A Somatically Acquired Enhancer of the Androgen Receptor Is a Noncoding Driver in Advanced Prostate Cancer.

Increased androgen receptor (AR) activity drives therapeutic resistance in advanced prostate cancer. The most common resistance mechanism is amplification of this locus presumably targeting the AR gene. Here, we identify and characterize a somatically acquired AR enhancer located 650 kb centromeric to the AR. Systematic perturbation of this enhancer using genome editing decreased proliferation by suppressing AR levels. Insertion of an additional copy of this region sufficed to increase proliferation under low androgen conditions and to decrease sensitivity to enzalutamide. Epigenetic data generated in localized prostate tumors and benign specimens support the notion that this region is a developmental enhancer. Collectively, these observations underscore the importance of epigenomic profiling in primary specimens and the value of deploying genome editing to functionally characterize noncoding elements. More broadly, this work identifies a therapeutic vulnerability for targeting the AR and emphasizes the importance of regulatory elements as highly recurrent oncogenic drivers.

SIX2 promotes cell plasticity via Wnt/ß-catenin signalling in androgen receptor independent prostate cancer.

The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting in resistance to AR-targeted therapies. In this study, we examined the chromatin landscape of AR-positive prostate cancer cells post-exposure to the AR inhibitor enzalutamide. We identified a novel regulator of cell plasticity, the homeobox transcription factor SIX2, whose motif is enriched in accessible chromatin regions after treatment. Depletion of SIX2 in androgen-independent PC-3 prostate cancer cells induced a switch from a stem-like to an epithelial state, resulting in reduced cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects were mediated through the downregulation of the Wnt/ß-catenin signalling pathway and subsequent reduction of nuclear ß-catenin. Collectively, our findings provide compelling evidence that the depletion of SIX2 may represent a promising strategy for overcoming the cell plasticity mechanisms driving antiandrogen resistance in prostate cancer.

ONECUT2 acts as a lineage plasticity driver in adenocarcinoma as well as neuroendocrine variants of prostate cancer.

Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 gene targets include the glucocorticoid receptor (GR; NR3C1) and the NE splicing factor SRRM4, which are key drivers of lineage plasticity. Thus, OC2, despite its previously described NEPC driver function, can indirectly activate a portion of the AR cistrome through epigenetic activation of GR. Mechanisms by which OC2 regulates gene expression include promoter binding, enhancement of genome-wide chromatin accessibility, and super-enhancer reprogramming. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC and support enhanced efforts to therapeutically target OC2 as a means of suppressing treatment-resistant disease.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate-resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage plasticity and phenotypic heterogeneity. Castrate-resistant prostate adenocarcinoma can transition to neuroendocrine (NE) and occasionally to amphicrine, co-expressed luminal and NE, phenotypes. We developed castrate-resistant prostate cancer (CRPC) patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and NE phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor (St/Pr) cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent St/Pr cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from St/Pr cells and self-renewing differentiated luminal cells. Neuroendocrine prostate cancer (NEPC) was composed almost exclusively of self-renewing St/Pr cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine St/Pr subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell (SC) characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate SCs.

CYCLIN K down-regulation induces androgen receptor gene intronic polyadenylation, variant expression and PARP inhibitor vulnerability in castration-resistant prostate cancer.

Androgen receptor (AR) messenger RNA (mRNA) alternative splicing variants (AR-Vs) are implicated in castration-resistant progression of prostate cancer (PCa), although the molecular mechanism underlying the genesis of AR-Vs remains poorly understood. The CDK12 gene is often deleted or mutated in PCa and CDK12 deficiency is known to cause homologous recombination repair gene alteration or BRCAness via alternative polyadenylation (APA). Here, we demonstrate that pharmacological inhibition or genetic inactivation of CDK12 induces AR gene intronic (intron 3) polyadenylation (IPA) usage, AR-V expression, and PCa cell resistance to the antiandrogen enzalutamide (ENZ). We further show that AR binds to the CCNK gene promoter and up-regulates CYCLIN K expression. In contrast, ENZ decreases AR occupancy at the CCNK gene promoter and suppresses CYCLIN K expression. Similar to the effect of the CDK12 inhibitor, CYCLIN K degrader or ENZ treatment promotes AR gene IPA usage, AR-V expression, and ENZ-resistant growth of PCa cells. Importantly, we show that targeting BRCAness induced by CYCLIN K down-regulation with the PARP inhibitor overcomes ENZ resistance. Our findings identify CYCLIN K down-regulation as a key driver of IPA usage, hormonal therapy-induced AR-V expression, and castration resistance in PCa. These results suggest that hormonal therapy-induced AR-V expression and therapy resistance are vulnerable to PARP inhibitor treatment.

Androgen receptor-mediated pharmacogenomic expression quantitative trait loci: implications for breast cancer response to AR-targeting therapy.

BACKGROUND: Endocrine therapy is the most important treatment modality of breast cancer patients whose tumors express the estrogen receptor α (ERα). The androgen receptor (AR) is also expressed in the vast majority (80-90%) of ERα-positive tumors. AR-targeting drugs are not used in clinical practice, but have been evaluated in multiple trials and preclinical studies. METHODS: We performed a genome-wide study to identify hormone/drug-induced single nucleotide polymorphism (SNP) genotype - dependent gene-expression, known as PGx-eQTL, mediated by either an AR agonist (dihydrotestosterone) or a partial antagonist (enzalutamide), utilizing a previously well characterized lymphoblastic cell line panel. The association of the identified SNPs-gene pairs with breast cancer phenotypes were then examined using three genome-wide association (GWAS) studies that we have published and other studies from the GWAS catalog. RESULTS: We identified 13 DHT-mediated PGx-eQTL loci and 23 Enz-mediated PGx-eQTL loci that were associated with breast cancer outcomes post ER antagonist or aromatase inhibitors (AI) treatment, or with pharmacodynamic (PD) effects of AIs. An additional 30 loci were found to be associated with cancer risk and sex-hormone binding globulin levels. The top loci involved the genes IDH2 and TMEM9, the expression of which were suppressed by DHT in a PGx-eQTL SNP genotype-dependent manner. Both of these genes were overexpressed in breast cancer and were associated with a poorer prognosis. Therefore, suppression of these genes by AR agonists may benefit patients with minor allele genotypes for these SNPs. CONCLUSIONS: We identified AR-related PGx-eQTL SNP-gene pairs that were associated with risks, outcomes and PD effects of endocrine therapy that may provide potential biomarkers for individualized treatment of breast cancer.

SYT4 binds to SNAP25 to facilitate exosomal secretion and prostate cancer enzalutamide resistance.

Prostate carcinoma represents a predominant malignancy affecting the male population, with androgen deprivation therapy (ADT) serving as a critical therapeutic modality for advanced disease states, but it often leads to the development of resistance. Enzalutamide (Enz), a second-generation antiandrogen drug, initially offers substantial therapeutic benefit, but its efficacy wanes as drug resistance ensues. In this study, we found that synaptotagmin 4 (SYT4) is an upregulated gene in enzalutamide-resistant (EnzR) cell lines. The downregulation of SYT4, in combination with enzalutamide therapy, substantially enhances the antiproliferative effect on resistant prostate cancer cells beyond the capacity of enzalutamide monotherapy. SYT4 promotes vesicle efflux by binding to the synaptosome-associated protein 25 (SNAP25), thereby contributing to cell resistance against enzalutamide. The elevated expression of SYT4 is mediated by bromodomain-containing protein 4 (BRD4), and BRD4 inhibition effectively suppressed the expression of SYT4. Treatment with a therapeutic dose of enzalutamide combined with ASO-1, an antisense oligonucleotide drug targeting SYT4, shows promising results in reversing the resistance of prostate cancer to enzalutamide.

Enhancing the anticancer effect of androgen deprivation therapy by monocarboxylate transporter 1 inhibitor in prostate cancer cells.

BACKGROUND: Tumor initiation and progression necessitate a metabolic shift in cancer cells. Consequently, the progression of prostate cancer (PCa), a leading cause of cancer-related deaths in males globally, involves a shift from lipogenic to glycolytic metabolism. Androgen deprivation therapy (ADT) serves as the standard treatment for advanced-stage PCa. However, despite initial patient responses, castrate resistance emerges ultimately, necessitating novel therapeutic approaches. Therefore, in this study, we aimed to investigate the role of monocarboxylate transporters (MCTs) in PCa post-ADT and evaluate their potential as therapeutic targets. METHODS: PCa cells (LNCaP and C4-2 cell line), which has high prostate-specific membrane antigen (PSMA) and androgen receptor (AR) expression among PCa cell lines, was used in this study. We assessed the expression of MCT1 in PCa cells subjected to ADT using charcoal-stripped bovine serum (CSS)-containing medium or enzalutamide (ENZ). Furthermore, we evaluated the synergistic anticancer effects of combined treatment with ENZ and SR13800, an MCT1 inhibitor. RESULTS: Short-term ADT led to a significant upregulation in folate hydrolase 1 (FOLH1) and solute carrier family 16 member 1 (SLC16A1) gene levels, with elevated PSMA and MCT1 protein levels. Long-term ADT induced notable changes in cell morphology with further upregulation of FOLH1/PSMA and SLC16A1/MCT1 levels. Treatment with ENZ, a nonsteroidal anti-androgen, also increased PSMA and MCT1 expression. However, combined therapy with ENZ and SR13800 led to reduced PSMA level, decreased cell viability, and suppressed expression of cancer stem cell markers and migration indicators. Additionally, analysis of human PCa tissues revealed a positive correlation between PSMA and MCT1 expression in tumor regions. CONCLUSIONS: Our results demonstrate that ADT led to a significant upregulation in MCT1 levels. However, the combination of ENZ and SR13800 demonstrated a promising synergistic anticancer effect, highlighting a potential therapeutic significance for patients with PCa undergoing ADT.

Loss of AR-regulated AFF3 contributes to prostate cancer progression and reduces ferroptosis sensitivity by downregulating ACSL4 based on single-cell sequencing analysis.

Prostate cancer (PCa) is one of the most common cancers affecting the health of men worldwide. Castration-resistant prostate cancer (CRPC), the advanced and refractory phase of prostate cancer, has multiple mechanisms of resistance to androgen deprivation therapy (ADT) such as AR mutations, aberrant androgen synthase, and abnormal expression of AR-related genes. Based on the research of the AR pathway, new drugs for the treatment of CRPC have been developed in clinical practice, such as Abiraterone and enzalutamide. However, many areas in this pathway are still worth exploring. In this study, single-cell sequencing analysis was utilized to scrutinize significant genes in the androgen receptor (AR) pathway related to CRPC. Our analysis of single-cell sequencing combined with bulk-cell sequencing revealed a substantial downregulation of AR-regulated AFF3 in CRPC. Overexpression of AFF3 restricted the proliferation and migration of prostate cancer cells whilst also increasing their sensitivity towards enzalutamide, while knockdown of AFF3 had the opposite effect. To elucidate the mechanism of tumor inhibition by AFF3, we applied GSVA and GSEA to investigate the metabolic pathways related to AFF3 and revealed that AFF3 had an impact on fatty acids metabolism and ferroptosis through the regulation of ACSL4 protein expression. Based on correlation analysis and flow cytometry, we can speculate that AFF3 can impact the sensitivity of the CRPC cell lines to the ferroptosis inducer (RSL3) by regulating ACSL4. Therefore, our findings may provide new insights into the mechanisms of drug resistance in CRPC, and AFF3 may serve as a novel prognostic biomarker in prostate cancer.

Androgen blockage impairs proliferation and function of Sertoli cells via Wee1 and Lfng.

BACKGROUND: Androgens are essential hormones for testicular development and the maintenance of male fertility. Environmental factors, stress, aging, and psychological conditions can disrupt androgen production, impacting the androgen signaling pathway and consequently spermatogenesis. Within the testes, testosterone is produced by Leydig cells and acts on Sertoli cells by activating the androgen receptor (AR), which then translocates to the nucleus to function as a transcription factor. Despite clinical correlations between low testosterone levels and diminished sperm quality, the precise mechanism remains unclear. METHODS: This study explores the hypothesis that reduced androgen levels impair Sertoli cell function by disrupting AR transcriptional regulation. Using an androgen blockade model with enzalutamide, we investigated the impact of low androgen levels on AR target genes in Sertoli cells through ChIP-seq and RNA-seq assays. RESULTS: Our results reveal that androgen blockage increases AR enrichment on the promoter region of Wee1, promoting Wee1 expression, while decreasing binding to the promoter region of Lfng, inhibiting its expression. Increased WEE1 protein inhibits Sertoli cell proliferation, whereas reduced LFNG affects Notch modification, leading to decreased production of glial cell line-derived neurotrophic factor (GDNF), a key growth factor for spermatogonial stem cell self-renewal. CONCLUSIONS: These findings provide new insights into the molecular mechanisms by which low androgen levels interfere with Sertoli cell functions, offering novel perspectives for the clinical treatment of male reproductive disorders.

MYO6 contributes to tumor progression and enzalutamide resistance in castration-resistant prostate cancer by activating the focal adhesion signaling pathway.

BACKGROUND: Enzalutamide (Enz) resistance is a poor prognostic factor for patients with castration-resistant prostate cancer (CRPC), which often involves aberrant expression of the androgen receptor (AR). Myosin VI (MYO6), one member of the myosin family, plays an important role in regulating cell survival and is highly expressed in prostate cancer (PCa). However, whether MYO6 is involved in Enz resistance in CRPC and its mechanism remain unclear. METHODS: Multiple open-access databases were utilized to examine the relationship between MYO6 expression and PCa progression, and to screen differentially expressed genes (DEGs) and potential signaling pathways associated with the MYO6-regulated Enz resistance. Both in vitro and in vivo tumorigenesis assays were employed to examine the impact of MYO6 on the growth and Enz resistance of PCa cells. Human PCa tissues and related clinical biochemical data were utilized to identify the role of MYO6 in promoting PCa progression and Enz resistance. The molecular mechanisms underlying the regulation of gene expression, PCa progression, and Enz resistance in CRPC by MYO6 were investigated. RESULTS: MYO6 expression increases in patients with PCa and is positively correlated with AR expression in PCa cell lines and tissues. Overexpression of AR increases MYO6 expression to promote PCa cell proliferation, migration and invasion, and to inhibit PCa cell apoptosis; whereas knockdown of MYO6 expression reverses these outcomes and enhances Enz function in suppressing the proliferation of the Enz- sensitive and resistant PCa cells both in vitro and in vivo. Mechanistically, AR binds directly to the promoter region (residues - 503 to - 283 base pairs) of MYO6 gene and promotes its transcription. Furthermore, MYO6 activates focal adhesion kinase (FAK) phosphorylation at tyrosine-397 through integrin beta 8 (ITGB8) modulation to promote PCa progression and Enz resistance. Notably, inhibition of FAK activity by Y15, an inhibitor of FAK, can resensitize CRPC cells to Enz treatment in cell lines and mouse xenograft models. CONCLUSIONS: MYO6 has pro-tumor and Enz-resistant effects in CRPC, suggesting that targeting MYO6 may be beneficial for ENZ-resistant CRPC therapy through the AR/MYO6/FAK signaling pathway.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer.

BACKGROUND: Endocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors. METHODS: Bioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth. RESULTS: RACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa. CONCLUSION: In summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Discovery of novel biphenyl derivatives as androgen receptor degraders for the treatment of enzalutamide-resistant prostate cancer.

Second-generation AR antagonists, such as enzalutamide, are the primary therapeutic agents for advanced prostate cancer. However, the development of both primary and secondary drug resistance leads to treatment failures and patient mortality. Bifunctional agents that simultaneously antagonize and degrade AR block the AR signaling pathway more completely and exhibit excellent antiproliferative activity against wild-type and drug-resistant prostate cancer cells. Here, we reported the discovery and optimization of a series of biphenyl derivatives as androgen receptor antagonists and degraders. These biphenyl derivatives exhibited potent antiproliferative activity against LNCaP and 22Rv1 cells. Our discoveries enrich the diversity of small molecule AR degraders and offer insights for the development of novel AR degraders for the treatment of enzalutamide-resistant prostate cancer.

IL-23 secreted by myeloid cells drives castration-resistant prostate cancer.

Patients with prostate cancer frequently show resistance to androgen-deprivation therapy, a condition known as castration-resistant prostate cancer (CRPC). Acquiring a better understanding of the mechanisms that control the development of CRPC remains an unmet clinical need. The well-established dependency of cancer cells on the tumour microenvironment indicates that the microenvironment might control the emergence of CRPC. Here we identify IL-23 produced by myeloid-derived suppressor cells (MDSCs) as a driver of CRPC in mice and patients with CRPC. Mechanistically, IL-23 secreted by MDSCs can activate the androgen receptor pathway in prostate tumour cells, promoting cell survival and proliferation in androgen-deprived conditions. Intra-tumour MDSC infiltration and IL-23 concentration are increased in blood and tumour samples from patients with CRPC. Antibody-mediated inactivation of IL-23 restored sensitivity to androgen-deprivation therapy in mice. Taken together, these results reveal that MDSCs promote CRPC by acting in a non-cell autonomous manner. Treatments that block IL-23 can oppose MDSC-mediated resistance to castration in prostate cancer and synergize with standard therapies.

METTL3-mediated m6A modification of circGLIS3 promotes prostate cancer progression and represents a potential target for ARSI therapy.

BACKGROUND: Circular RNAs (circRNAs) have been shown to be involved in tumorigenesis and progression. However, the role of circGLIS3 (hsa_circ_0002874) in prostate cancer (PCa) has yet not been reported. METHODS: Candidate circRNA were determined through comprehensive analysis of public datasets, PCa cell lines, and tissues data. A series of cellular functional assays, including CCK-8, colony formation, wound healing, and transwell assays were performed. Subsequently, RNA sequencing, RNA immunoprecipitation, methylated RNA immunoprecipitation, microRNA pulldown, luciferase reporter assay, and western blot were used to explore the underlying molecular mechanisms. Moreover, the xenograft tumor mouse model was established to elucidate the function of circGLIS3. RESULTS: CircGLIS3, derived from exon 2 of the parental GLIS3 gene, was identified as a novel oncogenic circRNA in PCa that was closely associated with the biochemical recurrence. Its expression levels were upregulated in PCa tissues and cell lines as well as enzalutamide high-resistant cells. The cellular functional assays revealed that circGLIS3 promoted PCa cell proliferation, migration, and invasion. METTL3-mediated N6-methyladenosine (m6A) modification maintained its upregulation by enhancing its stability. Mechanically, CircGLIS3 sponged miR-661 to upregulate MDM2, thus regulating the p53 signaling pathway to promote cell proliferation, migration, and invasion. Furthermore, in vitro and in vivo experiments, the knockdown of circGLIS3 improved the response of PCa cells to ARSI therapies such as enzalutamide. CONCLUSIONS: METTL3-mediated m6A modification of circGLIS3 regulates the p53 signaling pathway via the miR-661/MDM2 axis, thereby facilitating PCa progression. Meanwhile, this study unveils a promising potential target for ARSI therapy for PCa.

Estrogen receptor ß and treatment with a phytoestrogen are associated with inhibition of nuclear translocation of EGFR in the prostate.

Knockout of ERß in the mouse leads to nuclear expression of epidermal growth factor receptor (EGFR) in the prostate. To examine whether ERß plays a similar role in the human prostate, we used four cohorts of men: 1) a Swedish cohort of normal prostates and PCa (prostate cancer) of different Gleason grades; 2) men with benign prostatic hyperplasia (BPH) treated with the 5α-reductase inhibitor, finasteride, and finasteride together with the ERß agonists, soy isoflavones; 3) men with PCa above Gleason grade 4 (GG4), treated with ADT (androgen deprivation therapy) and abiraterone (AA), the blocker of androgen synthesis for different durations; and 4) men with GG4 PCa on ADT or ADT with the AR (androgen receptor) blocker, enzalutamide, for 4 mo to 6 mo. In men with BPH, finasteride treatment induced EGFR nuclear expression, but, when finasteride was combined with isoflavones, EGFR remained on the cell membrane. In GG4 patients, blocking of AR for 4 mo to 6 mo resulted in loss of ERß and PTEN expression and increase in patients with nuclear EGFR from 10 to 40%. In the men with GG4 PCa, blocking of adrenal synthesis of testosterone for 2 mo to 7 mo had the beneficial effect of increasing ERß expression, but, on treatment longer than 8 mo, ERß was lost and EGFR moved to the nucleus. Since nuclear EGFR is a predictor of poor outcome in PCa, addition of ERß agonists together with abiraterone should be considered as a treatment that might sustain expression of ERß and offer some benefit to patients.

Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity.

Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching.

New Marine Fungal Deoxy-14,15-Dehydroisoaustamide Resensitizes Prostate Cancer Cells to Enzalutamide.

Marine fungi serve as a valuable source for new bioactive molecules bearing various biological activities. In this study, we report on the isolation of a new indole diketopiperazine alkaloid deoxy-14,15-dehydroisoaustamide (1) from the marine-derived fungus Penicillium dimorphosporum KMM 4689 associated with a soft coral. The structure of this metabolite, including its absolute configuration, was determined by HR-MS, 1D and 2D NMR as well as CD data. Compound 1 is a very first deoxyisoaustamide alkaloid possessing two double bonds in the proline ring. The isolated compound was noncytotoxic to a panel of human normal and cancer cell lines up to 100 µM. At the same time, compound 1 resensitized prostate cancer 22Rv1 cells to androgen receptor (AR) blocker enzalutamide. The mechanism of this phenomenon was identified as specific drug-induced degradation of androgen receptor transcription variant V7 (AR-V7), which also resulted in general suppression of AR signaling. Our data suggest that the isolated alkaloid is a promising candidate for combinational therapy of castration resistant prostate cancer, including drug-resistant subtypes.

Enhanced Antitumor Efficacy of Radium-223 and Enzalutamide in the Intratibial LNCaP Prostate Cancer Model.

Radium-223 dichloride and enzalutamide are indicated for metastatic castration-resistant prostate cancer and their combination is currently being investigated in a large phase 3 clinical trial. Here, we evaluated the antitumor efficacy of radium-223, enzalutamide, and their combination in the intratibial LNCaP model mimicking prostate cancer metastasized to bone. In vitro experiments revealed that the combination of radium-223 and enzalutamide inhibited LNCaP cell proliferation and showed synergistic efficacy. The combination of radium-223 and enzalutamide also demonstrated enhanced in vivo antitumor efficacy, as determined by measuring serum PSA levels in the intratibial LNCaP model. A decreasing trend in the total area of tumor-induced abnormal bone was associated with the combination treatment. The serum levels of the bone formation marker PINP and the bone resorption marker CTX-I were lowest in the combination treatment group and markedly decreased compared with vehicle group. Concurrent administration of enzalutamide did not impair radium-223 uptake in tumor-bearing bone or the ability of radium-223 to inhibit tumor-induced abnormal bone formation. In conclusion, combination treatment with radium-223 and enzalutamide demonstrated enhanced antitumor efficacy without compromising the integrity of healthy bone. The results support the ongoing phase 3 trial of this combination.

Establishment of a five-enzalutamide-resistance-related-gene-based classifier for recurrence-free survival predicting of prostate cancer.

To identify prostate cancer (PCa) patients with a high risk of recurrence is critical before delivering adjuvant treatment. We developed a classifier based on the Enzalutamide treatment resistance-related genes to assist the currently available staging system in predicting the recurrence-free survival (RFS) prognosis of PCa patients. We overlapped the DEGs from two datasets to obtain a more convincing Enzalutamide-resistance-related-gene (ERRG) cluster. The five-ERRG-based classifier obtained good predictive values in both the training and validation cohorts. The classifier precisely predicted RFS of patients in four cohorts, independent of patient age, pathological tumour stage, Gleason score and PSA levels. The classifier and the clinicopathological factors were combined to construct a nomogram, which had an increased predictive accuracy than that of each variable alone. Besides, we also compared the differences between high- and low-risk subgroups and found their differences were enriched in cancer progression-related pathways. The five-ERRG-based classifier is a practical and reliable predictor, which adds value to the existing staging system for predicting the RFS prognosis of PCa after radical prostatectomy, enabling physicians to make more informed treatment decisions concerning adjuvant therapy.