Design, Construction, and Test of Compact, Distributed-Charge, X-Band Accelerator Systems that Enable Image-Guided, VHEE FLASH Radiotherapy.

The design and optimization of laser-Compton x-ray systems based on compact distributed charge accelerator structures can enable micron-scale imaging of disease and the concomitant production of beams of Very High Energy Electrons (VHEEs) capable of producing FLASH-relevant dose rates. The physics of laser-Compton x-ray scattering ensures that the scattered x-rays follow exactly the trajectory of the incident electrons, thus providing a route to image-guided, VHEE FLASH radiotherapy. The keys to a compact architecture capable of producing both laser-Compton x-rays and VHEEs are the use of X-band RF accelerator structures which have been demonstrated to operate with over 100 MeV/m acceleration gradients. The operation of these structures in a distributed charge mode in which each radiofrequency (RF) cycle of the drive RF pulse is filled with a low-charge, high-brightness electron bunch is enabled by the illumination of a high-brightness photogun with a train of UV laser pulses synchronized to the frequency of the underlying accelerator system. The UV pulse trains are created by a patented pulse synthesis approach which utilizes the RF clock of the accelerator to phase and amplitude modulate a narrow band continuous wave (CW) seed laser. In this way it is possible to produce up to 10 µA of average beam current from the accelerator. Such high current from a compact accelerator enables production of sufficient x-rays via laser-Compton scattering for clinical imaging and does so from a machine of "clinical" footprint. At the same time, the production of 1000 or greater individual micro-bunches per RF pulse enables > 10 nC of charge to be produced in a macrobunch of < 100 ns. The design, construction, and test of the 100-MeV class prototype system in Irvine, CA is also presented.

Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer.

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

Plexin D1 emerges as a novel target in the development of neural lineage plasticity in treatment-resistant prostate cancer.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) often arises from adenocarcinoma via lineage plasticity in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the specific regulators and targets involved in the transition to NEPC are not well understood. Plexin D1 (PLXND1) is a cellular receptor of the semaphorin (SEMA) family that plays important roles in modulating the cytoskeleton and cell adhesion. Here, we found that PLXND1 was highly expressed and positively correlated with neuroendocrine markers in patients with NEPC. High PLXND1 expression was associated with poorer prognosis in prostate cancer patients. Additionally, PLXND1 was upregulated and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells. Knockdown or knockout of PLXND1 inhibited neural lineage pathways, thereby suppressing NEPC cell proliferation, patient derived xenograft (PDX) tumor organoid viability, and xenograft tumor growth. Mechanistically, the heat shock protein 70 (HSP70) regulated PLXND1 protein stability through degradation, and inhibition of HSP70 decreased PLXND1 expression and NEPC organoid growth. In summary, our findings indicate that PLXND1 could serve as a promising therapeutic target and molecular marker for NEPC.

Plexin D1 emerges as a novel target in the development of neural lineage plasticity in treatment-resistant prostate cancer.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) often arises from adenocarcinoma via lineage plasticity in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the specific regulators and targets involved in the transition to NEPC are not well understood. Plexin D1 (PLXND1) is a cellular receptor of the semaphorin (SEMA) family that plays important roles in modulating the cytoskeleton and cell adhesion. Here, we found that PLXND1 is highly expressed and positively correlated with neuroendocrine markers in patients with NEPC. High PLXND1 expression is associated with poorer prognosis in prostate cancer patients. Additionally, PLXND1 was upregulated and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells. Knockdown or knockout of PLXND1 inhibit neural lineage pathways, suppressing NEPC cell proliferation, PDX tumor organoid viability, and xenograft tumor growth. Mechanistically, the chaperone protein HSP70 regulates PLXND1 protein stability through degradation, and inhibition of HSP70 decreases PLXND1 expression and NEPC organoid growth. In summary, our findings suggest that PLXND1 could be a new therapeutic target and molecular indicator for NEPC.

Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes.

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.

Androgen receptor blockade resistance with enzalutamide in prostate cancer results in immunosuppressive alterations in the tumor immune microenvironment.

BACKGROUND: Emerging data suggest that patients with enzalutamide-treated prostate cancer with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 clinical trial revealed that the combination of atezolizumab (a PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). However, the mechanisms underlying treatment failure remain unknown. METHODS: Human CRPC C4-2B cells and murine Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide and the cells resistant to enzalutamide were referred to as C4-2B MDVR and Myc-CaP MDVR, respectively. The mechanisms of action in drug-resistant prostate cancer cells were determined using RNA sequencing analyses, RNA interference, real-time PCR, western blotting, and co-culturing technologies. Myc-CaP and Myc-CaP MDVR tumors were established in syngeneic FVB mice, and tumor-infiltrating leukocytes were isolated after enzalutamide treatment. The stained immune cells were determined by flow cytometry, and the data were analyzed using FlowJo. RESULTS: Immune-related signaling pathways (interferon alpha/gamma response, inflammatory response, and cell chemotaxis) were suppressed in human enzalutamide-resistant prostate cancer cells. PD-L1 was overexpressed and negatively regulated by androgen receptor signaling in resistant cells and patient with CRPC cohorts. Enzalutamide treatment decreased CD8+ T-cell numbers but increased monocytic myeloid-derived suppressor cell (M-MDSC) populations and PD-L1 expression within murine Myc-CaP tumors. Similarly, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was also increased using enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and shifted towards M2 macrophage skewing. CONCLUSIONS: Our study suggests that immunosuppressive signaling can be promoted directly by enzalutamide-resistant prostate cancer cells and may be a potential means by which the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer is diminished.

Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models.

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

Novel inhibition of AKR1C3 and androgen receptor axis by PTUPB synergizes enzalutamide treatment in advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is the main driving force of mortality in prostate cancer patients. Among the parameters contributing to the progression of CRPC and treatment failure, elevation of the steroidogenic enzyme AKR1C3 and androgen receptor variant 7 (AR-V7) are frequently reported. The AKR1C3/AR-V7 complex has been recognized as a major driver for drug resistance in advanced prostate cancer. Herein we report that the level of AKR1C3 is reciprocally regulated by the full-length androgen receptor (AR-FL) through binding to the distal enhancer region of the AKR1C3 gene. A novel function of PTUPB in AKR1C3 inhibition was discovered and PTUPB showed more effectiveness than indomethacin and celecoxib in suppressing AKR1C3 activity and CRPC cell growth. PTUPB synergizes with enzalutamide treatment in tumor suppression and gene signature regulation. Combination treatments with PTUPB and enzalutamide provide benefits by blocking AR/AR-V7 signaling, which inhibits the growth of castration relapsed VCaP xenograft tumors and patient-derived xenograft organoids. Targeting of the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer.

Activation of neural lineage networks and ARHGEF2 in enzalutamide-resistant and neuroendocrine prostate cancer and association with patient outcomes.

Background: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed. Methods: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2. Results: We identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells. Conclusions: The 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Bioengineered BERA-Wnt5a siRNA Targeting Wnt5a/FZD2 Signaling Suppresses Advanced Prostate Cancer Tumor Growth and Enhances Enzalutamide Treatment.

The next-generation antiandrogen drugs such as enzalutamide and abiraterone extend survival times and improve quality of life in patients with advanced prostate cancer. However, resistance to both drugs occurs frequently through mechanisms that are incompletely understood. Wnt signaling, particularly through Wnt5a, plays vital roles in promoting prostate cancer progression and induction of resistance to enzalutamide and abiraterone. Development of novel strategies targeting Wnt5a to overcome resistance is an urgent need. In this study, we demonstrated that Wnt5a/FZD2-mediated noncanonical Wnt pathway is overexpressed in enzalutamide-resistant prostate cancer. In patient databases, both the levels of Wnt5a and FZD2 expression are upregulated upon the development of enzalutamide resistance and correlate with higher Gleason score, biochemical recurrence, and metastatic status, and with shortened disease-free survival duration. Blocking Wnt5a/FZD2 signal transduction not only diminished the activation of noncanonical Wnt signaling pathway, but also suppressed the constitutively activated androgen receptor (AR) and AR variants. Furthermore, we developed a novel bioengineered BERA-Wnt5a siRNA construct and demonstrated that inhibition of Wnt5a expression by the BERA-Wnt5a siRNA significantly suppressed tumor growth and enhanced enzalutamide treatment in vivo. These results indicate that Wnt5a/FZD2 signal pathway plays a critical role in promoting enzalutamide resistance, and targeting this pathway by BERA-Wnt5a siRNA can be developed as a potential therapy to treat advanced prostate cancer.

The Immunotherapy and Immunosuppressive Signaling in Therapy-Resistant Prostate Cancer.

Prostate cancer is one of the most common malignant tumors in men. Initially, it is androgen-dependent, but it eventually develops into castration-resistant prostate cancer (CRPC), which is incurable with current androgen receptor signaling target therapy and chemotherapy. Immunotherapy, specifically with immune checkpoint inhibitors, has brought hope for the treatment of this type of prostate cancer. Approaches such as vaccines, adoptive chimeric antigen receptor-T (CAR-T) cells, and immune checkpoint inhibitors have been employed to activate innate and adaptive immune responses to treat prostate cancer, but with limited success. Only Sipuleucel-T and the immune checkpoint inhibitor pembrolizumab are approved by the US FDA for the treatment of limited prostate cancer patients. Prostate cancer has a complex tumor microenvironment (TME) in which various immunosuppressive molecules and mechanisms coexist and interact. Additionally, prostate cancer is considered a "cold" tumor with low levels of tumor mutational burden, low amounts of antigen-presenting and cytotoxic T-cell activation, and high levels of immunosuppressive molecules including cytokines/chemokines. Thus, understanding the mechanisms of immunosuppressive signaling activation and immune evasion will help develop more effective treatments for prostate cancer. The purpose of this review is to summarize emerging advances in prostate cancer immunotherapy, with a particular focus on the molecular mechanisms that lead to immune evasion in prostate cancer. At the same time, we also highlight some potential therapeutic targets to provide a theoretical basis for the treatment of prostate cancer.

Resolving the structure of phage-bacteria interactions in the context of natural diversity.

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in the wild remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that lytic interactions in environmental interaction networks (as observed in agar overlay) are sparse-with phage predator loads being low for most bacterial strains, and phages being host-strain-specific. Paradoxically, we also find that although overlap in killing is generally rare between tailed phages, recombination is common. Together, these results suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it is important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on lytic host range.

ARVib suppresses growth of advanced prostate cancer via inhibition of androgen receptor signaling.

Targeting androgen signaling with the second-generation anti-androgen drugs, such as enzalutamide (Enza), abiraterone (Abi), apalutamide (Apal), and darolutamide (Daro), is the mainstay for the treatment of castration-resistant prostate cancer (CRPC). While these treatments are effective initially, resistance occurs frequently. Continued expression of androgen receptor (AR) and its variants such as AR-V7 despite AR-targeted therapy contributes to treatment resistance and cancer progression in advanced CRPC patients. This highlights the need for new strategies blocking continued AR signaling. Here, we identify a novel AR/AR-V7 degrader (ARVib) and found that ARVib effectively degrades AR/AR-V7 protein and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibits resistant prostate tumor growth and improves enzalutamide treatment in vitro and in vivo. These data suggest that ARVib has potential for development as an AR/AR-V7 degrader to treat resistant CRPC.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p = 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

Targeting Feedforward Loops Formed by Nuclear Receptor RORγ and Kinase PBK in mCRPC with Hyperactive AR Signaling.

Metastatic castration-resistant prostate cancer (mCRPC) is a highly aggressive disease with few therapeutic options. Hyperactive androgen receptor (AR) signaling plays a key role in CRPC progression. Previously, we identified RAR-related orphan receptor gamma (RORγ) as a novel key driver of AR gene overexpression and increased AR signaling. We report here that several RORγ antagonists/inverse agonists including XY018 and compound 31 were orally effective in potent inhibition of the growth of tumor models including patient-derived xenograft (PDX) tumors. RORγ controls the expression of multiple aggressive-tumor gene programs including those of epithelial-mesenchymal transition (EMT) and invasion. We found that PDZ binding kinase (PBK), a serine/threonine kinase, is a downstream target of RORγ that exerts the cellular effects. Alterations of RORγ expression or function significantly downregulated the mRNA and protein level of PBK. Our further analyses demonstrated that elevated PBK associates with and stabilizes RORγ and AR proteins, thus constituting novel, interlocked feed-forward loops in hyperactive AR and RORγ signaling. Indeed, dual inhibition of RORγ and PBK synergistically inhibited the expression and function of RORγ, AR, and AR-V7, and the growth and survival of CRPC cells. Therefore, our study provided a promising, new strategy for treatment of advanced forms of prostate cancer.

Psychological reactions to COVID-19: Survey data assessing perceived susceptibility, distress, mindfulness, and preventive health behaviors.

The COVID-19 pandemic created a complex psychological environment for persons in America. A total of 450 USA MTurk workers completed measures of: (a) basic demographic characteristics; (b) health risk factors for COVID-19; (c) perceived susceptibility variables related to COVID-19; (d) COVID-19 preventive health behaviors; and (e) distress, physical symptoms, and quality of life measures. The surveys were completed between April 9, 2020 and April 18, 2020. This recruitment period corresponded to the first 2-3 weeks of lockdown in most of the USA. Follow-up surveys were completed by 151 of the USA participants between June 19, 2020 and July 11, 2020 (approximately 2 months after the first measurement). These data permit evaluation of relationships among demographic variables, COVID-19 stress and coping, COVID-19 preventive health behavior, and the role of mindfulness as a possible moderator of distress as well as a predictor of preventive health behavior. The availability of follow-up data permit longitudinal analyses that provide a stronger basis for causal inference.

The RING domain of TRIM69 promotes higher-order assembly.

Members of the TRIM protein family have been shown to inhibit a range of viral infections. Recently, TRIM69 was identified as a potent inhibitor of Vesicular stomatitis Indiana virus infection, with its inhibition being dependent upon multimerization. Using SEC-MALLS analysis, it is demonstrated that the assembly of TRIM69 is mediated through the RING domain and not the Bbox domain as has been shown for other TRIM proteins. Using X-ray crystallography, the structure of the TRIM69 RING domain has been determined to a resolution of 2.1 Å, the oligomerization interface has been identified and regions outside the four-helix bundle have been observed to form interactions that are likely to support assembly.

Steroid Sulfatase Stimulates Intracrine Androgen Synthesis and is a Therapeutic Target for Advanced Prostate Cancer.

PURPOSE: Most patients with prostate cancer receiving enzalutamide or abiraterone develop resistance. Clinical evidence indicates that serum levels of dehydroepiandrosterone sulfate (DHEAS) and biologically active DHEA remain in the high range despite antiandrogen treatment. The conversion of DHEAS into DHEA by steroid sulfatase (STS) may contribute to sustained intracrine androgen synthesis. Here, we determine the contribution of STS to treatment resistance and explore the potential of targeting STS to overcome resistance in prostate cancer. EXPERIMENTAL DESIGN: STS expression was examined in patients and cell lines. In vitro, STS activity and expression were modulated using STS-specific siRNA or novel STS inhibitors (STSi). Cell growth, colony formation, androgen production, and gene expression were examined. RNA-sequencing analysis was conducted on VCaP cells treated with STSi. Mice were treated with STSis with or without enzalutamide to determine their effects in vivo. RESULTS: STS is overexpressed in patients with castration-resistant prostate cancer (CRPC) and resistant cells. STS overexpression increases intracrine androgen synthesis, cell proliferation, and confers resistance to enzalutamide and abiraterone. Inhibition of STS using siRNA suppresses prostate cancer cell growth. Targeting STS activity using STSi inhibits STS activity, suppresses androgen receptor transcriptional activity, and reduces the growth of resistant C4-2B and VCaP prostate cancer cells. STSis significantly suppress resistant VCaP tumor growth, decrease serum PSA levels, and enhance enzalutamide treatment in vitro and in vivo. CONCLUSIONS: These studies suggest that STS drives intracrine androgen synthesis and prostate cancer proliferation. Targeting STS represents a therapeutic strategy to treat CRPC and improve second-generation antiandrogen therapy.

Cross-Resistance Among Next-Generation Antiandrogen Drugs Through the AKR1C3/AR-V7 Axis in Advanced Prostate Cancer.

The next-generation antiandrogen drugs, XTANDI (enzalutamide), ZYTIGA (abiraterone acetate), ERLEADA (apalutamide) and NUBEQA (darolutamide) extend survival times and improve quality of life in patients with advanced prostate cancer. Despite these advances, resistance occurs frequently and there is currently no definitive cure for castration-resistant prostate cancer. Our previous studies identified that similar mechanisms of resistance to enzalutamide or abiraterone occur following treatment and cross-resistance exists between these therapies in advanced prostate cancer. Here, we show that enzalutamide- and abiraterone-resistant prostate cancer cells are further cross-resistant to apalutamide and darolutamide. Mechanistically, we have determined that the AKR1C3/AR-V7 axis confers this cross-resistance. Knockdown of AR-V7 in enzalutamide-resistant cells resensitize cells to apalutamide and darolutamide treatment. Furthermore, targeting AKR1C3 resensitizes resistant cells to apalutamide and darolutamide treatment through AR-V7 inhibition. Chronic apalutamide treatment in C4-2B cells activates the steroid hormone biosynthesis pathway and increases AKR1C3 expression, which confers resistance to enzalutamide, abiraterone, and darolutamide. In conclusion, our results suggest that apalutamide and darolutamide share similar resistant mechanisms with enzalutamide and abiraterone. The AKR1C3/AR-V7 complex confers cross-resistance to second-generation androgen receptor-targeted therapies in advanced prostate cancer.

GnRH Antagonists Have Direct Inhibitory Effects On Castration-Resistant Prostate Cancer Via Intracrine Androgen and AR-V7 Expression.

Hormone therapy is currently the mainstay in the management of locally advanced and metastatic prostate cancer. Degarelix (Firmagon), a gonadotropin-releasing hormone (GnRH) receptor antagonist differs from luteinizing hormone-releasing hormone (LHRH) agonists by avoiding "testosterone flare" and lower follicle-stimulating hormone (FSH) levels. The direct effect of degarelix and leuprolide on human prostate cancer cells was evaluated. In LNCaP, C4-2BMDVR, and CWR22Rv1 cells, degarelix significantly reduced cell viability compared with the controls (P ≤ 0.01). Leuprolide was stimulatory in the same cell lines. In C4-2B MDVR cells, degarelix alone or combined with abiraterone or enzalutamide reduced the AR-V7 protein expression compared with the control group. SCID mice bearing VCaP xenograft tumors were divided into 4 groups and treated with surgical castration, degarelix, leuprolide, or buffer alone for 4 weeks. Leuprolide slightly suppressed tumor growth compared with the vehicle control group (P > 0.05). Tumors in degarelix-treated mice were 67% of those in the leuprolide-treatment group but 170% larger than in surgically castrated ones. Measurements of intratumoral steroids in serum, tumor samples, or treated cell pellets by LC/MS confirmed that degarelix better decreased the levels of testosterone and steroidogenesis pathway intermediates, comparable to surgical castration, whereas leuprolide had no inhibitory effect. Collectively, our results suggested a selective mechanism of action of degarelix against androgen steroidogenesis and AR-variants. This study provides additional molecular insights regarding the mechanism of degarelix compared with GnRH agonist therapy, which may have clinical implications.