Castration Resistance Research Articles

Clinical aspects of prostate cancer in patients with germinal and somatic mutations in DNA homologous recombination repair genes

Background. In clinical practice, there is a need to predict clinical behavior of prostate cancer with germinal and somatic mutations in DNA homologous recombination repair (HRR) genes due to an atypical response to standard treatment methods. Also, the expediency of testing the mutational status of HRR genes is dictated by the possibility of using the PARP-inhibition strategy in metastatic castration-resistant prostate cancer (mCRPC). In addition to expanding the possibilities for targeted therapy the necessity to inform the relatives of mutation carriers is underestimated. It is also important to realize the fact of accumulation of somatic changes both in the primary tumor and in the metastatic lesion during tumor evolution and under treatment, which dictates the possibility of repeated biopsy with exhausted therapy possibilities.Aim. Evaluation of prostate cancer clinical behavior features and response to drug therapy depending on the identified mutations in the HRR genes.Materials and methods. The study was performed at the Clinical Oncological Dispensary No. 1 (Krasnodar). Clinical and morphological data of 27 patients with prostate cancer and identified germinal and somatic mutations in HRR genes (BRCA1, BRCA2, ATM, BARD, BRIP1, CDK12, CHEK1, CHEK2, PALB2, RAD51B, RAD51C, RAD54L, FANCL) were retrospectively analyzed. Statistical analysis was performed using the IBM SPSS Statistics v.22 statistical package.Results and conclusion. The median age of patients was 61 years. The most frequent were mutations in the BRCA2 (37 %), CHEK2 (18.5 %), ATM (14.8 %) genes. More than half of the patients (69 %) had primary metastatic disease. The differentiation grade of G2 and G3 according to the classification of the International Society of Urological Pathologists (ISUP) with Gleason score of 7 (3 + 4) and 7 (4 + 3) were both detected in 27 % of cases. The type of mutation did not affect the time of castration resistance development (p = 0.216). The time to castration resistance increased close to statistical significance in the case of primary stage of T3–4N0M0 compared to other stages (log-rank p = 0.092). Progression-free survival (PFS) with docetaxel monochemotherapy was significantly longer when prescribed for metastatic hormone-sensitive prostate cancer with mutations in HRR genes compared to mCRPC (p = 0.061) and to primary metastatic disease (p = 0.04). At the same time, the risk of progression during therapy was higher for presence of regional lymph node metastases with primary advancement (p = 0.005; hazard ratio 1.167; 95 % confidence interval 2.765–267). There was also an advantage in PFS when prescribing docetaxel for BRCA1/2 and ATM mutations in comparison with other mutations (p = 0.038). When prescribing therapy with 2nd generation antiandrogens or abiraterone, progression-free survival is higher in the group of patients with prostate cancer with Gleason score of 7 (4 + 3) compared to cohort with other morphological types, and this difference is almost statistically significant (log-rank p = 0.091, Breslow p = 0.076, Taron-Ware p = 0.074). Targeted therapy with the PARP inhibitor Olaparib in the performed trial was received by 10 patients with HRR mutations. At the same time, according to the data of the PROfound trial, the advantage of Olaparib in radiological PFS was shown in germinal and somatic mutations in group A (BRCA1, BRCA2, ATM) and in the general group (A and B – other HRR mutations).

Apalutamide plus androgen deprivation therapy in clinical subgroups of patients with metastatic castration-sensitive prostate cancer: A subgroup analysis of the randomised clinical TITAN study

BackgroundWhether disease burden in patients with metastatic castration-sensitive prostate cancer (mCSPC) predicts treatment outcomes is unknown. We assessed apalutamide treatment effect in TITAN patients with mCSPC by disease volume, metastasis number and timing of metastasis presentation. MethodsThese protocol-defined and post hoc analyses of the phase III randomised TITAN study evaluated clinical outcomes in patients receiving 240 mg/day apalutamide (n = 525) or placebo (n = 527) plus androgen-deprivation therapy (ADT). Subgroups were defined by volume (high: visceral and ≥1 bone metastases or ≥4 bone lesions with ≥1 beyond vertebral column/pelvis), development of metastases per conventional imaging (synchronous: at initial diagnosis; metachronous: after localised disease) and oligometastases (≤5 bone-only metastases) or polymetastases (>5 in bone ± other locations or ≤5 in bone plus other locations). Overall survival (OS), radiographic or second progression-free survival, and time to prostate-specific antigen progression or castration resistance were assessed using Cox proportional hazards models. ResultsOf 1052 patients, 63%, 81%, 54%, 27%, 5.7%, and 8.0% had high-volume, synchronous, synchronous/high-volume, synchronous/low-volume, metachronous/high-volume, and metachronous/low-volume disease, respectively. The OS benefit favoured apalutamide plus ADT versus ADT alone in synchronous/high-volume (hazard ratio = 0.68 [95% confidence interval: 0.53–0.87]; p = 0.002), synchronous/low-volume (0.65 [0.40–1.05]; p = 0.08), metachronous/high-volume (0.69 [0.33–1.44]; p = 0.32) and metachronous/low-volume (0.22 [0.09–0.55]; p = 0.001) subgroups. Apalutamide improved other clinical outcomes regardless of subgroup, with similar safety profiles. Most favourable outcomes were observed in oligometastatic disease. ConclusionTITAN patients derived a robust benefit with apalutamide plus ADT regardless of disease volume and timing of metastasis presentation without differences in safety, supporting early apalutamide intensification in mCSPC. Clinical Trial RegistrationNCT02489318.

RNA-Sequencing Analysis Indicates That N-Cadherin Promotes Prostate Cancer Progression by the Epigenetic Modification of Key Genes.

N-cadherin (cadherin-2 [CDH2]) is widely known as the promoter of prostate cancer (PCa) invasion and castration resistance. However, the biological mechanism of N-cadherin in PCa progression is unclear. In this study, we overexpressed N-cadherin in LNCaP cells and downregulated N-cadherin in PC3 cells by lentiviral transduction. Then, differentially expressed genes (DEGs) and dysregulated biological functions were investigated through RNA sequencing (RNA-seq) analyses. We found 13 long noncoding RNA (lncRNA) transcripts, 72 messenger RNA (mRNA) transcripts, and 3 integrated genes were dysregulated by N-cadherin. In the disease enrichment, bone cancer, and neurodegenerative and nervous system diseases were associated with N-cadherin in the circular RNA (circRNA; PC3 versus [vs.,/] LNCaP [PC3/LNCaP] comparison) and DEG analysis (LNCaP_oe_CDH2 vs. LNCaP_oe_NC [LNCaP_oe_CDH2/NC] comparison). Epigenetic reprogramming, such as nucleic acid binding, and chromatin and histone modifications, was enriched in Gene Ontology (GO) analysis (DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2, and host genes of circRNA in PC3/LNCaP). Transcriptional misregulation in cancer, post-translational protein modification, gene expression, and generic transcription pathways were dysregulated in the pathway enrichment analysis (host genes of circRNA in PC3/LNCaP, and DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2). Verifying DEGs through TCGA-PRAD dataset revealed six oncogenes (ARHGEF1, GRAMD1A, GTF2H4, MAPK8IP3, POLD1, and PTBP1) that were commonly upregulated by N-cadherin and in advanced PCa stages. In summary, we identified several oncogenes and biological functions associated with N-cadherin expression in PCa cells. N-cadherin may trigger epigenetic reprogramming in PCa cells to promote tumor progression.

Performance of clinical risk scores and prediction models to identify pathogenic germline variants in patients with advanced prostate cancer

PurposeDetermining the frequency and distribution of pathogenic germline variants (PGVs) in Austrian prostate cancer (PCa) patients and to assess the accuracy of different clinical risk scores to correctly predict PGVs.MethodsThis cross-sectional study included 313 men with advanced PCa. A comprehensive personal and family history was obtained based on predefined questionnaires. Germline DNA sequencing was performed between 2019 and 2021 irrespective of family history, metastatic or castration status or age at diagnosis. Clinical risk scores for hereditary cancer syndromes were evaluated and a PCa-specific score was developed to assess the presence of PGVs.ResultsPGV presence was associated with metastasis (p = 0.047) and castration resistance (p = 0.011), but not with personal cancer history or with relatives with any type of cancer. Clinical risk scores (Manchester score, PREMM5 score, Amsterdam II criteria or Johns Hopkins criteria) showed low sensitivities (3.3–20%) for assessing the probability of PGV presence. A score specifically designed for PCa patients stratifying patients into low- or high-risk regarding PGV probability, correctly classified all PGV carriers as high-risk, whereas a third of PCa patients without PGVs was classified as low risk of the presence of PGVs.ConclusionApplication of common clinical risk scores based on family history are not suitable to identify PCa patients with high PGV probabilities. A PCa-specific score stratified PCa patients into low- or high-risk of PGV presence with sufficient accuracy, and germline DNA sequencing may be omitted in patients with a low score. Further studies are needed to evaluate the score.

Androgen Receptor Variants Confer Castration Resistance in Prostate Cancer by Counteracting Antiandrogen-Induced Ferroptosis.

Upregulation of SLC7A11 can be induced by androgen receptor variants to inhibit antiandrogen-induced prostate cancer cell ferroptosis and to drive castration resistance in prostate cancer.

A Multivalent Peptoid Conjugate Modulates Androgen Receptor Transcriptional Activity to Inhibit Therapy-resistant Prostate Cancer.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Methods for Evaluating the Efficacy of Medical Castration: A Systematic Review.

Measuring serum testosterone determination during medical castration is recommended by prostate cancer (PCa) guidelines to assess its efficacy and define castration resistance. It has been suggested that other biochemical compounds, such as free testosterone or luteinising hormone (LH), could also assess castration efficacy. We aimed to analyse the current evidence for serum biochemical compounds that could be appropriate candidates for evaluating medical castration efficacy. A systematic review was conducted after two investigators independently searched the literature in the PubMed, Cochrane Library, and EMBASE databases published between January 1980 and February 2023. Their searches used the medical subject headings 'prostatic neoplasms', 'testosterone and androgen antagonists', 'gonadotropin-releasing hormone/analogues and derivatives', 'free testosterone', and 'luteinising hormone'. Studies were selected according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria, and their eligibility was based on the Participants, Intervention, Comparator, and Outcome strategy. The search was limited to original articles published in English. Among the 6599 initially identified titles, 15 original studies analysing the clinical impact of serum testosterone levels in PCa patients undergoing androgen deprivation therapy (ADT) were selected for evidence acquisition. The risk of bias in individual studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. All selected studies used immunoassays to measure serum testosterone, although only methods based on liquid or gas chromatography and mass spectrometry are recommended to measure low testosterone concentrations. The reported series were not uniform in clinical stage, ADT types, and the time or number of serum testosterone measurements. Only some studies found low serum testosterone levels (<20 or <32 ng/dL) associated with greater survival free of biochemical progression and castration resistance. We conclude that little current evidence justifies the measurement of serum testosterone during ADT using no appropriate methods. No reported longitudinal studies have examined the clinical impact of serum testosterone measured using liquid chromatography with tandem mass spectrometry (LC-MSMS), free testosterone, or LH in PCa patients undergoing medical castration. We conclude that well-designed longitudinal studies examining the clinical impact of serum testosterone measured with LC-MSMS, serum-free testosterone, and LH on biochemical progression and castration resistance in PCa patients undergoing neo-adjuvant castration in radiation therapy or continuous castration are needed.

Androgen-regulated stromal complement component 7 (C7) suppresses prostate cancer growth.

The complement system is a major component of the innate immune system that works through the cytolytic effect of the membrane attack complex (MAC). Complement component 7 (C7) is essential for MAC assembly and its precisely regulated expression level is crucial for the cytolytic activity of MAC. We show that C7 is specifically expressed by the stromal cells in both mouse and human prostates. The expression level of C7 inversely correlates with clinical outcomes in prostate cancer. C7 is positively regulated by androgen signaling in the mouse prostate stromal cells. The androgen receptor directly transcriptionally regulates the mouse and human C7. Increasing C7 expression in the C57Bl/6 syngeneic RM-1 and Pten-Kras allografts suppresses tumor growth in vivo. Conversely, C7 haploinsufficiency promotes tumor growth in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Interestingly, replenishing C7 in androgen-sensitive Pten-Kras tumors during androgen depletion only slightly enhances cellular apoptosis, highlighting the diverse mechanisms employed by tumors to counteract complement activity. Collectively, our research indicates that augmenting complement activity could be a promising therapeutic approach to impede the development of castration resistance in prostate cancer.

Antiandrogen treatment induces stromal cell reprogramming to promote castration resistance in prostate cancer.

Lineage plasticity causes therapeutic resistance; however, it remains unclear how the fate conversion and phenotype switching of cancer-associated fibroblasts (CAFs) are implicated in disease relapse. Here, we show that androgen deprivation therapy (ADT)-induced SPP1+ myofibroblastic CAFs (myCAFs) are critical stromal constituents that drive the development of castration-resistant prostate cancer (CRPC). Our results reveal that SPP1+ myCAFs arise from the inflammatory CAFs in hormone-sensitive PCa; therefore, they represent two functional states of an otherwise ontogenically identical cell type. Antiandrogen treatment unleashes TGF-β signaling, resulting in SOX4-SWI/SNF-dependent CAF phenotype switching. SPP1+ myCAFs in turn render PCa refractory to ADT via an SPP1-ERK paracrine mechanism. Importantly, these sub-myCAFs are associated with inferior therapeutic outcomes, providing the rationale for inhibiting polarization or paracrine mechanisms to circumvent castration resistance. Collectively, our results highlight that therapy-induced phenotypic switching of CAFs is coupled with disease progression and that targeting this stromal component may restrain CRPC.

Lineage plasticity and treatment resistance in prostate cancer: the intersection of genetics, epigenetics, and evolution.

Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Immunotherapy in Prostate Cancer: State of Art and New Therapeutic Perspectives.

Prostate cancer (PC) is the most common type of tumor in men. In the early stage of the disease, it is sensitive to androgen deprivation therapy. In patients with metastatic castration-sensitive prostate cancer (mHSPC), chemotherapy and second-generation androgen receptor therapy have led to increased survival. However, despite advances in the management of mHSPC, castration resistance is unavoidable and many patients develop metastatic castration-resistant disease (mCRPC). In the past few decades, immunotherapy has dramatically changed the oncology landscape and has increased the survival rate of many types of cancer. However, immunotherapy in prostate cancer has not yet given the revolutionary results it has in other types of tumors. Research into new treatments is very important for patients with mCRPC because of its poor prognosis. In this review, we focus on the reasons for the apparent intrinsic resistance of prostate cancer to immunotherapy, the possibilities for overcoming this resistance, and the clinical evidence and new therapeutic perspectives regarding immunotherapy in prostate cancer with a look toward the future.

The Molecular Basis and Clinical Consequences of Chronic Inflammation in Prostatic Diseases: Prostatitis, Benign Prostatic Hyperplasia, and Prostate Cancer.

Chronic inflammation is now recognized as one of the major risk factors and molecular hallmarks of chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate tumorigenesis. However, the molecular mechanisms by which chronic inflammation signaling contributes to the pathogenesis of these prostate diseases are poorly understood. Previous efforts to therapeutically target the upstream (e.g., TLRs and IL1-Rs) and downstream (e.g., NF-κB subunits and cytokines) inflammatory signaling molecules in people with these conditions have been clinically ambiguous and unsatisfactory, hence fostering the recent paradigm shift towards unraveling and understanding the functional roles and clinical significance of the novel and relatively underexplored inflammatory molecules and pathways that could become potential therapeutic targets in managing prostatic diseases. In this review article, we exclusively discuss the causal and molecular drivers of prostatitis, BPH, and prostate tumorigenesis, as well as the potential impacts of microbiome dysbiosis and chronic inflammation in promoting prostate pathologies. We specifically focus on the importance of some of the underexplored druggable inflammatory molecules, by discussing how their aberrant signaling could promote prostate cancer (PCa) stemness, neuroendocrine differentiation, castration resistance, metabolic reprogramming, and immunosuppression. The potential contribution of the IL1R-TLR-IRAK-NF-κBs signaling molecules and NLR/inflammasomes in prostate pathologies, as well as the prospective benefits of selectively targeting the midstream molecules in the various inflammatory cascades, are also discussed. Though this review concentrates more on PCa, we envision that the information could be applied to other prostate diseases. In conclusion, we have underlined the molecular mechanisms and signaling pathways that may need to be targeted and/or further investigated to better understand the association between chronic inflammation and prostate diseases.

Apalutamide for metastatic castration-sensitive prostate cancer: final analysis of the Asian subpopulation in the TITAN trial.

The final analysis of the phase 3 Targeted Investigational Treatment Analysis of Novel Anti-androgen (TITAN) trial showed improvement in overall survival (OS) and other efficacy endpoints with apalutamide plus androgen deprivation therapy (ADT) versus ADT alone in patients with metastatic castration-sensitive prostate cancer (mCSPC). As ethnicity and regional differences may affect treatment outcomes in advanced prostate cancer, a post hoc final analysis was conducted to assess the efficacy and safety of apalutamide in the Asian subpopulation. Event-driven endpoints were OS, and time from randomization to initiation of castration resistance, prostate-specific antigen (PSA) progression, and second progression-free survival (PFS2) on first subsequent therapy or death. Efficacy endpoints were assessed using the Kaplan-Meier method and Cox proportional-hazards models without formal statistical testing and adjustment for multiplicity. Participating Asian patients received once-daily apalutamide 240 mg (n = 111) or placebo (n = 110) plus ADT. After a median follow-up of 42.5 months and despite crossover of 47 placebo recipients to open-label apalutamide, apalutamide reduced the risk of death by 32% (hazard ratio [HR]: 0.68; 95% confidence interval [CI]: 0.42-1.13), risk of castration resistance by 69% (HR: 0.31; 95% CI: 0.21-0.46), PSA progression by 79% (HR: 0.21; 95% CI: 0.13-0.35) and PFS2 by 24% (HR: 0.76; 95% CI: 0.44-1.29) relative to placebo. The outcomes were comparable between subgroups with low- and high-volume disease at baseline. No new safety issues were identified. Apalutamide provides valuable clinical benefits to Asian patients with mCSPC, with an efficacy and safety profile consistent with that in the overall patient population.

Abstract A071: SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Currently, the development of efficacious agents against metastatic castration resistance prostate cancer is hampered by the lack of preclinical models that accurately reflect disease heterogeneity and treatment response. To overcome this barrier, we have developed a preclinical metastasis model using LuCap136 (PDM136), an androgen receptor (AR) positive patient-derived xenograft (PDX) that forms metastases in bone and soft tissues 3 weeks after intracardiac injection. With a combined PTEN −/−, TP53 −/mut, RB1 −/− genotype, PDM136 represents a subgroup of difficult-to-treat mPCRC. PDM136 is organotropic for bone. Mice usually reach the end point after 6-7 weeks due to paraplegia and/or cachexia. Metastatic cells maintain the expression of nuclear AR and epithelial marker cytokeratin 8. Surgical castration significantly inhibits metastatic progression and prolonged the survival rate, however, tumor will eventually progress to castration resistance. To our knowledge, PDM136 is the first AR-positive prostate cancer metastasis model that recapitulates the disease progress of mCRPC. To define the molecular mechanisms of castration resistance in PDM136, we performed RNA expression profiling on bone and soft tissue metastasis collected from intact and castrated mice. Differential expression analysis revealed over 500 transcripts were significantly altered after castration, with SOX2 and ASCL1 among the top up-regulated genes. Gene set enrichment analysis showed decreased AR signaling and increased neuroepithelium transformation in the castrated group. To validate the RNA-seq data, we performed IHC on proteins whose genes were identified by RNAseq including SOX2, ASCL1, as well as androgen receptor (AR), glucocorticoid receptor (GR) and neuroendocrine marker SYP. Surprisingly, we found that the expression of the five markers is organ-specific upon castration. Unsupervised clustering of the IHC stained markers from the bone of castrated mice revealed that AR and GR were undetectable in majority of foci whereas substantial numbers of foci in soft tissue metastasis expressed AR and GR at moderate intensity. Furthermore, we found that AR and GR expression were mutually exclusive in many foci from soft tissue metastasis. SOX2 and ASCL1 were mainly expressed in AR negative tumors, while SYP was expressed in a subpopulation of SOX2+ positive foci. Notably, liver metastasis had the most SYP expressing foci among all organs suggesting liver microenvironment promotes neuroendocrine differentiation. In summary, our IHC results revealed inter-tumor and organ-specific cellular heterogeneity induced by castration. PDM136 is a platform to understand the mechanism of responsible for mCRPC, but also a valuable tool for anti-mCRPC drug discovery. The co-existence of multiple phenotypes indicated that combination therapies targeting multiple pathways are essential to harness this disease. Citation Format: Anson Ku, Juan Yin, Adam G. Sowalsky, Kathleen Kelly. SOX2 induced plasticity and microenvironment lead to intratumoral and tissue-specific phenotypic heterogeneity in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A071.

Abstract A012: Adaptive and non-adaptive gene expression responses in prostate cancer during androgen deprivation

Abstract Development of novel and efficient treatments for advanced prostate cancer (PCa) requires better understanding of the cellular mechanisms behind the transition to castration resistant prostate cancer (CRPC). We created long-term cell cultures to model the development of castration resistance. Using a low passage VCaP cells as an initial model, we first created a highly testosterone-dependent PCa cell line (VCaP-T) and then a subclone cell line developing the ability to grow at low testosterone concentration (VCaP-CT). Adaptation to low testosterone was solely due to long term culturing and experimental work confirmed for example higher AR expression and antiandrogen resistance in VCaP-CT compared to VCaP-T. With the developed models we aimed to uncover persistent and adaptive responses to testosterone level and to identify genes that potentially contribute to transition and proliferation of the castration resistant cells through androgen receptor (AR) regulation. Expression levels were measured using RNA-seq. We defined AR-associated genes as the ones with changes due to testosterone depletion in VCaP-T (418 genes) and further adaptive genes as the ones that restored expression level in VCaP-CT (134 genes). Interestingly, we identified 151 genes that were non-AR-associated in VCaP-T but gained the association in VCaP-CT cells. Genes gaining the AR-association were also hypothesized to be important for CRPC progression and castration resistant cells. We validated and assessed the clinical significance of the model and the findings using The Cancer Genome Atlas Prostate Adenocarcinoma data and multivariate Cox regression analysis. Expressions of 47 AR-associated or association gaining genes were statistically significant markers for progression-free survival. These included genes related to immune response, adhesion and transport. Genes ABCC5, SPRED3, TMPRSS11F, SPARC, SLC1A1, SATB1, PDGFRB and C1QTNF6 showed strong evidence being associated with tumor stage and aggressiveness in addition to progression-free survival. Taken together, we identified and validated multiple genes being linked with progression of prostate cancer and proposed several novel risk genes. These findings increase knowledge on mechanisms of castration resistance and may open new possibilities for treatments. Identified genes have potential to be used as biomarkers or therapeutic targets after further studies. Citation Format: Reetta Nätkin, Pasi Pennanen, Heimo Syvälä, Merja Bläuer, Juha Kesseli, Teuvo L.J. Tammela, Matti Nykter, Teemu J. Murtola. Adaptive and non-adaptive gene expression responses in prostate cancer during androgen deprivation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A012.

Abstract B018: Loss of androgen receptor-mediated repression leads to EphA2 overexpression that promotes cellular dedifferentiation and castration resistance of prostate cancer through noncanonical signaling

Abstract As a member of Eph subfamily receptor tyrosine kinases, EphA2 has been extensively studied in cancer. It is frequently overexpressed in many different types of human cancer, which is often correlated with tumor progression. We reported recently that EphA2 has diametrically opposite roles in regulating PCa cell differentiation, proliferation, migration, and invasion. In the presence of ligands called ephrin-As, EphA2 canonical signaling inhibits Ras/ERK and PI3K/Akt pathways and suppressed cell proliferation and migration. In contrast, in the absence of ligands EphA2 mediates noncanonical signaling, which promotes chemotactic migration and invasion and accentuates the tumor cell stem properties. Interestingly the ligand-independent noncanonical signaling is correlated with phosphorylation of EphA2 on serine 897 (S897). S897A mutation abolished this ligand-independent effect on cell differentiation and migration and suppressed tumor development in PC3 xenograft model. In mouse prostate, EphA2 is expressed in the stem cell niche together with its cognate ephrin-A ligands in the peri-urethra region but is repressed in AR-positive, differentiated prostate epithelial cells. During progression toward CRPC, ephrin-ligand expression is reduced while EphA2 is overexpressed, facilitating ligand-independent noncanonical signaling by EphA2. Consistent with the negative roles of AR in regulating EphA2 expression, treatment with anti-AR enzalutamide promotes EphA2 expression. In contrast, activating AR with DHT strongly suppressed EphA2 expression. Knockout of EphA2 in the AR+ LAPC4 cells, delayed progression towards castration resistance in vivo. Our results unveiled the previously unknown functional crosstalk between AR and EphA2, and the important role of EphA2 in the malignant progression of prostate cancer. Citation Format: Ryan Lingerak, Bingcheng Wang. Loss of androgen receptor-mediated repression leads to EphA2 overexpression that promotes cellular dedifferentiation and castration resistance of prostate cancer through noncanonical signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B018.

Abstract B020: BCL2 expression is enriched in AR-independent advanced prostate cancer

Abstract Background The widespread use of highly potent androgen receptor (AR) inhibitors has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC). AR independence, typically driven by loss of AR expression and/or trans-differentiation to a neuroendocrine or basal phenotype, lacks effective therapeutic strategies. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine tumours and has been proposed as a therapeutic target for this subset of advanced prostate cancers. Objective To characterise AR expression in advanced PC, to evaluate its association with BCL2, to elucidate their clinical significance, and to explore whether BCL2 may represent a valid therapeutic target and/or biomarker. Methods AR and BCL2 expression were immunohistochemically scored in two independent CRPC cohorts (431 biopsies from 247 patients), including 62 matched same-patient castration-sensitive PC (CSPC) biopsies. Scores were associated with retrospectively collected clinical data including overall survival (OS) and response to AR-targeting therapy. BCL2 protein expression was also determined in 28 primary prostate samples (19 patients). Transcriptome data from two independent CRPC cohorts were analysed, including for associations between BCL2 expression and gene expression signatures. A patient-derived xenograft-organoid (PDX-O) CRPC model with high expression of BCL2 was treated with multiple BH3 mimetics. Results and limitations We demonstrate that AR protein loss emerges with castration resistance and occurs in subset of metastatic CRPC (mCRPC) (5%) associating with worse OS (51 vs 18 months from CRPC diagnosis, hazard ratio [HR] 5.52 [2.83-10.78], p&amp;lt;0.001). BCL2 protein expression is enriched in AR-negative mCRPC (p&amp;lt;0.001), associating with worse OS (53 vs 19 months from CRPC diagnosis, HR 2.80 [1.40-5.57], p=0.002) and resistance to AR-targeting therapy (≥50% PSA response: 48% vs 13%), irrespective of AR expression status. BCL2 mRNA expression associates with several key signalling pathways known to promote stemness and lineage plasticity, including EMT and IL6/JAK/STAT3. Furthermore, BCL2 is enriched in basal prostate cells, whose expression profile has been linked with aggressive subtypes of mCRPC, including small cell neuroendocrine CPRC. We show that a high BCL2-expressing PDX-O is resistant to BCL2 inhibition, with functional redundancy between the anti-apoptotic BCL2 family proteins. BCLXL and MCL1 are highly expressed in BCL2-positive mCRPC and may drive resistance to BCL2 targeting. Utilising a post-mortem mCRPC biopsy cohort, we show intra-patient heterogeneity in BCL2 expression. These findings highlight the need for combination therapies. Further studies are required to dissect the interaction between AR and BCL2, and to probe the functional role of BCL2 in this subset of mCRPC. Conclusions BCL2-positivity associates with AR-independence, worse OS, and resistance to AR-targeting in CRPC. Combination strategies are required to optimise response to BCL2 inhibition and to combat heterogeneity. Citation Format: Daniel Westaby, Juan M. Jimenez-Vacas, Ines Figueiredo, Claire Pettinger, Bora Gurel, Denisa Bogdan, Jan Rekowski, Lorenzo Buroni, Antje Neeb, Ruth Riisnaes, Mateus Crespo, Susana Miranda, Ana Ferreira, Daniel Nava Rodrigues, George Seed, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Christina Guo, Juliet Carmichael, Rafael Grochot, Khobe Chandran, Andreas Varkaris, Steven P. Balk, Wei Yuan, Suzanne Carreira, Peter S. Nelson, Michael Haffner, Eva Corey, Johann de Bono, Adam Sharp. BCL2 expression is enriched in AR-independent advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B020.

Abstract B058: Exploring the oncogenic role of six-transmembrane epithelial antigen of the prostate in the context of androgen receptor signaling

Abstract Prostate cancer is the second leading cause of death among American men, where pathogenesis is predominantly driven by dysregulation of the Androgen Receptor (AR). The current standard of care for metastatic disease includes a regime of androgen deprivation therapy (ADT) and AR antagonists, which increases survival to 24-48 months. Selective pressure of the therapy often leads to resurgence of AR activity resulting in castrate resistant disease, for which there are no curative treatment options. Moreover, once the disease has progressed to castrate resistance, AR directed therapies are no longer viable as a sole treatment option. Six-Transmembrane Epithelial Antigen of the Prostate 1-4 (STEAP) proteins are a family of ferric metalloreductases involved in maintaining iron homeostasis. STEAP expression is enriched in androgen sensitive and hormone refractory disease states, where increased expression correlates with high Gleason Score. STEAP proteins display greater intratumoral homogeneity at both primary and metastatic sites than the canonically used Prostate Specific Membrane Antigen (PSMA), indicating attractive diagnostic and therapeutic potential. Differential expression profiles have been observed among the STEAP family members depending on AR status in-vitro, suggesting a dependence on the AR signaling network. Previous literature indicates silencing of STEAPs in-vitro decreased cell viability and reduced proliferative, invasive, and migratory phenotypes; moreover in-vivo xenografts in which STEAPs are knocked down exhibited a significant reduction in tumor growth. Preliminary data suggests that treatment with Enzalutamide reduces the expression of STEAP proteins, implying a mechanistic link to the AR signaling axis. AMG509, a bispecific STEAP1-CD3 antibody, is the only STEAP directed therapeutic actively in clinical trials for prostate cancer (NCT04221542). There is limited mechanistic data regarding how STEAP directed therapies exert their anti-tumor function. Despite sharing similar architecture, localization, and biological implications, each STEAP protein retains unique functional roles within the cell. We plan to perform a systematic investigation on the oncogenic roles of individual STEAP protein in the context of AR dependent and independent prostate cancer models. In-vitro pharmacological AR manipulations will be used to evaluate changes in STEAP expression in multiple disease models. Genetic manipulations of STEAPs will be carried out under normal growth and ADT conditions to investigate the effects on growth, migration, oxidative stress, and cell cycle regulation. Given their principal role as ferric reductases, iron supplementation and iron drop-out will be examined in parallel. Novel treatment regimens will be explored using a combination of AR and STEAP directed therapeutics. Collectively, this study aims to uncover both the overlapping and distinct functions of STEAP proteins within the AR signaling axis and to illuminate their usefulness as a therapeutic target. Citation Format: Candice Bizzaro, Latese Evens, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Exploring the oncogenic role of six-transmembrane epithelial antigen of the prostate in the context of androgen receptor signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B058.

Abstract A002: A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer

Abstract Introduction: A leading contributor to the significant mortality burden of prostate cancer (PC) is the resistance to androgen deprivation therapy (ADT). Although most PCs are initially sensitive to ADT, the duration of response is variable, and relapse invariably occurs in the transition to metastatic castration-resistant prostate cancer (mCRPC), the lethal form of the disease. Therefore, it is essential to discover and validate early resistance mechanisms that initially protect castration sensitive prostate cancer (CSPC) cells from ADT and to develop novel combination therapies that can block or delay the ADT-induced shift from CSPC to mCRPC. Overexpression of the hallmark antiapoptotic gene B-cell lymphoma 2 (BCL2) is a potential mechanism of resistance to ADT. Methods: To discover the molecular drivers of castration resistance, we analyzed the tumor transcriptomic profile of a cohort of localized PC patients (N=58) treated with neoadjuvant-intensive ADT (NCT00924469; Taplin et al, 2014, Sowalsky et al, 2018). Experimentally, we treated CSPC cells and genetically engineered mice (GEM) prostate tumors with ADT and analyzed the signaling pathways, which were enriched post-ADT treatment compared untreated samples. Results: We discovered ~10-fold increased mRNA expression of BCL2 (p&amp;lt;0.001) in ADT-treated localized PC patients compared to untreated (standard of care surgery) patients in NCT00924469 samples. Our experimental study showed that treatment with AR inhibitors strongly augments BCL2 expression in human CSPC cell lines and GEM PC model, indicating possible direct negative regulation of BCL2 by the AR-signaling pathway. Mechanistically, our preliminary data reveal a striking induction of cell metabolism pathways (fatty acid and xenobiotic metabolism), including activation of PI3K/AKT signaling in BCL2-overexpressing CSPC cells. We used siRNA-mediated BCL2 knockdown in LNCaP cells and showed that loss of BCL2 strongly suppressed ADT-induced AKT- phosphorylation. We also showed increased Bcl2 mRNA expression in PTEN-knockout mice-derived prostate organoids compared to the wild-type control. Furthermore, we observed that AKT kinase -inhibitor MK2206 strongly inhibits enzalutamide-induced BCL2 expression in LNCaP cells. Conclusion: Together our data indicate a non-canonical role of BCL2 in activating PI3K/AKT signaling in prostate cancer. For the first time, we demonstrate the crucial importance of AR-BCL2-AKT signaling pathway crosstalk in CSPC biology and reveal this complex signaling crosstalk may act as a key driver of CSPC transformation to lethal CRPC. Citation Format: Goutam Chakraborty, Rahim Hirani, Subhiksha Nandakumar, Nabila Zaman, Teja Muralidhar Kalidindi, Sai Harisha Rajanala, Gwo-Shu M. Lee, Konrad H. Stopsack, Naga Vara Kishore Pillarsetty, Lorelei A. Mucci, Daniel C. Danila, Philip W. Kantoff. A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A002.