benefits are limited by the development of drug resistance. Adaptive resistance of metastatic prostate cancer to enzalutamide treatment can be due to the activation of both androgen receptor (AR)-dependent pathways (expression of constitutively active AR splice variants, AR point mutations, gene amplification and overexpression) and mechanisms independent of AR signaling pathway (altered steroidogenesis, upregulation of the glucocorticoid receptor, epithelial–mesenchymal transition, neuroendocrine transformation, autophagy and activation of the immune system). In this review, we focus on resistance mechanisms to enzalutamide, exploring how we could overcome them through novel therapeutic options.

The high expression of FOXA1 is correlated with a favourable prognosis in salivary duct carcinomas: a study of 142 cases.

Salivary duct carcinoma (SDC) is an uncommon, aggressive tumour that, histologically, resembles high-grade mammary ductal carcinoma, and is characterised by the expression of androgen receptor (AR). The androgen signalling pathway, a potential therapeutic target, can be regulated by FOXA1. This study aimed to evaluate the clinicopathological implications of FOXA1 in SDC. We examined the relationship between the immunoexpression of FOXA1 and FOXA1 mutations and clinicopathological factors, including the biomarker status and clinical outcome, in 142 SDCs. FOXA1 was expressed in 128 SDCs (90.1%); the immunoexpression was heterogeneous. SDCs with a higher FOXA1 labelling index (LI) (≥20%) more frequently showed less advanced tumors on T classification (P=0.002). FOXA1 LI was correlated positively with the AR expression value (r=0.430, P<0.001). PI3K and p-mTOR positivity, and intact-PTEN, were associated with a higher FOXA1 LI. Twenty-two of 121 SDCs (18.2%) harboured FOXA1 gene mutations at the flanking regions in and around the forkhead DNA binding domain; however, the given gene mutation and the expression of FOXA1 were not significantly correlated. A multivariate analysis revealed that SDCs with a higher FOXA1 LI were associated with longer overall survival and progression-free survival (P=0.029 and 0.016, respectively). In SDC, FOXA1, which may biologically interact with the AR and PI3K signalling pathways, is a putative biomarker that may be associated with a favourable prognosis. Further studies are needed to apply the findings to the development of targeted personalised therapy for patients with SDC.

JQ1: a novel potential therapeutic target.

The bromo- and extra-terminal domain (BET) signaling pathway plays an important role in cell proliferation, immune responses, and pro-inflammatory events. JQ1 as a first-in-class potent and selective inhibitor of the BRD4 signaling pathway is widely used for tumor biology studies. It was found that JQ1 could potently reduce cancer cell viability in vitro and in vivo. The underlying mechanisms include an effect on cell cycle arrest in the G1 phase and a decrease in the percentage of cells in the S phase. Furthermore, JQ1 could alter cytokines expressions not only in T cells but also in dendritic cells (DCs). Apoptosis of tumor cells was induced by JQ1 through downregulation of E2f1 protein expression. In addition, JQ1 exhibited a potent suppressive effect on ERα and androgen receptor (AR) signaling pathways in breast and prostate cancers. Accumulating evidence supports the notion of BRD4 suppression as a target of therapeutic intervention in clinical oncology. Our present review article advances the understanding of the role of the JQ1 / BRD4 protein.

Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Abstract Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.

Abstract A078: Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism

Abstract An estimated 1 in 7 men will develop prostate cancer (PCa) with many progressing to advanced castrate-resistant disease. Unlike other tissue types, normal prostate cell growth and development is heavily dependent on the androgen receptor (AR) signaling pathway. While the introduction of novel AR antagonists for clinical treatment has improved outcomes, most castration-resistant prostate cancer (CRPC) patients ultimately develop resistance to these therapies. A need exists to better understand the mechanisms that control the transition of prostate cells from a hormone-dependent to castrate-resistant state. Androgens strongly influence the metabolic state of PCa cells to favor sustained cellular growth. We hypothesize there are effectors working in conjunction with AR to coordinate alterations to androgen-dependent metabolism that are linchpins in the orchestration of the transition to CRPC. Leading candidates are members of phosphoinositol (PI) pathways, which have a high frequency of alteration in PCa (i.e phosphoinositide 3-kinase (PI3K)). Herein we explore a family of poorly understood lipid kinases called the type II phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks) and predict them to be critical regulators of cancer cell survival. PI5P4Ks are druggable targets that act by phosphorylating the lipid phosphatidylinositol-5-phosphate (PI 5-P) at the 4 position of the inositol ring to generate phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2; PIP2). We implicate the three PI5P4K isoforms (PI5P4Kα, PI5P4Kβ, and PI5P4Kγ) encoded by the genes PIP4K2A, B, and C, to be important regulators of cancer metabolism that play a role in the maintenance of prostate biology and oncogenesis. Analysis of transcript data revealed expression of PIP4K2A, B, and C in primary PCa patient samples, which was correlated with an AR activation gene signature and hotspot tumor suppressor deletion. As well, isoform expression was assessed for differential expression in relation to an integrated neuroendocrine prostate cancer mRNA score (TCGA; n=333). PI5P4Kα and PI5P4Kβ protein was detected in primary and advanced prostate cancer using optimized antibodies of patient tissue TMAs (n= 72). Using in vitro LNCaP cell models, siRNA knockdown systems were tested to evaluate the molecular consequence of targeting PIP4K2A and PIP4K2B in androgen-dependent systems. Stable knockdown using fluorescently labeled lentiviral shRNA constructs significantly reduced proliferation of shPIP4K2 treated cells. As well, we have produced a prostate-specific PI5P4K knockout mouse model by expressing probasin-driven Cre in a homozygous 129/SvEv Pip4k2aflx/flx murine strain. Finally, implementation of a discovery-based metabolomic platform (Metabolon HD4) was used to profile the overall shift in metabolite species that results from downregulating the expression of PIP4K2A in androgen-dependent cell models. In summary, we have developed novel insights into the role of a family of noncanonical PI kinases in prostate biology. There are a growing number of PI3K/AKT inhibitors being tested in combination with androgen deprivation therapy in clinical trials, but there is still almost nothing known about the potential crosstalk of the greater PI kinase network. These data convincingly implicate a fundamental role for PI5P4Ks in PCa androgen signaling and metabolism, as well as lay the foundation of phenotypic understanding of what PI5P4K is responsible for in the prostate. Citation Format: Joanna Triscott, Matteo Benelli, Verena Sailer, Davide Prandi, Brooke Emerling, Francesca Demichelis, Lewis Cantley, Mark A. Rubin. Towards understanding noncanonical phosphatidylinositol kinases in the maintenance of prostate metabolism [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A078.

Precision medicine based on tumorigenic signaling pathways for triple-negative breast cancer.

As a clinically heterogeneous subtype of breast cancer, triple-negative breast cancer (TNBC) is associated with a poor clinical outcome and a high relapse rate. Conventional chemotherapy and radiotherapy are effective treatments for patients with TNBC. However, the prognosis of TNBC remains unsatisfactory. Therefore, a large volume of research has explored the molecular markers and oncogenic signaling pathways associated with TNBC, including the cell cycle, DNA damage response and androgen receptor (AR) signaling pathways, to identify more efficient targeted therapies. However, whether these predicted pathways are effective targets has yet to be confirmed. In the present review, potentially carcinogenic signaling pathways in TNBCs from previous reports were considered, and ultimately five tumorigenic signaling pathways were selected, specifically receptor tyrosine kinases and downstream signaling pathways, the epithelial-to-mesenchymal transition and associated pathways, the immunoregulatory tumor microenvironment, DNA damage repair pathways, and AR and coordinating pathways. The conclusions of the preclinical and clinical trials of each pathway were then consolidated. Although a number of signaling pathways in TNBC have been considered in preclinical and clinical trials, the aforementioned pathways account for the majority of the malignant behaviors of TNBC. Identifying the alterations to different carcinogenic signaling pathways and their association with the heterogeneity of TNBC may facilitate the development of optimal precision medical approaches for patients with TNBC, potentially improving the efficiency of anticancer therapy.

High-Content Screening Campaign to Identify Compounds That Inhibit or Disrupt Androgen Receptor-Transcriptional Intermediary Factor 2 Protein-Protein Interactions for the Treatment of Prostate Cancer.

Twenty percent of prostate cancer (PCa) patients develop a noncurable drug-resistant form of the disease termed castration-resistant prostate cancer (CRPC). Overexpression of Androgen Receptor (AR) coactivators such as transcriptional intermediary factor 2 (TIF2) is associated with poor CRPC patient outcomes. We describe the implementation of the AR-TIF2 protein-protein interaction biosensor (PPIB) assay in a high-content screening (HCS) campaign of 143,535 compounds. The assay performed robustly and reproducibly and enabled us to identify compounds that inhibited dihydrotestosterone (DHT)-induced AR-TIF2 protein-protein interaction (PPI) formation or disrupted preexisting AR-TIF2 PPIs. We used multiparameter HCS data z-scores to identify and deprioritize cytotoxic or autofluorescent outliers and confirmed the resulting qualified actives in triplicate. None of the confirmed AR-TIF2 PPIB inhibitors/disruptors exhibited activity in a p53-hDM2 PPIB counter screen, indicating that they were unlikely to be either nonselective PPI inhibitors or to interfere with the biosensor assay format. However, eight confirmed AR-TIF2 PPIB actives also inhibited the glucocorticoid receptor (GR) nuclear translocation counter screen by >50%. These compounds were deprioritized because they either lacked AR specificity/selectivity, or they inhibited a shared component of the AR and GR signaling pathways. Twenty-nine confirmed AR-TIF2 PPIB actives also inhibited the AR nuclear localization counter screen, suggesting that they might indirectly inhibit the AR-TIF2 PPIB assay rather than directly blocking/disrupting PPIs. A total of 62.2% of the confirmed actives inhibited the DHT-induced AR-TIF2 PPI formation in a concentration-dependent manner with IC50s < 40 μM, and 59.4% also disrupted preexisting AR-TIF2 PPI complexes. Overall, the hit rate for the AR-TIF2 PPIB HCS campaign was 0.12%, and most hits inhibited AR-TIF2 PPI formation and disrupted preexisting AR-TIF2 complexes with similar AR-red fluorescent protein distribution phenotypes. Further secondary and tertiary hit characterization assays are underway to select AR-TIF2 PPI inhibitor/disruptor hits suitable for medicinal chemistry lead optimization and development into novel PCa/CRPC therapeutics.

Clinical Development of Darolutamide: A Novel Androgen Receptor Antagonist for the Treatment of Prostate Cancer.

Prostate cancer (PC) is the second most common cancer in men and is the fifth leading cause of cancer-related deaths among men. Androgen receptor (AR) signaling plays a key role in PC tumor growth and progression, with androgens stimulating PC proliferation and survival. Castration-resistant PC (CRPC) is characterized by increasing levels of prostate-specific antigen or radiographic progression despite androgen-deprivation therapy (ADT). In most patients, castration resistance results from aberrations in AR or the AR signaling pathway. Up to one-third of patients with localized high-risk PC will have disease progression on local therapy and develop CRPC. This review summarizes the key clinical data, including ongoing trials, for hormonal therapies in CRPC and provides an overview of the clinical development of darolutamide, a novel, nonsteroidal AR antagonist currently in phase III development for the treatment of nonmetastatic CRPC and metastatic hormone-sensitive PC. In phase I/II trials, darolutamide has demonstrated a favorable safety profile, antitumor activity, and significant decreases in prostate-specific antigen in patients with metastatic CRPC. In the phase III ARAMIS (NCT02200614; A Multinational, Randomized, Double-Blind, Placebo-Controlled, Phase III Efficacy and Safety Study of Darolutamide [ODM-201] in Men With High-Risk Non-metastatic Castration-Resistant Prostate Cancer) study, metastasis-free survival is being evaluated in men with nonmetastatic CRPC who will receive ADT in combination with darolutamide or placebo. The ARASENS (NCT02799602; A Randomized, Double-Blind, Placebo Controlled Phase III Study of Darolutamide [ODM-201] Versus Placebo in Addition to Standard Androgen Deprivation Therapy and Docetaxel in Patients With Metastatic Hormone Sensitive Prostate Cancer) study is a placebo-controlled trial assessing whether the addition of darolutamide to ADT and docetaxel significantly prolongs overall survival in men with metastatic hormone-sensitive PC.

A critical review of the environmental occurrence and potential effects in aquatic vertebrates of the potent androgen receptor agonist 17β-trenbolone.

Trenbolone acetate is widely used in some parts of the world for its desirable anabolic effects on livestock. Several metabolites of the acetate, including 17β-trenbolone, have been detected at low nanograms per liter concentrations in surface waters associated with animal feedlots. The 17β-trenbolone isomer can affect androgen receptor signaling pathways in various vertebrate species at comparatively low concentrations/doses. The present article provides a comprehensive review and synthesis of the existing literature concerning exposure to and biological effects of 17β-trenbolone, with an emphasis on potential risks to aquatic animals. In vitro studies indicate that, although 17β-trenbolone can activate several nuclear hormone receptors, its highest affinity is for the androgen receptor in all vertebrate taxa examined, including fish. Exposure of fish to nanograms per liter water concentrations of 17β-trenbolone can cause changes in endocrine function in the short term, and adverse apical effects in longer exposures during development and reproduction. Impacts on endocrine function typically are indicative of inappropriate androgen receptor signaling, such as changes in sex steroid metabolism, impacts on gonadal stage, and masculinization of females. Exposure of fish to 17β-trenbolone during sexual differentiation in early development can greatly skew sex ratios, whereas adult exposures can adversely impact fertility and fecundity. To fully assess ecosystem-level risks, additional research is warranted to address uncertainties as to the degree/breadth of environmental exposures and potential population-level effects of 17β-trenbolone in sensitive species. Environ Toxicol Chem 2018;37:2064-2078. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.