Tea polyphenol-engineered hybrid cellular nanovesicles for cancer immunotherapy and androgen deprivation therapy.

Androgen deprivation therapy (ADT) is a crucial and effective strategy for prostate cancer, while systemic administration may cause profound side effects on normal tissues. More importantly, the ADT can easily lead to resistance by involving the activation of NF-κB signaling pathway and high infiltration of M2 macrophages in tumor microenvironment (TME). Herein, we developed a biomimetic nanotherapeutic platform by deriving cell membrane nanovesicles from cancer cells and probiotics to yield the hybrid cellular nanovesicles (hNVs), loading flutamide (Flu) into the resulting hNVs, and finally modifying the hNVs@Flu with Epigallocatechin-3-gallate (EGCG). In this nanotherapeutic platform, the hNVs significantly improved the accumulation of hNVs@Flu-EGCG in tumor sites and reprogramed immunosuppressive M2 macrophages into antitumorigenic M1 macrophages, the Flu acted on androgen receptors and inhibited tumor proliferation, and the EGCG promoted apoptosis of prostate cancer cells by inhibiting the NF-κB pathway, thus synergistically stimulating the antitumor immunity and reducing the side effects and resistance of ADT. In a prostate cancer mouse model, the hNVs@Flu-EGCG significantly extended the lifespan of mice with tumors and led to an 81.78% reduction in tumor growth compared with the untreated group. Overall, the hNVs@Flu-EGCG are safe, modifiable, and effective, thus offering a promising platform for effective therapeutics of prostate cancer.

The antiviral potential of the antiandrogen enzalutamide and the viral-androgen signaling interplay in seasonal coronaviruses.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.

NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers.

BACKGROUND: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization. METHODS: Our goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions. RESULTS: Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus. CONCLUSIONS: We showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Analysis of the responsiveness to antiandrogens in multiple breast cancer cell lines.

Antiandrogens were originally developed as therapeutic agents for prostate cancer but are also expected to be effective for breast cancer. However, the role of androgen signaling in breast cancer has long been controversial due to the limited number of experimental models. Our study aimed to comprehensively investigate the efficacy of antiandrogens on breast cancer. In the present study, a total of 18 breast cancer cell lines were treated with the agonist or antagonists of the androgen receptor (AR). Among the 18 cell lines tested, only T-47D cells proliferated in an androgen-dependent manner, while the other cell lines were almost irresponsive to AR stimulation. On the other hand, treatment with AR antagonists at relatively high doses suppressed the proliferation of not only T-47D cells but also some other cell lines including AR-low/negative cells. In addition, expression of the full-length AR and constitutively active AR splice variants, AR-V7 and ARV567es, was not correlated with sensitivity to AR antagonists. These data suggest that the antiproliferative effect of AR antagonists is AR-independent in some cases. Consistently, proliferation of AR-knockout BT-549 cells was inhibited by AR antagonists. Identification of biomarkers would be necessary to determine which breast cancer patients will benefit from these drugs.

Discovery of a novel androgen receptor antagonist, MEL-6, with stereoselective activity and optimization of its metabolic stability.

A new chemical scaffold with antagonistic activity towards the androgen receptor (AR) was identified. The parent compound, (3-Methoxy-N-[1-methyl-2-(4-phenyl-1-piperazinyl)-2-(2-thienyl)ethyl]benzamide) referred to as MEL-6, binds in the ligand binding pocket of AR and induces an antagonistic conformation of the ligand binding domain, even in presence of the antagonist-to-agonist switch mutations W741C, T877A and F876L-T877A. MEL-6 has antiproliferative effects on several AR positive prostate cancer cell lines. We further identified AR as the specific target of MEL-6 since it demonstrates little effect on other steroid receptors. In LNCaP cells it also inhibits the androgen-regulated transcriptome. These findings identify MEL-6 as a promising candidate for treatment of patients with prostate tumors that have become resistant to current clinically used AR antagonists. Analytical studies on the chemical composition of MEL-6 identified the presence of four isomers (two enantiomeric pairs), among which one isomer is responsible for the antiandrogenic activity. We therefore developed a synthetic route towards the selective preparation of the active enantiomeric pair. Various MEL-6-like analogues had improved metabolic stability while maintaining antiandrogenic activity. Metabolite identification of MEL-6 derivatives pinpointed N-dealkylation of the piperazine as the main mode for inactivation by liver enzymes. For further structural optimization, MEL-6 derivatives were purchased or synthesized having alterations on the N-phenyl group of the piperazine, the benzoyl group and additionally substituting the thiophen-2-yl ring of MEL-6 to a phenyl ring. This optimization process resulted in compound 12b with sustained AR inhibition and a 4-fold increased half-life due to the 1-(5-chloro-2-methylphenyl)-piperazine substitution, thienyl-to-phenyl substitution and chloro in para-position of the benzoyl group.

Androgen deprivation induces double-null prostate cancer via aberrant nuclear export and ribosomal biogenesis through HGF and Wnt activation.

Androgen deprivation therapy (ADT) targeting androgen/androgen receptor (AR)- signaling pathways is the main therapy for advanced prostate cancer (PCa). However, ADT eventually fails in most patients who consequently develop castration-resistant prostate cancer (CRPC). While more potent AR antagonists and blockers for androgen synthesis were developed to improve clinical outcomes, they also show to induce more diverse CRPC phenotypes. Specifically, the AR- and neuroendocrine-null PCa, DNPC, occurs in abiraterone and enzalutamide-treated patients. Here, we uncover that current ADT induces aberrant HGF/MET signaling activation that further elevates Wnt/ß-catenin signaling in human DNPC samples. Co-activation of HGF/MET and Wnt/ß-catenin axes in mouse prostates induces DNPC-like lesions. Single-cell RNA sequencing analyses identify increased expression and activity of XPO1 and ribosomal proteins in mouse DNPC-like cells. Elevated expression of XPO1 and ribosomal proteins is also identified in clinical DNPC specimens. Inhibition of XPO1 and ribosomal pathways represses DNPC growth in both in vivo and ex vivo conditions, evidencing future therapeutic targets.

Design and Synthesis of Dual-Target Inhibitors Targeting Androgen Receptors and Glucocorticoid Receptors to Overcome Antiandrogen Resistance in Castration-Resistant Prostate Cancer.

Androgen receptor (AR) antagonists play important roles in the treatment of castration-resistant prostate cancer (CRPC). The glucocorticoid receptor (GR) upregulation leads to drug resistance for clinically used antiandrogens. Therefore, blocking AR/GR signaling simultaneously has become an efficient strategy to overcome the drug resistance of CRPC. Our previous work indicated that Z19 could inhibit the activity of both AR and GR. Herein, we optimized the structure of Z19 and identified GA32 as a potent AR/GR dual inhibitor. GA32 efficiently reduced the mRNA and protein levels of AR/GR downstream genes. GA32 efficiently inhibited the proliferation of enzalutamide resistance CRPC both in vitro and in vivo. GA32 could directly bind to AR and GR, and the predicted binding modes for GA32 with AR/GR suggested that GA32 binds to the AR or GR hormone binding pocket. This work provides a potential lead compound with dual AR/GR inhibitory activity to conquer the drug resistance of CRPC.

Structural Development of Androgen Receptor Antagonists Using Phenylferrocene Framework as a Hydrophobic Pharmacophore.

We previously identified nitrophenylferrocenes and cyanophenylferrocenes as promising lead structures of novel androgen receptor (AR) antagonists, based on the structural similarity between ferrocene and the steroidal skeleton. In the present research, we explored the structure-activity relationship (SAR) of phenylferrocene derivatives. Introduction of a hydrophobic substituent such as a chlorine atom at the 2-position or 3-position of phenylferrocene derivatives significantly increased the antagonistic activity toward wild-type AR, and among the synthesized compounds, 3-chloro-4-cyanophenylferrocene (29) exhibited the most potent anti-proliferative activity toward the androgen-dependent growth of SC-3 cells expressing wild-type AR (IC50 14 nM). Like conventional antiandrogens such as hydroxyflutamide, the major active metabolite of flutamide, compound 29 exhibited agonistic activity toward T877A-AR, a mutant AR expressed in human prostate cancer cell line LNCaP. Notably, however, the 2-chloro isomer 27 showed potent antagonistic activity toward wild-type AR (IC50 49 nM) and also exhibited antagonistic activity toward T877A-AR. Our SAR data should prove helpful for the development of new-generation AR antagonists based on phenylferrocene as candidate agents to treat drug-resistant prostate cancer.

Evaluation of the anti-androgenic and cytotoxic effects of benzophenone-3 in male Sprague-Dawley rats.

Benzophenone-3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) is one of the most widely used types of benzophenone organic sunscreen. However, this compound is a potentially harmful toxicant. The aim of this study was 2-fold to: (1) utilize a Hershberger bioassay in vivo in castrated male Sprague-Dawley rats to investigate the anti-androgenic activities of BP-3, and (2) use in vitro a methyl tetrazolium assay to compare the toxicity between Leydig cells (TM3 cells) and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were divided into 6 groups (each of n = 7): a vehicle control, negative control, positive control, PB-3 low (40 mg/kg), BP-3 intermediate (200 mg/kg), and BP-3 high (1000 mg/kg)-dose. The weight of the ventral prostate was significantly decreased at BP-3 doses of 200 or 1,000 mg/kg/day. In addition, the levator anibulbocavernosus muscle weights were also significantly reduced at BP-3 doses of 40, 200, or 1,000 mg/kg/day. In the MTT assay, the viability of NIH-3T3 mouse fibroblast cells was within the normal range. However, the TM3 mouse testis Leydig cell viability was significantly lowered in a concentration-dependent manner. Therefore, data indicate that BP-3 might exert in vivo anti-androgenic and in vitro cytotoxic effects in cells associated with the male reproductive system compared to normal non-reproductive cells.Abbreviation: BP-3: benzophenone-3; CG: Cowper's gland; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator anibulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate.

Inhibition of the Cyclin K-CDK12 complex induces DNA damage and increases the effect of androgen deprivation therapy in prostate cancer.

Androgen deprivation therapy (ADT) is the mainstay of the current first-line treatment concepts for patients with advanced prostate carcinoma (PCa). However, due to treatment failure and recurrence investigation of new targeted therapeutics is urgently needed. In this study, we investigated the suitability of the Cyclin K-CDK12 complex as a novel therapeutic approach in PCa using the new covalent CDK12/13 inhibitor THZ531. Here we show that THZ531 impairs cellular proliferation, induces apoptosis, and decreases the expression of selected DNA repair genes in PCa cell lines, which is associated with an increasing extent of DNA damage. Furthermore, combination of THZ531 and ADT leads to an increase in these anti-tumoral effects in androgen-sensitive PCa cells. The anti-proliferative and pro-apoptotic activity of THZ531 in combination with ADT was validated in an ex vivo PCa tissue culture model. In a retrospective immunohistochemical analysis of 300 clinical tissue samples we show that Cyclin K (CycK) but not CDK12 expression correlates with a more aggressive type of PCa. In conclusion, this study demonstrates the clinical relevance of the CycK-CDK12 complex as a promising target for combinational therapy with ADT in PCa and its importance as a prognostic biomarker for patients with PCa.

Cyanidin-3-O-glucoside and its derivative vitisin A alleviate androgenetic alopecia by exerting anti-androgen effect and inhibiting dermal papilla cell apoptosis.

Androgenetic alopecia (AGA), one of the most common forms of hair loss, lacks satisfactory treatment methods in modern society. This study employed an experimental design combining in vitro and in vivo approaches to explore the effects of Cyanidin-3-O-glucoside (C3G) and Carboxypyranocyanidin-3-O-glucoside (Vitisin A) on AGA. In human dermal papilla cells (HDPCs), both anthocyanins demonstrated inhibitory effects on androgen receptors, significantly reduced dihydrotestosterone (DHT) induced apoptosis of HDPCs, and regulated the secretion of Fibroblast growth factor 7 and Transforming growth factor beta 1. In vitro transdermal experiment revealed that both C3G and Vitisin A could penetrate mice skin, aided by the application of cream. Furthermore, in vivo experiments with mice indicated that application of C3G or Vitisin A cream effectively improved hair follicles miniaturization, regression, and apoptosis caused by DHT. The repression of Wnt10b and ß-catenin expression induced by DHT was prevented by C3G and Vitisin A in both cell and mouse model. Consequently, these findings suggest that C3G and Vitisin A could be considered as alternative methods for alleviating AGA.

Chromatin accessibility and pioneer factor FOXA1 restrict glucocorticoid receptor action in prostate cancer.

Treatment of prostate cancer relies predominantly on the inhibition of androgen receptor (AR) signaling. Despite the initial effectiveness of the antiandrogen therapies, the cancer often develops resistance to the AR blockade. One mechanism of the resistance is glucocorticoid receptor (GR)-mediated replacement of AR function. Nevertheless, the mechanistic ways and means how the GR-mediated antiandrogen resistance occurs have remained elusive. Here, we have discovered several crucial features of GR action in prostate cancer cells through genome-wide techniques. We detected that the replacement of AR by GR in enzalutamide-exposed prostate cancer cells occurs almost exclusively at pre-accessible chromatin sites displaying FOXA1 occupancy. Counterintuitively to the classical pioneer factor model, silencing of FOXA1 potentiated the chromatin binding and transcriptional activity of GR. This was attributed to FOXA1-mediated repression of the NR3C1 (gene encoding GR) expression via the corepressor TLE3. Moreover, the small-molecule inhibition of coactivator p300's enzymatic activity efficiently restricted GR-mediated gene regulation and cell proliferation. Overall, we identified chromatin pre-accessibility and FOXA1-mediated repression as important regulators of GR action in prostate cancer, pointing out new avenues to oppose steroid receptor-mediated antiandrogen resistance.

Endosomal recycling inhibitors downregulate the androgen receptor and synergise with enzalutamide.

Prostate cancer is the second most frequent cancer diagnosed in men, and accounts for one-fifth of cancer associated deaths worldwide. Despite the availability of effective prostate cancer therapies, if it is not cured by radical local treatment, progression to drug resistant metastatic prostate cancer is inevitable. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. We have recently published research demonstrating that targeting the endosomal recycling pathway, a membrane transport pathway that recycles internalised cell surface proteins back to the plasma membrane, may be a novel means to downregulate clinically relevant cell surface proteins and potentially overcome drug resistance. A reverse phase protein array (RPPA) assay of breast cancer cells treated with an endosomal recycling inhibitor identified the androgen receptor (AR) as one of the top downregulated proteins. We confirmed that endosomal recycling inhibitors also downregulated AR in prostate cancer cells and show that this occurs at the transcriptional level. We also found that endosomal recycling inhibitors synergise with enzalutamide, a standard-of-care therapy for prostate cancer. Our data suggest that combining recycling inhibitors with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

UBE2J1 is the E2 ubiquitin-conjugating enzyme regulating androgen receptor degradation and antiandrogen resistance.

Prostate cancer (PCa) is primarily driven by aberrant Androgen Receptor (AR) signaling. Although there has been substantial advancement in antiandrogen therapies, resistance to these treatments remains a significant obstacle, often marked by continuous or enhanced AR signaling in resistant tumors. While the dysregulation of the ubiquitination-based protein degradation process is instrumental in the accumulation of oncogenic proteins, including AR, the molecular mechanism of ubiquitination-driven AR degradation remains largely undefined. We identified UBE2J1 as the critical E2 ubiquitin-conjugating enzyme responsible for guiding AR ubiquitination and eventual degradation. The absence of UBE2J1, found in 5-15% of PCa patients, results in disrupted AR ubiquitination and degradation. This disruption leads to an accumulation of AR proteins, promoting resistance to antiandrogen treatments. By employing a ubiquitination-based AR degrader to adeptly restore AR ubiquitination, we reestablished AR degradation and inhibited the proliferation of antiandrogen-resistant PCa tumors. These findings underscore the fundamental role of UBE2J1 in AR degradation and illuminate an uncharted mechanism through which PCa maintains heightened AR protein levels, fostering resistance to antiandrogen therapies.

Vortioxetine Reverses Impairment of Visuospatial Memory and Cognitive Flexibility Induced by Degarelix as a Model of Androgen Deprivation Therapy in Rats.

INTRODUCTION: Androgen deprivation therapy (ADT) is a mainstay treatment for prostate cancer, but many patients experience cognitive impairment in domains mediated by the medial prefrontal cortex (mPFC) and hippocampus. Prostate cancer typically occurs in older patients (>65 years). As age is often accompanied by cognitive decline, it may impact the efficacy of any treatment aimed at restoring cognitive impairment induced by ADT. Vortioxetine, a multimodal antidepressant that improves cognition in depression, has been shown to be efficacious in elderly patients. Therefore, vortioxetine may improve cognition in older patients who experience cognitive decline after ADT. METHODS: Young (3 months) and middle-aged (13 months) rats were used to investigate the influence of age on treating ADT-induced cognitive decline. As our previous studies used surgical castration, we tested if vortioxetine would reverse cognitive deficits associated with more translationally relevant chemical castration using degarelix. Vortioxetine was given in the diet for 21 days. Animals underwent behavioral testing to assess visuospatial memory mediated by the hippocampus and cognitive flexibility mediated by the mPFC. We also investigated changes in afferent-evoked responses in these regions in middle-aged rats. RESULTS: Degarelix induced impairments in both visuospatial memory and cognitive flexibility that were reversed by vortioxetine. Vortioxetine also rescued afferent-evoked responses in the mPFC and hippocampus. However, modest age-related reductions in baseline visuospatial memory limited our ability to detect further decreases induced by degarelix in middle-aged rats due to a floor effect. CONCLUSION: These results suggest that vortioxetine may be a treatment option for older prostate cancer patients who experience cognitive decline after ADT.

Prostate lineage-specific metabolism governs luminal differentiation and response to antiandrogen treatment.

Lineage transitions are a central feature of prostate development, tumourigenesis and treatment resistance. While epigenetic changes are well known to drive prostate lineage transitions, it remains unclear how upstream metabolic signalling contributes to the regulation of prostate epithelial identity. To fill this gap, we developed an approach to perform metabolomics on primary prostate epithelial cells. Using this approach, we discovered that the basal and luminal cells of the prostate exhibit distinct metabolomes and nutrient utilization patterns. Furthermore, basal-to-luminal differentiation is accompanied by increased pyruvate oxidation. We establish the mitochondrial pyruvate carrier and subsequent lactate accumulation as regulators of prostate luminal identity. Inhibition of the mitochondrial pyruvate carrier or supplementation with exogenous lactate results in large-scale chromatin remodelling, influencing both lineage-specific transcription factors and response to antiandrogen treatment. These results establish reciprocal regulation of metabolism and prostate epithelial lineage identity.

Androgen receptor inhibition suppresses anti-tumor neutrophil response against bone metastatic prostate cancer via regulation of TßRI expression.

Bone metastatic disease of prostate cancer (PCa) is incurable and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone neutrophils initially inhibit bone metastatic PCa growth yet metastatic PCa becomes resistant to neutrophil response. Further, neutrophils isolated from tumor-bone lost their ability to suppress tumor growth through unknown mechanisms. With this study, our goal was to define the impact of metastatic PCa on neutrophil function throughout tumor progression and to determine the potential of neutrophils as predictive biomarkers of metastatic disease. Using patient peripheral blood polymorphonuclear neutrophils (PMNs), we identified that PCa progression dictates PMN cell surface markers and gene expression, but not cytotoxicity against PCa. Importantly, we also identified a novel phenomenon in which second generation androgen deprivation therapy (ADT) suppresses PMN cytotoxicity via increased transforming growth factor beta receptor I (TßRI). High dose testosterone and genetic or pharmacologic TßRI inhibition rescued androgen receptor-mediated neutrophil suppression and restored neutrophil anti-tumor immune response. These studies highlight the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa.

Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

Intratumoral androgen biosynthesis associated with 3ß-hydroxysteroid dehydrogenase 1 promotes resistance to radiotherapy in prostate cancer.

Half of all men with advanced prostate cancer (PCa) inherit at least 1 copy of an adrenal-permissive HSD3B1 (1245C) allele, which increases levels of 3ß-hydroxysteroid dehydrogenase 1 (3ßHSD1) and promotes intracellular androgen biosynthesis. Germline inheritance of the adrenally permissive allele confers worse outcomes in men with advanced PCa. We investigated whether HSD3B1 (1245C) drives resistance to combined androgen deprivation and radiotherapy. Adrenally permissive 3ßHSD1 enhanced resistance to radiotherapy in PCa cell lines and xenograft models engineered to mimic the human adrenal/gonadal axis during androgen deprivation. The allele-specific effects on radiosensitivity were dependent on availability of DHEA, the substrate for 3ßHSD1. In lines expressing the HSD3B1 (1245C) allele, enhanced expression of DNA damage response (DDR) genes and more rapid DNA double-strand break (DSB) resolution were observed. A correlation between androgen receptor (AR) expression and increased DDR gene expression was confirmed in 680 radical prostatectomy specimens. Treatment with the nonsteroidal antiandrogen enzalutamide reversed the resistant phenotype of HSD3B1 (1245C) PCa in vitro and in vivo. In conclusion, 3ßHSD1 promotes prostate cancer resistance to combined androgen deprivation and radiotherapy by upregulating DNA DSB repair. This work supports prospective validation of early combined androgen blockade for high-risk men harboring the HSD3B1 (1245C) allele.

Monitoring Glucocorticoid Receptor in Plasma-derived Extracellular Vesicles as a Marker of Resistance to Androgen Receptor Signaling Inhibition in Prostate Cancer.

Disease progression following androgen ablation was shown to be associated with upregulation of the glucocorticoid receptor (GR). Longitudinal monitoring of GR expression in circulating extracellular vesicles (EV) may reflect changes in the tumor cell and facilitates detection of acquired resistance. We utilized LNCaP, LREX cells and a patient-derived xenograft, MDA PDX 322-2-6a, for in vitro and in vivo experiments. Plasma-derived EVs were isolated from patients with localized high-risk prostate cancer undergoing androgen ablation. The mRNA levels of GR in EVs and their responsive genes were detected by transcriptome analysis, qRT-PCR and the protein levels by Western blot analysis. We detected changes in GR expression at mRNA and protein levels in EVs derived from LNCaP and LREX cells in in vitro studies. In in vivo experiments, LNCaP and the PDX MDA 322-2-6a-bearing mice were treated with enzalutamide. GR levels in plasma-derived EVs were increased only in those tumors that did not respond to enzalutamide. Treatment of mice bearing enzalutamide-resistant tumors with a GR inhibitor in combination with enzalutamide led to a transient pause in tumor growth in a subset of tumors and decreased GR levels intracellular and in plasma-derived EVs. In a subgroup of patients with high-risk localized prostate cancer treated with androgen signaling inhibition, GR was found upregulated in matching tissue and plasma EVs. These analyses showed that GR levels in plasma-derived EVs may be used for monitoring the transition of GR expression allowing for early detection of resistance to androgen ablation treatment. SIGNIFICANCE: Longitudinal monitoring of GR expression in plasma-derived EVs from patients with prostate cancer treated with androgen signaling inhibitors facilitates early detection of acquisition of resistance to androgen receptor signaling inhibition in individual patients.