Androgen Receptor Signaling Inhibitors Research Articles

PSA-Targeted Alpha-, Beta-, and Positron-Emitting Immunotheranostics in Murine Prostate Cancer Models and Nonhuman Primates.

Most patients with prostate cancer treated with androgen receptor (AR) signaling inhibitors develop therapeutic resistance due to restoration of AR functionality. Thus, there is a critical need for novel treatment approaches. Here we investigate the theranostic potential of hu5A10, a humanized mAb specifically targeting free PSA (KLK3). LNCaP-AR (LNCaP with overexpression of wildtype AR) xenografts (NSG mice) and KLK3_Hi-Myc transgenic mice were imaged with 89Zr- or treated with 90Y- or 225Ac-labeled hu5A10; biodistribution and subcellular localization were analyzed by gamma counting, PET, autoradiography, and microscopy. Therapeutic efficacy of [225Ac]hu5A10 and [90Y]hu5A10 in LNCaP-AR tumors was assessed by tumor volume measurements, time to nadir (TTN), time to progression (TTP), and survival. Pharmacokinetics of [89Zr]hu5A10 in nonhuman primates (NHP) were determined using PET. Biodistribution of radiolabeled hu5A10 constructs was comparable in different mouse models. Specific tumor uptake increased over time and correlated with PSA expression. Treatment with [90Y]/[225Ac]hu5A10 effectively reduced tumor burden and prolonged survival (P ≤ 0.0054). Effects of [90Y]hu5A10 were more immediate than [225Ac]hu5A10 (TTN, P < 0.0001) but less sustained (TTP, P < 0.0001). Complete responses were observed in 7 of 18 [225Ac]hu5A10 and 1 of 9 mice [90Y]hu5A10. Pharmacokinetics of [89Zr]hu5A10 were consistent between NHPs and comparable with those in mice. [89Zr]hu5A10-PET visualized the NHP-prostate over the 2-week observation period. We present a complete preclinical evaluation of radiolabeled hu5A10 in mouse prostate cancer models and NHPs, and establish hu5A10 as a new theranostic agent that allows highly specific and effective downstream targeting of AR in PSA-expressing tissue. Our data support the clinical translation of radiolabeled hu5A10 for treating prostate cancer.

Regulation of carcinogenesis and mediation through Wnt/\u03b2-catenin signaling by 3,3\u2032-diindolylmethane in an enzalutamide-resistant prostate cancer cell line

Enzalutamide (ENZ) is an important drug used to treat castration-resistant prostate cancer (CRPC), which inhibits androgen receptor (AR) signaling. Previous study showed that 3,3′-diindolylmethane (DIM) is an AR antagonist that also inhibits Wnt signaling and epithelial-mesenchymal transition (EMT). To investigate whether combined treatment with ENZ and DIM can overcome ENZ resistance by regulating Wnt signaling to inhibit AR signaling and EMT in ENZ-resistant prostate cancer cells, 22Rv1 cells were cultured in normal medium and treated with ENZ, DIM, and DIM with ENZ. Exposure of ENZ-resistant cells to both DIM and ENZ significantly inhibited cell proliferation without cytotoxicity and invasion in comparison with the control. DIM significantly increased the E-cadherin expression and inhibited the expressions of Vimentin and Fibronectin, subsequently inhibiting EMT. Co-treatment with ENZ and DIM significantly increased the expressions of GSK3β and APC and decreased the β-catenin protein expression, causing inhibition of Wnt signaling and AR expression, it also significantly decreased the AR-v7 expression and down-regulated AR signaling. Via suppression of Wnt and AR signaling, co-treatment increased the E-cadherin and decreased the Vimentin and Fibronectin RNA and protein expressions, then inhibited EMT. Co-treatment with DIM and ENZ regulated Wnt signaling to reduce not only the AR expression, but also the AR-v7 expression, indicating suppression of EMT that inhibits cancer cell proliferation, invasion and migration to ameliorate ENZ resistance.

Selective targeting of the androgen receptor-DNA binding domain by the novel antiandrogen SBF-1 and inhibition of the growth of prostate cancer cells.

Prostate cancers are reliant on androgens for growth and survival. Clinicians and researchers are looking for potent treatments for the resistant forms of prostate cancer; however, a handful of small molecules used in the treatment of castration-resistant prostate cancer have not shown potent effects owing to the mutations in the AR (Androgen Receptor). We used SBF-1, a well-characterized antitumor agent with potent cytotoxic effects against different kinds of cancers and investigated its effect on human prostate cancer. SBF-1 substantially inhibited the proliferation, induced apoptosis, and caused cell cycle arrest in LNCaP and PC3/AR+ prostate cancer cell lines. SBF-1 inhibited the activation of the IGF-1-PNCA pathway, as demonstrated by decreased expression of IGF-1 (insulin-like growth factor 1), proliferating cell nuclear antigen (PCNA), and its downstream Bcl-2 protein. Using microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) assays, we observed a direct binding of SBF-1 to the AR. SBF-1 binds to the AR-DBD (DNA-binding domain) and blocks the transcription of its target gene. SBF-1 demonstrated a potent antitumor effect in vivo; it inhibited AR signaling and suppressed tumor growth in animals. Our study suggests that SBF-1 is an inhibitor of the AR and might be used in the treatment of prostate cancer.

Dynamic Plasticity of Prostate Cancer Intermediate Cells During Androgen Receptor Targeted Therapy

The prevalence of treatment induced small cell neuroendocrine prostate cancer (t-SCNC) is increasing due to the extended use of potent AR signaling inhibitors. Understanding the kinetics and potential for reversibility of t-SCNC progression has been hampered by the lack of model systems suitable for studying intermediate tumor cell states. Here, we define a transcriptional reporter model that provides dynamic measures of treatment induced changes in human prostate cancer cell subpopulations. With this system we have identified intermediate tumor cells, defined as double high in expression for prostate specific antigen (PSA) and neuron specific enolase (NSE) expression. This tumor cell subpopulation functions as a necessary and sufficient component for the cellular conversion to an NE transcriptional signature when under the selective pressure of androgen ablation or AR signaling inhibitors. Such transcriptional plasticity was found to be reversible in a hormone and genetic driver dependent manner. Our data provide novel and mechanistic in site for understanding the contribution of transition cell populations to the emergence of therapeutic resistance in advanced castration resistant prostate cancer (CRPC).

A Clinical Evaluation of Enzalutamide in Metastatic Castration-Sensitive Prostate Cancer: Guiding Principles for Treatment Selection and Perspectives on Research.

Enzalutamide was the first novel androgen receptor signaling inhibitor to demonstrate an overall survival benefit in non-metastatic and metastatic castration-sensitive prostate cancer (CSPC). It has emerged as one of the most commonly prescribed oral prostate cancer therapies (ARSI) by medical oncologists and urologists. Amongst a panoply of treatment options for metastatic CSPC, safe and effective utilization of enzalutamide dictates a detailed understanding of alternative therapy options and competing toxicity profiles. Ongoing research supports the potential for expanded enzalutamide use in earlier disease states, in combination with other systemic agents and as monotherapy (without androgen deprivation therapy). Optimal application of enzalutamide will ultimately require greater insight and attention to mitigating strategies for treatment-associated fatigue, cognitive impairment, and functional decline. This publication will comprehensively analyze the clinical evidence and guiding principles of enzalutamide use in CSPC. We will also provide a critical review of ongoing and future ARSI research focusing on pharmacologic approaches to overcome treatment resistance and strategies to improve treatment-associated functional impairment.

Automated enumeration and phenotypic characterization of CTCs and tdEVs in patients with metastatic castration resistant prostate cancer

BackgroundAlthough most patients with metastatic castration-resistant prostate cancer (mCRPC) initially benefit from treatment with androgen receptor signaling inhibitors (ARSi), resistance inevitably occurs. Hence, we investigated the prognostic value of automated circulating tumor cell (CTC) and tumor-derived extracellular vesicle (tdEV) enumeration and their dynamics, in patients with mCRPC in the context of the initiation of treatment with ARSi. Furthermore, we hypothesize that CTC phenotypic heterogeneity might serve as a measurable biomarker under these circumstances.MethodsUsing an image analysis tool, we reanalyzed all CellSearch images previously acquired in the context of a prospective, multicenter clinical study for patients with mCRPC (n = 170) starting a new line of ARSi, for CTC and tdEV detection and enumeration. CTC (n = 19 129) phenotypic diversity was quantified by the Shannon index (SI). Progression-free survival (PFS) and overall survival (OS) were compared between groups of patients stratified according to CTC, tdEV, and SI levels.ResultsAutomated CTC enumeration provided similar clinical prognostication compared with operator-based counts. Patients demonstrating high CTC phenotypic heterogeneity before therapy had a shorter median PFS (4.82 vs. 8.49 months, HR 1.79; P = 0.03) and OS (12.6 months vs. not reached, HR 2.32; P = 0.03), compared to patients with low diversity, irrespective of CTC level. Multivariable analysis showed how the prognostic value of the baseline SI was lost by pretreatment chemotherapy status, CTC counts, and PSA levels.ConclusionsAutomated CTC counts are a reliable substitute for reviewer-based enumeration, as they are equally informative for prognosis assessment in patients with mCRPC. Beyond enumeration, we demonstrated the added value of studying CTC phenotypic diversity for patient prognostication, warranting future investigation.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

SummaryAnti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Morphology-Predicted Large-Scale Transition Number in Circulating Tumor Cells Identifies a Chromosomal Instability Biomarker Associated with Poor Outcome in Castration-Resistant Prostate Cancer.

Chromosomal instability (CIN) increases a tumor cell's ability to acquire chromosomal alterations, a mechanism by which tumor cells evolve, adapt, and resist therapeutics. We sought to develop a biomarker of CIN in circulating tumor cells (CTC) that are more likely to reflect the genetic diversity of patient's disease than a single-site biopsy and be assessed rapidly so as to inform treatment management decisions in real time. Large-scale transitions (LST) are genomic alterations defined as chromosomal breakages that generate chromosomal gains or losses of greater than or equal to10 Mb. Here we studied the relationship between the number of LST in an individual CTC determined by direct sequencing and morphologic features of the cells. This relationship was then used to develop a computer vision algorithm that utilizes CTC image features to predict the presence of a high (9 or more) versus low (8 or fewer) LST number in a single cell. As LSTs are a primary functional component of homologous recombination deficient cellular phenotypes, the image-based algorithm was studied prospectively on 10,240 CTCs in 367 blood samples obtained from 294 patients with progressing metastatic castration-resistant prostate cancer taken prior to starting a standard-of-care approved therapy. The resultant computer vision-based biomarker of CIN in CTCs in a pretreatment sample strongly associated with poor overall survival times in patients treated with androgen receptor signaling inhibitors and taxanes. SIGNIFICANCE: A rapidly assessable biomarker of chromosomal instability in CTC is associated with poor outcomes when detected in men with progressing mCRPC.

A Systematic Review and Meta-Analysis on the Predictive Value of Cell-Free DNA-Based Androgen Receptor Copy Number Gain in Patients With Castration-Resistant Prostate Cancer.

It has been suggested that androgen receptor copy number gain (AR gain) detected in cell-free DNA (cfDNA) can predict treatment response to androgen receptor signaling inhibitors (ARSIs) in patients with castration-resistant prostate cancer (CRPC). But it is unclear whether cfDNA-based AR gain is a true resistance mechanism to ARSIs or mainly a reflection of the tumor burden. In this systematic review, we aim to summarize current literature and comment on the potential of cfDNA-based AR gain as a predictive biomarker to guide therapy choices. A literature search was conducted in PubMed/Medline, Cochrane, Embase, and Web of Science databases. Sixteen articles published before November 2019 were selected for the meta-analysis, representing more than 1,000 patients. By using a random effects model, the progression-free survival (PFS) and overall survival (OS) were compared between patients with and without cfDNA-based AR gain who had been treated with ARSIs or with taxane chemotherapy. Upon treatment with ARSIs, the PFS (hazard ratio [HR], 2.33; 95% CI, 2.00 to 2.72; P < .0001) and the OS (HR, 3.83; 95% CI, 3.11 to 4.70; P < .0001) were worse for patients with cfDNA-based AR gain, independent of the line and type of ARSIs. The OS and PFS in patients treated with first-line docetaxel or second-line or third-line cabazitaxel seemed to be unaffected by AR gain, despite a higher disease burden in patients with AR gain. AR gain was associated with reduced response with later lines of docetaxel. In patients with CRPC, cfDNA-based AR gain is associated with a worse response to ARSIs. The effect on patients who are receiving taxane chemotherapy seems to be dependent on the type and line, although data are limited. Future prospective studies are essential to assess the true potential of cfDNA-based AR gain as a minimally invasive biomarker to guide therapy choice.

RNA-binding protein DDX3 mediates posttranscriptional regulation of androgen receptor: A mechanism of castration resistance

Prostate cancer (CaP) driven by androgen receptor (AR) is treated with androgen deprivation; however, therapy failure results in lethal castration-resistant prostate cancer (CRPC). AR-low/negative (ARL/-) CRPC subtypes have recently been characterized and cannot be targeted by hormonal therapies, resulting in poor prognosis. RNA-binding protein (RBP)/helicase DDX3 (DEAD-box helicase 3 X-linked) is a key component of stress granules (SG) and is postulated to affect protein translation. Here, we investigated DDX3-mediated posttranscriptional regulation of AR mRNA (messenger RNA) in CRPC. Using patient samples and preclinical models, we objectively quantified DDX3 and AR expression in ARL/- CRPC. We utilized CRPC models to identify DDX3:AR mRNA complexes by RNA immunoprecipitation, assess the effects of DDX3 gain/loss-of-function on AR expression and signaling, and address clinical implications of targeting DDX3 by assessing sensitivity to AR-signaling inhibitors (ARSI) in CRPC xenografts in vivo. ARL/- CRPC expressed abundant AR mRNA despite diminished levels of AR protein. DDX3 protein was highly expressed in ARL/- CRPC, where it bound to AR mRNA. Consistent with a repressive regulatory role, DDX3 localized to cytoplasmic puncta with SG marker PABP1 in CRPC. While induction of DDX3-nucleated SGs resulted in decreased AR protein expression, inhibiting DDX3 was sufficient to restore 1) AR protein expression, 2) AR signaling, and 3) sensitivity to ARSI in vitro and in vivo. Our findings implicate the RBP protein DDX3 as a mechanism of posttranscriptional regulation for AR in CRPC. Clinically, DDX3 may be targetable for sensitizing ARL/- CRPC to AR-directed therapies.

Inhibition of Androgen Receptor Signaling Promotes Prostate Cancer Cell Migration via Upregulation of Annexin A1 Expression

Recent studies indicate that androgen deprivation therapy (ADT), the main therapeutic approach for metastatic prostate cancer (PCa), accelerates PCa invasion and metastasis. Annexin A1 (ANXA1) is a Ca2+-regulated phospholipid-binding protein that can promote PCa migration and invasion. The aim of this study is to determine whether ANXA1 is regulated by ADT and participates in PCa progression after ADT, and to explore the possible mechanism of ANXA1-mediated PCa migration. Expression of ANXA1 and androgen receptor (AR) in PCa cell lines and tissues was detected, and the association between these two proteins were analyzed. Expression of ANXA1 was evaluated after AR knockdown or AR inhibition in PCa cell lines. Cell migration of PCa cell liness after ANXA1 knockdown or overexpression was determined by invitro migration assay. Transcriptome analysis was used to explore the possible mechanism of ANXA1-mediated PCa migration. ANXA1 expression in PCa cell lines and tissues was reversely associated with AR. Invitro studies revealed an increase in ANXA1 expression after AR knockdown or treatment with AR antagonist. Moreover, functional assays indicated that ANXA1 knockdown in PCa cells significantly inhibited cell migration, while ANXA1 overexpression in PCa cells significantly accelerated cell migration. Transcriptome analysis showed that ANXA1 regulated multiple genes involved in cell junction organization, such as CADM1, LIMCH1 and PPM1F. Our results indicate that ADT might accelerate PCa metastasis via ANXA1 expression and PCa cell migration.

Recent Discoveries in the Androgen Receptor Pathway in Castration-Resistant Prostate Cancer.

The androgen receptor (AR) is the main therapeutic target in advanced prostate cancer, because it regulates the growth and progression of prostate cancer cells. Patients may undergo multiple lines of AR-directed treatments, including androgen-deprivation therapy, AR signaling inhibitors (abiraterone acetate, enzalutamide, apalutamide, or darolutamide), or combinations of these therapies. Yet, tumors inevitably develop resistance to the successive lines of treatment. The diverse mechanisms of resistance include reactivation of the AR and dysregulation of AR cofactors and collaborative transcription factors (TFs). Further elucidating the nexus between the AR and collaborative TFs may reveal new strategies targeting the AR directly or indirectly, such as targeting BET proteins or OCT1. However, appropriate preclinical models will be required to test the efficacy of these approaches. Fortunately, an increasing variety of patient-derived models, such as xenografts and organoids, are being developed for discovery-based research and preclinical drug screening. Here we review the mechanisms of drug resistance in the AR signaling pathway, the intersection with collaborative TFs, and the use of patient-derived models for novel drug discovery.

Impact of mutations in homologous recombination repair genes on treatment outcomes for metastatic castration resistant prostate cancer.

IntroductionA significant proportion of patients with metastatic castration-resistant prostate cancer (mCRPC) harbor mutations in homologous recombination (HR) repair genes, with some of these mutations associating with increased tumor susceptibility to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy. While mutations in some HR repair genes (e.g., BRCA1/2) have been associated with a more aggressive clinical course, prior studies correlating HR mutational status with treatment response to androgen receptor (AR) signaling inhibitors (ARSIs) or taxane-based chemotherapy have yielded conflicting results.MethodsWe conducted a single-center retrospective analysis to assess clinical outcomes to conventional, regulatory-approved therapies in mCRPC patients with somatic (monoallelic and biallelic) and/or germline HR repair mutations compared to patients without alterations as determined by clinical-grade next-generation sequencing assays. The primary endpoint was PSA30/PSA50 response, defined as ≥30%/≥50% prostate-specific antigen (PSA) reduction from baseline. Secondary endpoints of PSA progression-free survival (pPFS) and clinical/radiographic progression-free survival (crPFS) were estimated using Kaplan-Meier methods.ResultsA total of 90 consecutively selected patients were included in this analysis, of which 33 (37%) were identified to have HR repair gene mutations. Age, race, Gleason score, prior surgery, and receipt of prior radiation therapy were comparable between carriers and non-carriers. There was no evidence that PSA30/PSA50 differed by HR gene mutational status. Median pPFS and crPFS ranged 3–14 months across treatment modalities, but there was no evidence either differed by HR gene mutational status (all p>0.05). There was also no difference in outcomes between those with BRCA2 or PALB2 mutations (n = 17) compared to those without HR repair mutations.ConclusionHR gene mutational status was associated with comparable clinical outcomes following treatment with ARSIs or taxane-based chemotherapy. Additional prospective studies are needed to confirm these findings.

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).

Role of androgen receptor splice variant-7 (AR-V7) in prostate cancer resistance to 2nd-generation androgen receptor signaling inhibitors.

The role of truncated androgen receptor splice variant-7 (AR-V7) in prostate cancer biology is an unresolved question. Is it simply a marker of resistance to 2nd generation androgen receptor signaling inhibitors (ARSi) like Abiraterone Acetate (Abi) and Enzalutamide (Enza) or a functional driver of lethal resistance via its ligand-independent transcriptional activity? To resolve this question, the correlation between resistance to ARSi and genetic chances and expression of full length AR (AR-FL) vs. AR-V7 were evaluated in a series of independent patient-derived xenografts (PDXs). While all PDXs lack PTEN expression, there is no consistent requirement for mutation in TP53, RB1, BRCA2, PIK3CA, or MSH2, or expression of SOX2 or ERG and ARSi-resistance. Elevated expression of AR-FL alone is sufficient for Abi- but not Enza-resistance, even if AR-FL is gain-of-function (GOF) mutated. Enza-resistance is consistently correlated with enhanced AR-V7 expression. In vitro and in vivo growth responses of Abi-/Enza-resistant LNCaP-95 cells in which CRISPR-Cas9 was used to knockout AR-FL or AR-V7 alone or in combination were evaluated. Combining these growth responses with RNAseq analysis demonstrates that both AR-FL and AR-V7 dependent transcriptional complementation are needed for Abi/Enza resistance.

Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

Negative cross talk between LIMK2 and PTEN promotes castration resistant prostate cancer pathogenesis in cells and in vivo.

Androgen deprivation therapy (ADT) and androgen receptor (AR) signaling inhibitors are front-line treatments for highly aggressive prostate cancer. However, prolonged inhibition of AR triggers a compensatory activation of PI3K pathway, most often due to the genomic loss of tumor suppressor PTEN, driving progression to the castration-resistant prostate cancer (CRPC) stage, which has very poor prognosis. We uncovered a novel mechanism of PTEN downregulation triggered by LIMK2, which contributes significantly to CRPC pathogenesis. LIMK2 is a CRPC-specific target. Its depletion fully reverses tumorigenesis in vivo. LIMK2 phosphorylates PTEN at five sites, degrading and inhibiting its activity, thereby driving highly aggressive oncogenic phenotypes in cells and in vivo. PTEN also degrades LIMK2 in a feedback loop, which was confirmed in prostates from PTEN-/- and PTEN+/+ mice. LIMK2 is also the missing link between hypoxia and PTEN degradation in CRPC. This is the first study to show a feedback loop between PTEN and its regulator. Uncovering the LIMK2-PTEN loop provides a powerful therapeutic opportunity to retain the activity and stability of PTEN protein by inhibiting LIMK2, thereby halting the progression to CRPC, ADT-resistance and drug-resistance.

Androgen receptor signalling in macrophages promotes TREM-1-mediated prostate cancer cell line migration and invasion

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.

BRCA Mutations in Prostate Cancer: Prognostic and Predictive Implications.

Despite chemotherapy and novel androgen-receptor signalling inhibitors (ARSi) have been approved during the last decades, metastatic castration-resistant prostate cancer (mCRPC) remains a lethal disease with poor clinical outcomes. Several studies found that germline or acquired DNA damage repair (DDR) defects affect a high percentage of mCRPC patients. Among DDR defects, BRCA mutations show relevant clinical implications. BRCA mutations are associated with adverse clinical features in primary tumors and with poor outcomes in patients with mCRPC. In addition, BRCA mutations predict good response to poly-ADP ribose polymerase (PARP) inhibitors, such as olaparib, rucaparib, and niraparib. However, concerns still remain on the role of extensive mutational testing in prostate cancer patients, given the implications for patients and for their progeny. The present comprehensive review attempts to provide an overview of BRCA mutations in prostate cancer, focusing on their prognostic and predictive roles.

1971P Neuroendocrine (NE) expression profiling in non-castrate tumours is associated with poor therapy benefit and adverse clinical outcome in metastatic castration-resistant prostate cancer (mCRPC) patients

NE dedifferentiation is associated to clinical aggressiveness and resistance to androgen receptor (AR) inhibition in prostate cancer. We analyzed the correlation of a NE expression signature in non-castrate tumors with the clinical outcome of mCRPC patients treated with taxanes or AR signaling inhibitors (ARSI; abiraterone or enzalutamide). Patients with mCRPC were retrospectively tested for a customized signature of 45 NE-related genes in total RNA from formalin-fixed paraffin-embedded non-castrate tumor samples by the nCounter platform (Nanostring Technologies). Patients were grouped as high and low NE profile according to the unsupervised clustering of gene expression data and correlated with treatment response and clinical outcome. Ninety-six patients were included, with a median follow-up of 20 (1.17-81.3) months. Median age was 69.2 (55.8-87.1) years. All patients received taxanes (68 docetaxel, 10 cabazitaxel and 18 both), 29 received ARSI as first line in CRPC and 53 received ARSI after taxanes. Patients classified as high NE expression profile (59 vs. 37 patients) were associated with a shorter time of CRPC development (median 12.6 vs. 30.8 mo, HR 3.3, 95%CI 2-5.5, P<0.001) and shorter OS from CRPC diagnosis (median 24.3 vs. 42.2 mo, HR 2.2, 95%CI 1.4-3.4, P=0.001). Regarding taxanes outcome, high NE profile patients correlated with lower PSA-PFS (HR 1.7, 95%CI 1.1-2.6, P=0.011), radiologic (RX)-PFS (HR 1.8, 95%CI 1.1-2.8, P=0.012) and overall survival (OS) (HR 1.7, 95%CI 1.1-2.6, P=0.009), and they were independently associated with shorter PSA-PFS (HR 1.8, 95%CI 1.1-3, P=0.023). In first-line ARSI-treated patients, high NE profile patients (15 vs. 14 patients) correlated with shorter PSA-PFS (HR 3, 95%CI 1.3-7, P=0.011), RX-PFS (HR 2.5, 95%CI 1-5.9, P=0.042) and OS (HR 5.8, 95%CI 2.3-14.9, P<0.001). NE-related gene expression in non-castrate tumor samples is associated with adverse clinical outcome and both poor taxanes and ARSI benefit in mCRPC patients. Thus, molecular characterization of primary tumors may be useful to guide treatment strategies in mCRPC.