Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC..

BACKGROUNDClinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and preclinically sensitizes to PARP inhibition. The current study reports on RNASEH2B protein loss in advanced prostate cancer and its association with RB1 protein loss, clinical outcome, and clonal dynamics during treatment with PARP inhibition in a prospective clinical trial.METHODSWhole tumor biopsies from multiple cohorts of patients with advanced prostate cancer were interrogated using whole-exome sequencing (WES), RNA-Seq (bulk and single nucleus), and IHC for RNASEH2B and RB1. Biopsies from patients treated with olaparib in the TOPARP-A and TOPARP-B clinical trials were used to evaluate RNASEH2B clonal selection during olaparib treatment.RESULTSShallow codeletion of RNASEH2B and adjacent RB1 - colocated at chromosome 13q14 - was common, deep codeletion infrequent, and gene loss associated with lower mRNA expression. In castration-resistant prostate cancer (CRPC) biopsies, RNASEH2B and RB1 mRNA expression correlated, but single nucleus RNA-Seq indicated discordant loss of expression. IHC studies showed that loss of the 2 proteins often occurred independently, arguably due to stochastic second allele loss. Pre- and posttreatment metastatic CRPC (mCRPC) biopsy studies from BRCA1/2 WT tumors, treated on the TOPARP phase II trial, indicated that olaparib eradicated RNASEH2B-loss tumor subclones.CONCLUSIONPARP inhibition may benefit men suffering from mCRPC by eradicating tumor subclones with RNASEH2B loss.TRIAL REGISTRATIONClinicaltrials.gov NCT01682772.FUNDINGAstraZeneca; Cancer Research UK; Medical Research Council; Cancer Research UK; Prostate Cancer UK; Movember Foundation; Prostate Cancer Foundation..

BACKGROUND: Though cancer is more prevalent in the older population, this patient group are underrepresented in phase I oncology trials. AIMS: We evaluated the use of a geriatric screening tool (SAOP3) in patients of 70 years of age or older who attended a Phase I Clinical Trials Unit, with the aim of assessing the feasibility of the tool and identifying potential unmet needs in this patient group. METHODS: Twenty-two patients over the age of 70 completed the SAOP3 questionnaire. Geriatric impairments and needs were analysed with descriptive statistics. Qualitative responses were grouped in themes using structured thematic analysis. RESULTS: All of patients triggered at least 1 geriatric domain, most commonly mobility. Six core themes were identified as being important to the patient including family, friends and positivity. On cognition assessment over 20% of patients triggered as requiring further cognitive assessment. The group had a relatively high screen fail risk. CONCLUSION: In conclusion, routine geriatric screening withSAOP3 was feasible and identified areas of patient need. Results highlight the prevalence of psychological distress and cognitive impairment. Geriatric screening offers an opportunity for prehabilitation prior to trial and support during trial participation to optimise safety and improve trial access..

Castration resistant prostate cancer (CRPC) remains an incurable disease stage with ineffective treatments options. Here, the androgen receptor (AR) coactivators CBP/p300, which are histone acetyltransferases, were identified as critical mediators of DNA damage repair (DDR) to potentially enhance therapeutic targeting of CRPC. Key findings demonstrate that CBP/p300 expression increases with disease progression and selects for poor prognosis in metastatic disease. CBP/p300 bromodomain inhibition enhances response to standard of care therapeutics. Functional studies, CBP/p300 cistrome mapping, and transcriptome in CRPC revealed that CBP/p300 regulates DDR. Further mechanistic investigation showed that CBP/p300 attenuation via therapeutic targeting and genomic knockdown decreases homologous recombination (HR) factors in vitro, in vivo, and in human prostate cancer (PCa) tumors ex vivo. Similarly, CBP/p300 expression in human prostate tissue correlates with HR factors. Lastly, targeting CBP/p300 impacts HR-mediate repair and patient outcome. Collectively, these studies identify CBP/p300 as drivers of PCa tumorigenesis and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed and DDR therapies..

The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset. There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/or biomarker with clinical utility. Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs. Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity. We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1. Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC models, highlighting the need for combination strategies to enhance tumor cell apoptosis and enrich response..

(1) Background: We assessed the test-re-test repeatability of radiomics in metastatic castration-resistant prostate cancer (mCPRC) bone disease on whole-body diffusion-weighted (DWI) and T1-weighted Dixon MRI. (2) Methods: In 10 mCRPC patients, 1.5 T MRI, including DWI and T1-weighted gradient-echo Dixon sequences, was performed twice on the same day. Apparent diffusion coefficient (ADC) and relative fat-fraction-percentage (rFF%) maps were calculated. Per study, up to 10 target bone metastases were manually delineated on DWI and Dixon images. All 106 radiomic features included in the Pyradiomics toolbox were derived for each target volume from the ADC and rFF% maps. To account for inter- and intra-patient measurement repeatability, the log-transformed individual target measurements were fitted to a hierarchical model, represented as a Bayesian network. Repeatability measurements, including the intraclass correlation coefficient (ICC), were derived. Feature ICCs were compared with mean ADC and rFF ICCs. (3) Results: A total of 65 DWI and 47 rFF% targets were analysed. There was no significant bias for any features. Pairwise correlation revealed fifteen ADC and fourteen rFF% feature sub-groups, without specific patterns between feature classes. The median intra-patient ICC was generally higher than the inter-patient ICC. Features that describe extremes in voxel values (minimum, maximum, range, skewness, and kurtosis) showed generally lower ICCs. Several mostly shape-based texture features were identified, which showed high inter- and intra-patient ICCs when compared with the mean ADC or mean rFF%, respectively. (4) Conclusions: Pyradiomics texture features of mCRPC bone metastases varied greatly in inter- and intra-patient repeatability. Several features demonstrated good repeatability, allowing for further exploration as diagnostic parameters in mCRPC bone disease..

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression..

UNLABELLED: The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor-positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor-positive (ER+) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein-protein interactions such as BAG1-mortalin (GRP75) interaction as well as wild-type p53-mortalin or mutant p53-BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER+ breast cancers. SIGNIFICANCE: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER+ breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis..

Cancer is highly infiltrated by myeloid-derived suppressor cells (MDSCs). Currently available immunotherapies do not completely eradicate MDSCs. Through a genome-wide analysis of the translatome of prostate cancers driven by different genetic alterations, we demonstrate that prostate cancer rewires its secretome at the translational level to recruit MDSCs. Among different secreted proteins released by prostate tumor cells, we identified Hgf, Spp1 and Bgn as the key factors that regulate MDSC migration. Mechanistically, we found that the coordinated loss of Pdcd4 and activation of the MNK/eIF4E pathways regulate the mRNAs translation of Hgf, Spp1 and Bgn. MDSC infiltration and tumor growth were dampened in prostate cancer treated with the MNK1/2 inhibitor eFT508 and/or the AKT inhibitor ipatasertib, either alone or in combination with a clinically available MDSC-targeting immunotherapy. This work provides a therapeutic strategy that combines translation inhibition with available immunotherapies to restore immune surveillance in prostate cancer..

BCL-2-associated athanogene-1L (BAG-1L) is a critical co-regulator that binds to and enhances the transactivation function of the androgen receptor, leading to prostate cancer development and progression. Studies investigating the clinical importance of BAG-1L protein expression in advanced prostate cancer have been limited by the paucity of antibodies that specifically recognize the long isoform. In this study, we developed and validated a new BAG-1L-specific antibody using multiple orthogonal methods across several cell lines with and without genomic manipulation of BAG-1L and all BAG-1 isoforms. Following this, we performed exploratory immunohistochemistry to determine BAG-1L protein expression in normal human, matched castration-sensitive prostate cancer (CSPC) and castration-resistant prostate cancer (CRPC), unmatched primary and metastatic CRPC, and early breast cancer tissues. We demonstrated higher BAG-1L protein expression in CRPC metastases than in unmatched, untreated, castration-sensitive prostatectomies from men who remained recurrence-free for 5 years. In contrast, BAG-1L protein expression did not change between matched, same patient, CSPC and CRPC biopsies, suggesting that BAG-1L protein expression may be associated with more aggressive biology and the development of castration resistance. Finally, in a cohort of patients who universally developed CRPC, there was no association between BAG-1L protein expression at diagnosis and time to CRPC or overall survival, and no association between BAG-1L protein expression at CRPC biopsy and clinical outcome from androgen receptor targeting therapies or docetaxel chemotherapy. The limitations of this study include the requirement to validate the reproducibility of the assay developed, the potential influence of pre-analytical factors, timing of CRPC biopsies, relatively small patient numbers, and heterogenous therapies on BAG-1L protein expression, and the clinical outcome analyses performed. We describe a new BAG-1L-specific antibody that the research community can further develop to elucidate the biological and clinical significance of BAG-1L protein expression in malignant and nonmalignant diseases..

Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC..

Owing to the development of multiple novel therapies, there has been major progress in the treatment of advanced prostate cancer over the last two decades; however, the disease remains invariably fatal. Androgens and the androgen receptor (AR) play a critical role in prostate carcinogenesis, and targeting the AR signaling axis with abiraterone, enzalutamide, darolutamide, and apalutamide has improved outcomes for men with this lethal disease. Targeting the AR and elucidating mechanisms of resistance to these agents remain central to drug development efforts. This review provides an overview of the evolution and current approaches for targeting the AR in advanced prostate cancer. It describes the biology of AR signaling, explores AR-targeting resistance mechanisms, and discusses future perspectives and promising novel therapeutic strategies..

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease in which molecular stratification is needed to improve clinical outcomes. The identification of predictive biomarkers can have a major impact on the care of these patients, but the availability of metastatic tissue samples for research in this setting is limited. OBJECTIVE: To study the prevalence of immune biomarkers of potential clinical utility to immunotherapy in mCRPC and to determine their association with overall survival (OS). DESIGN, SETTING, AND PARTICIPANTS: From 100 patients, mCRPC biopsies were assayed by whole exome sequencing, targeted next-generation sequencing, RNA sequencing, tumor mutational burden, T-cell-inflamed gene expression profile (TcellinfGEP) score (Nanostring), and immunohistochemistry for programmed cell death 1 ligand 1 (PD-L1), ataxia-telangiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), SRY homology box 2 (SOX2), and the presence of neuroendocrine features. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The phi coefficient determined correlations between biomarkers of interest. OS was assessed using Kaplan-Meier curves and adjusted hazard ratios (aHRs) from Cox regression. RESULTS AND LIMITATIONS: PD-L1 and SOX2 protein expression was detected by immunohistochemistry (combined positive score ≥1 and >5% cells, respectively) in 24 (33%) and 27 (27%) mCRPC biopsies, respectively; 23 (26%) mCRPC biopsies had high TcellinfGEP scores (>-0.318). PD-L1 protein expression and TcellinfGEP scores were positively correlated (phi 0.63 [0.45; 0.76]). PD-L1 protein expression (aHR: 1.90 [1.05; 3.45]), high TcellinfGEP score (aHR: 1.86 [1.04; 3.31]), and SOX2 expression (aHR: 2.09 [1.20; 3.64]) were associated with worse OS. CONCLUSIONS: PD-L1, TcellinfGEP score, and SOX2 are prognostic of outcome from the mCRPC setting. If validated, predictive biomarker studies incorporating survival endpoints need to take these findings into consideration. PATIENT SUMMARY: This study presents an analysis of immune biomarkers in biopsies from patients with metastatic prostate cancer. We describe tumor alterations that predict prognosis that can impact future studies..

PURPOSE: Prostate-specific membrane antigen (PSMA) targeting therapies such as Lutetium-177 (177Lu)-PSMA-617 are affecting outcomes from metastatic castration-resistant prostate cancer (mCRPC). However, a significant subset of patients have prostate cancer cells lacking PSMA expression, raising concerns about treatment resistance attributable at least in part to heterogeneous PSMA expression. We have previously demonstrated an association between high PSMA expression and DNA damage repair defects in mCRPC biopsies and therefore hypothesized that DNA damage upregulates PSMA expression. EXPERIMENTAL DESIGN: To test this relationship between PSMA and DNA damage we conducted a screen of 147 anticancer agents (NCI/NIH FDA-approved anticancer "Oncology Set") and treated tumor cells with repeated ionizing irradiation. RESULTS: The topoisomerase-2 inhibitors, daunorubicin and mitoxantrone, were identified from the screen to upregulate PSMA protein expression in castration-resistant LNCaP95 cells; this result was validated in vitro in LNCaP, LNCaP95, and 22Rv1 cell lines and in vivo using an mCRPC patient-derived xenograft model CP286 identified to have heterogeneous PSMA expression. As double-strand DNA break induction by topoisomerase-2 inhibitors upregulated PSMA, we next studied the impact of ionizing radiation on PSMA expression; this also upregulated PSMA protein expression in a dose-dependent fashion. CONCLUSIONS: The results presented herein are the first, to our knowledge, to demonstrate that PSMA is upregulated in response to double-strand DNA damage by anticancer treatment. These data support the study of rational combinations that maximize the antitumor activity of PSMA-targeted therapeutic strategies by upregulating PSMA..

PURPOSE: Therapies targeting the androgen receptor (AR) have improved the outcome for patients with castration-sensitive prostate cancer (CSPC). Expression of the constitutively active AR splice variant-7 (AR-V7) has shown clinical utility as a predictive biomarker of AR-targeted therapy resistance in castration-resistant prostate cancer (CRPC), but its importance in CSPC remains understudied. EXPERIMENTAL DESIGN: We assessed different approaches to quantify AR-V7 mRNA and protein in prostate cancer cell lines, patient-derived xenograft (PDX) models, publicly available cohorts, and independent institutional clinical cohorts, to identify reliable approaches for detecting AR-V7 mRNA and protein and its association with clinical outcome. RESULTS: In CSPC and CRPC cohorts, AR-V7 mRNA was much less abundant when detected using reads across splice boundaries than when considering isoform-specific exonic reads. The RM7 AR-V7 antibody had increased sensitivity and specificity for AR-V7 protein detection by immunohistochemistry (IHC) in CRPC cohorts but rarely identified AR-V7 protein reactivity in CSPC cohorts, when compared with the EPR15656 AR-V7 antibody. Using multiple CRPC PDX models, we demonstrated that AR-V7 expression was exquisitely sensitive to hormonal manipulation. In CSPC institutional cohorts, AR-V7 protein quantification by either assay was associated neither with time to development of castration resistance nor with overall survival, and intense neoadjuvant androgen-deprivation therapy did not lead to significant AR-V7 mRNA or staining following treatment. Neither pre- nor posttreatment AR-V7 levels were associated with volumes of residual disease after therapy. CONCLUSIONS: This study demonstrates that further analytical validation and clinical qualification are required before AR-V7 can be considered for clinical use in CSPC as a predictive biomarker..

BACKGROUND: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. OBJECTIVE: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. DESIGN, SETTING, AND PARTICIPANTS: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. RESULTS AND LIMITATIONS: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. CONCLUSIONS: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. PATIENT SUMMARY: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR..

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995..

BACKGROUND: CD38, a druggable ectoenzyme, is involved in the generation of adenosine, which is implicated in tumour immune evasion. Its expression and role in prostate tumour-infiltrating immune cells (TIICs) have not been elucidated. OBJECTIVE: To characterise CD38 expression on prostate cancer (PC) epithelial cells and TIICs, and to associate this expression with clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: RNAseq from 159 patients with metastatic castration-resistant prostate cancer (mCRPC) in the International Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF) cohort and 171 mCRPC samples taken from 63 patients in the Fred Hutchinson Cancer Research Centre cohort were analysed. CD38 expression was immunohistochemically scored by a validated assay on 51 castration-resistant PC (CRPC) and matching, same-patient castration-sensitive PC (CSPC) biopsies obtained between 2016 and 2018, and was associated with retrospectively collected clinical data. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: mCRPC transcriptomes were analysed for associations between CD38 expression and gene expression signatures. Multiplex immunofluorescence determined CD38 expression in PC biopsies. Differences in CD38+ TIIC densities between CSPC and CRPC biopsies were analysed using a negative binomial mixed model. Differences in the proportions of CD38+ epithelial cells between non-matched benign prostatic epithelium and PC were compared using Fisher's exact test. Differences in the proportions of biopsies containing CD38+ tumour epithelial cells between matched CSPC and CRPC biopsies were compared by McNemar's test. Univariable and multivariable survival analyses were performed using Cox regression models. RESULTS AND LIMITATIONS: CD38 mRNA expression in mCRPC was most significantly associated with upregulated immune signalling pathways. CD38 mRNA expression was associated with interleukin (IL)-12, IL-23, and IL-27 signalling signatures as well as immunosuppressive adenosine signalling and T cell exhaustion signatures. CD38 protein was frequently expressed on phenotypically diverse TIICs including B cells and myeloid cells, but largely absent from tumour epithelial cells. CD38+ TIIC density increased with progression to CRPC and was independently associated with worse overall survival. Future studies are required to dissect TIIC CD38 function. CONCLUSIONS: CD38+ prostate TIICs associate with worse survival and immunosuppressive mechanisms. The role of CD38 in PC progression warrants investigation as insights into its functions may provide rationale for CD38 targeting in lethal PC. PATIENT SUMMARY: CD38 is expressed on the surface of white blood cells surrounding PC cells. These cells may impact PC growth and treatment resistance. Patients with PC with more CD38-expressing white blood cells are more likely to die earlier..

Targeting the androgen receptor by depriving testosterone with gonadotropin-releasing hormone agonists or antagonists, or surgical castration, has been the backbone of metastatic prostate cancer treatment. Although most prostate cancers initially respond to androgen deprivation, metastatic castration-resistant prostate cancer evolves into a heterogeneous disease with diverse drivers of progression and mechanisms of therapeutic resistance. Development of castrate resistance phenotype is associated with lethality despite the recent noteworthy strides gained via increase in therapeutic options. Identification of novel therapeutics to further improve survival and achieve durable responses in metastatic castration-resistant prostate cancer is a clinical necessity. In this review, we outline the existing avengers for treatment of metastatic castration-resistant prostate cancer by clinical presentation, placing into context the clinical state of the patient, such as burden of disease and symptoms. Doing so might aid in the ability to optimize the sequence of agents and allow for maximal exposure to life-prolonging therapeutics. Realizing the limitations of the androgen signaling inhibition, we explore the androgen-indifferent prostate cancer: the mutants. Classically, these subtypes have been associated with variant histology, but androgen-indifferent prostate cancer features are now frequently observed in association with heterogeneous morphologies, including double-negative prostate cancers, lacking both androgen receptor and neuroendocrine features, or clinicopathologic criteria, such as the aggressive variant prostate cancer criteria. The framework of new avengers against metastatic castration-resistant prostate cancer based on mechanism, including DNA repair, immune checkpoint inhibition, PTEN/PI3K/AKT pathway, prostate-specific membrane antigen targets, bispecific T-cell engagers, and radionuclide therapies, is summarized in this review..

BACKGROUND: Better blood tests to elucidate the behaviour of metastatic castration-resistant prostate cancer (mCRPC) are urgently needed to drive therapeutic decisions. Plasma cell-free DNA (cfDNA) comprises normal and circulating tumour DNA (ctDNA). Low-pass whole-genome sequencing (lpWGS) of ctDNA can provide information on mCRPC behaviour. OBJECTIVE: To validate and clinically qualify plasma lpWGS for mCRPC. DESIGN, SETTING, AND PARTICIPANTS: Plasma lpWGS data were obtained for mCRPC patients consenting to optional substudies of two prospective phase 3 trials (FIRSTANA and PROSELICA). In FIRSTANA, chemotherapy-naïve patients were randomised to treatment with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2). In PROSELICA, patients previously treated with docetaxel were randomised to 20 or 25 mg/m2 cabazitaxel. lpWGS data were generated from 540 samples from 188 mCRPC patients acquired at four different time points (screening, cycle 1, cycle 4, and end of study). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: lpWGS data for ctDNA were evaluated for prognostic, response, and tumour genomic measures. Associations with response and survival data were determined for tumour fraction. Genomic biomarkers including large-scale transition (LST) scores were explored in the context of prior treatments. RESULTS AND LIMITATIONS: Plasma tumour fraction was prognostic for overall survival in univariable and stratified multivariable analyses (hazard ratio 1.75, 95% confidence interval 1.08-2.85; p = 0.024) and offered added value compared to existing biomarkers (C index 0.722 vs 0.709; p = 0.021). Longitudinal changes were associated with drug response. PROSELICA samples were enriched for LSTs (p = 0.029) indicating genomic instability, and this enrichment was associated with prior abiraterone and enzalutamide treatment but not taxane or radiation therapy. Higher LSTs were correlated with losses of RB1/RNASEH2B, independent of BRCA2 loss. CONCLUSIONS: Plasma lpWGS of ctDNA describes CRPC behaviour, providing prognostic and response data of clinical relevance. The added prognostic value of the ctDNA fraction over established biomarkers should be studied further. PATIENT SUMMARY: We studied tumour DNA in blood samples from patients with prostate cancer. We found that levels of tumour DNA in blood were indicative of disease prognosis, and that changes after treatment could be detected. We also observed a "genetic scar" in the results that was associated with certain previous treatments. This test allows an assessment of tumour activity that can complement existing tests, offer insights into drug response, and detect clinically relevant genetic changes..

Circulating tumor cell (CTC) enumeration and changes following treatment have been demonstrated to be superior to PSA response in determining mCRPC outcome in patients receiving AR signaling inhibitors but not taxanes. We carried out a pooled analysis of two prospective studies in mCRPC patients treated with docetaxel. CTCs were measured at baseline and 3-6 weeks post treatment initiation. Cox regression models were constructed to compare 6-month radiographical progression-free survival (rPFS), CTCs and PSA changes predicting outcome. Among the subjects, 80 and 52 patients had evaluable baseline and post-treatment CTC counts, respectively. A significant association of higher baseline CTC count with worse overall survival (OS), PFS and time to PSA progression (TTPP) was observed. While CTC response at 3-6 weeks (CTC conversion (from ≥5 to <5 CTCs), CTC30 (≥30% decline in CTC) or CTC0 (decline to 0 CTC)) and 6-month rPFS were significantly associated with OS (all p < 0.005), the association was not significant for PSA30 or PSA50 response. CTC and PSA response were discordant in over 50% of cases, with outcome driven by CTC response in these patients. The c-index values for OS were superior for early CTC changes compared to PSA response endpoints, and similar to 6-month rPFS. Early CTC declines were good predictors of improved outcomes in mCRPC patients treated with docetaxel in this small study, offering a superior and/or earlier estimation of docetaxel benefit in comparison to PSA or rPFS that merits further confirmation in larger studies..

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials..

PURPOSE: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. EXPERIMENTAL DESIGN: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. RESULTS: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. CONCLUSIONS: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer..

Endocrine resistance (EnR) in advanced prostate cancer is fatal. EnR can be mediated by androgen receptor (AR) splice variants, with AR splice variant 7 (AR-V7) arguably the most clinically important variant. In this study, we determined proteins key to generating AR-V7, validated our findings using clinical samples, and studied splicing regulatory mechanisms in prostate cancer models. Triangulation studies identified JMJD6 as a key regulator of AR-V7, as evidenced by its upregulation with in vitro EnR, its downregulation alongside AR-V7 by bromodomain inhibition, and its identification as a top hit of a targeted siRNA screen of spliceosome-related genes. JMJD6 protein levels increased (P < 0.001) with castration resistance and were associated with higher AR-V7 levels and shorter survival (P = 0.048). JMJD6 knockdown reduced prostate cancer cell growth, AR-V7 levels, and recruitment of U2AF65 to AR pre-mRNA. Mutagenesis studies suggested that JMJD6 activity is key to the generation of AR-V7, with the catalytic machinery residing within a druggable pocket. Taken together, these data highlight the relationship between JMJD6 and AR-V7 in advanced prostate cancer and support further evaluation of JMJD6 as a therapeutic target in this disease. SIGNIFICANCE: This study identifies JMJD6 as being critical for the generation of AR-V7 in prostate cancer, where it may serve as a tractable target for therapeutic intervention..

PURPOSE: Cyclin-dependent kinase 12 (CDK12) aberrations have been reported as a biomarker of response to immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). Herein, we characterize CDK12-mutated mCRPC, presenting clinical, genomic, and tumor-infiltrating lymphocyte (TIL) data. EXPERIMENTAL DESIGN: Patients with mCRPC consented to the molecular analyses of diagnostic and mCRPC biopsies. Genomic analyses involved targeted next-generation (MiSeq; Illumina) and exome sequencing (NovaSeq; Illumina). TILs were assessed by validated immunocytochemistry coupled with deep learning-based artificial intelligence analyses including multiplex immunofluorescence assays for CD4, CD8, and FOXP3 evaluating TIL subsets. The control group comprised a randomly selected mCRPC cohort with sequencing and clinical data available. RESULTS: Biopsies from 913 patients underwent targeted sequencing between February 2015 and October 2019. Forty-three patients (4.7%) had tumors with CDK12 alterations. CDK12-altered cancers had distinctive features, with some revealing high chromosomal break numbers in exome sequencing. Biallelic CDK12-aberrant mCRPCs had shorter overall survival from diagnosis than controls [5.1 years (95% confidence interval (CI), 4.0-7.9) vs. 6.4 years (95% CI, 5.7-7.8); hazard ratio (HR), 1.65 (95% CI, 1.07-2.53); P = 0.02]. Median intratumoral CD3+ cell density was higher in CDK12 cancers, although this was not statistically significant (203.7 vs. 86.7 cells/mm2; P = 0.07). This infiltrate primarily comprised of CD4+FOXP3- cells (50.5 vs. 6.2 cells/mm2; P < 0.0001), where high counts tended to be associated with worse survival from diagnosis (HR, 1.64; 95% CI, 0.95-2.84; P = 0.077) in the overall population. CONCLUSIONS: CDK12-altered mCRPCs have worse prognosis, with these tumors surprisingly being primarily enriched for CD4+FOXP3- cells that seem to associate with worse outcome and may be immunosuppressive.See related commentary by Lotan and Antonarakis, p. 380..

Despite major improvements in the management of advanced prostate cancer over the last 20 years, the disease remains invariably fatal, and new effective therapies are required. The development of novel hormonal agents and taxane chemotherapy has improved outcomes, although primary and acquired resistance remains problematic. Inducing cancer cell death via apoptosis has long been an attractive goal in the treatment of cancer. Apoptosis, a form of regulated cell death, is a highly controlled process, split into two main pathways (intrinsic and extrinsic), and is stimulated by a multitude of factors, including cellular and genotoxic stress. Numerous therapeutic strategies targeting the intrinsic apoptosis pathway are in clinical development, and BH3 mimetics have shown promising efficacy for hematological malignancies. Utilizing these agents for solid malignancies has proved more challenging, though efforts are ongoing. Molecular characterization and the development of predictive biomarkers is likely to be critical for patient selection, by identifying tumors with a vulnerability in the intrinsic apoptosis pathway. This review provides an up-to-date overview of cell death and apoptosis, specifically focusing on the intrinsic pathway. It summarizes the latest approaches for targeting the intrinsic apoptosis pathway with BH3 mimetics and discusses how these strategies may be leveraged to treat prostate cancer..

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The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti-CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors..

INTRODUCTION: The 2019 Coffey-Holden Prostate Cancer Academy (CHPCA) Meeting, "Prostate Cancer Research: The Next Generation," was held 20 to 23 June, 2019, in Los Angeles, California. METHODS: The CHPCA Meeting is an annual conference held by the Prostate Cancer Foundation, that is uniquely structured to stimulate intense discussion surrounding topics most critical to accelerating prostate cancer research and the discovery of new life-extending treatments for patients. The 7th Annual CHPCA Meeting was attended by 86 investigators and concentrated on many of the most promising new treatment opportunities and next-generation research technologies. RESULTS: The topics of focus at the meeting included: new treatment strategies and novel agents for targeted therapies and precision medicine, new treatment strategies that may synergize with checkpoint immunotherapy, next-generation technologies that visualize tumor microenvironment (TME) and molecular pathology in situ, multi-omics and tumor heterogeneity using single cells, 3D and TME models, and the role of extracellular vesicles in cancer and their potential as biomarkers. DISCUSSION: This meeting report provides a comprehensive summary of the talks and discussions held at the 2019 CHPCA Meeting, for the purpose of globally disseminating this knowledge and ultimately accelerating new treatments and diagnostics for patients with prostate cancer..

Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA polymerase II-associated (RNA Pol II-associated) chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins - CTCF and cohesins - and are regulated by the cooperative action of master transcription factors, such as the androgen receptor (AR) and FOXA1. By combining analyses from metastatic castration-resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional upregulation of the AR gene by increasing the total number of chromatin interaction modules comprising the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes - VPS53, FAM57A, and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa..

BACKGROUND: Activation of the PI3K/AKT/mTOR pathway through loss of phosphatase and tensin homolog (PTEN) occurs in approximately 50% of patients with metastatic castration-resistant prostate cancer (mCRPC). Recent evidence suggests that combined inhibition of the androgen receptor (AR) and AKT may be beneficial in mCRPC with PTEN loss. PATIENTS AND METHODS: mCRPC patients who previously failed abiraterone and/or enzalutamide, received escalating doses of AZD5363 (capivasertib) starting at 320 mg twice daily (b.i.d.) given 4 days on and 3 days off, in combination with enzalutamide 160 mg daily. The co-primary endpoints were safety/tolerability and determining the maximum tolerated dose and recommended phase II dose; pharmacokinetics, antitumour activity, and exploratory biomarker analysis were also evaluated. RESULTS: Sixteen patients were enrolled, 15 received study treatment and 13 were assessable for dose-limiting toxicities (DLTs). Patients were treated at 320, 400, and 480 mg b.i.d. dose levels of capivasertib. The recommended phase II dose identified for capivasertib was 400 mg b.i.d. with 1/6 patients experiencing a DLT (maculopapular rash) at this level. The most common grade ≥3 adverse events were hyperglycemia (26.7%) and rash (20%). Concomitant administration of enzalutamide significantly decreased plasma exposure of capivasertib, though this did not appear to impact pharmacodynamics. Three patients met the criteria for response (defined as prostate-specific antigen decline ≥50%, circulating tumour cell conversion, and/or radiological response). Responses were seen in patients with PTEN loss or activating mutations in AKT, low or absent AR-V7 expression, as well as those with an increase in phosphorylated extracellular signal-regulated kinase (pERK) in post-exposure samples. CONCLUSIONS: The combination of capivasertib and enzalutamide is tolerable and has antitumour activity, with all responding patients harbouring aberrations in the PI3K/AKT/mTOR pathway. CLINICAL TRIAL NUMBER: NCT02525068..

BACKGROUND: In men with castration-sensitive prostate cancer (CSPC), the HSD3B1 c.1245A>C variant has been reported to be associated with shorter responses to first-line androgen-deprivation therapy (ADT). Here, we evaluated the association between the inherited HSD3B1 c.1245A>C variant and outcomes from metastatic castration-resistant prostate cancer (mCRPC) after first-line treatment with abiraterone (Abi) or enzalutamide (Enza). PATIENTS AND METHODS: Patients with mCRPC (n = 266) were enrolled from two centers at the time of starting first-line Abi/Enza. Outcomes after Abi/Enza included best prostate-specific antigen (PSA) response, treatment duration, and overall survival (OS). Outcomes after first-line ADT were determined retrospectively, and included treatment duration and OS. As was prespecified, we compared patients with the homozygous variant HSD3B1 genotype (CC genotype) versus the combined group with the heterozygous (AC) and homozygous wild-type (AA) genotypes. RESULTS: Among the 266 patients, 22 (8.3%) were homozygous for the HSD3B1 variant (CC). The CC genotype had no association with PSA response rate; the median Abi/Enza treatment duration was 7.1 months for the CC group and 10.3 months for the AA/AC group (log rank P = 0.34). Patients with the CC genotype had significantly worse OS, with median survival at 23.6 months for the CC group and 30.7 months for the AA/AC group (log rank P = 0.02). In multivariable analysis adjusting for age, Gleason score, PSA, prior chemotherapy, and M1 disease, the association between the CC genotype and OS remained significant (hazard ratio 1.78, 95% confidence interval 1.03-3.07, P = 0.04). Poor outcome after first-line ADT in the CC group was also observed when evaluating retrospective ADT duration data for the same combined cohort. CONCLUSIONS: In this large two-center study evaluating the HSD3B1 c.1245 genotype and outcomes after first-line Abi/Enza, homozygous variant (CC) HSD3B1 genotype was associated with worse outcomes. Novel therapeutic strategies are needed to enable treatment selection based on this genetic marker..

Platinum-based regimens have not been proved to increase survival from advanced prostate cancer (PCa). Incontrovertible evidence that a proportion of prostate cancers have homologous recombination DNA (HRD) repair defects, and that such genomic aberrations are synthetically lethal with both poly(ADP)-ribose polymerase inhibition and platinum, has increased interest in the utilisation of these drugs against a subset of these diseases. Here in we report three patients with advanced castration-resistant PCa with HRD defects having exceptional responses to carboplatin..

BACKGROUND: Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival from endocrine therapies in castration-resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue. METHODS: Following generation and validation of a potentially novel AR-V7 antibody for IHC, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full-length androgen receptor (AR-FL) expression, response to therapy, and gene expression were determined. RESULTS: We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed, suggesting that mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient, although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical coregulator of AR-V7 function. Finally, AR-V7-negative disease associated with better prostate-specific antigen (PSA) responses (100% vs. 54%, P = 0.03) and overall survival (74.3 vs. 25.2 months, hazard ratio 0.23 [0.07-0.79], P = 0.02) from endocrine therapies (pre-chemotherapy). CONCLUSION: This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology and to confirm the clinical significance of AR-V7. FUNDING: Work at the University of Washington and in the Plymate and Nelson laboratories is supported by the Department of Defense Prostate Cancer Research Program (W81XWH-14-2-0183, W81XWH-12-PCRP-TIA, W81XWH-15-1-0430, and W81XWH-13-2-0070), the Pacific Northwest Prostate Cancer SPORE (P50CA97186), the Institute for Prostate Cancer Research, the Veterans Affairs Research Program, the NIH/National Cancer Institute (P01CA163227), and the Prostate Cancer Foundation. Work in the de Bono laboratory was supported by funding from the Movember Foundation/Prostate Cancer UK (CEO13-2-002), the US Department of Defense (W81XWH-13-2-0093), the Prostate Cancer Foundation (20131017 and 20131017-1), Stand Up To Cancer (SU2C-AACR-DT0712), Cancer Research UK (CRM108X-A25144), and the UK Department of Health through an Experimental Cancer Medicine Centre grant (ECMC-CRM064X)..

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced 'castrate-resistant' disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3'UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets..

BACKGROUND: Prostate-specific membrane antigen (PSMA; folate hydrolase) prostate cancer (PC) expression has theranostic utility. OBJECTIVE: To elucidate PC PSMA expression and associate this with defective DNA damage repair (DDR). DESIGN, SETTING, AND PARTICIPANTS: Membranous PSMA (mPSMA) expression was scored immunohistochemically from metastatic castration-resistant PC (mCRPC) and matching, same-patient, diagnostic biopsies, and correlated with next-generation sequencing (NGS) and clinical outcome data. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Expression of mPSMA was quantitated by modified H-score. Patient DNA was tested by NGS. Gene expression and activity scores were determined from mCRPC transcriptomes. Statistical correlations utilised Wilcoxon signed rank tests, survival was estimated by Kaplan-Meier test, and sample heterogeneity was quantified by Shannon's diversity index. RESULTS AND LIMITATIONS: Expression of mPSMA at diagnosis was associated with higher Gleason grade (p=0.04) and worse overall survival (p=0.006). Overall, mPSMA expression levels increased at mCRPC (median H-score [interquartile range]: castration-sensitive prostate cancer [CSPC] 17.5 [0.0-60.0] vs mCRPC 55.0 [2.8-117.5]). Surprisingly, 42% (n=16) of CSPC and 27% (n=16) of mCRPC tissues sampled had no detectable mPSMA (H-score <10). Marked intratumour heterogeneity of mPSMA expression, with foci containing no detectable PSMA, was observed in all mPSMA expressing CSPC (100%) and 37 (84%) mCRPC biopsies. Heterogeneous intrapatient mPSMA expression between metastases was also observed, with the lowest expression in liver metastases. Tumours with DDR had higher mPSMA expression (p=0.016; 87.5 [25.0-247.5] vs 20 [0.3-98.8]; difference in medians 60 [5.0-95.0]); validation cohort studies confirmed higher mPSMA expression in patients with deleterious aberrations in BRCA2 (p<0.001; median H-score: 300 [165-300]; difference in medians 195.0 [100.0-270.0]) and ATM (p=0.005; 212.5 [136.3-300]; difference in medians 140.0 [55.0-200]) than in molecularly unselected mCRPC biopsies (55.0 [2.75-117.5]). Validation studies using mCRPC transcriptomes corroborated these findings, also indicating that SOX2 high tumours have low PSMA expression. CONCLUSIONS: Membranous PSMA expression is upregulated in some but not all PCs, with mPSMA expression demonstrating marked inter- and intrapatient heterogeneity. DDR aberrations are associated with higher mPSMA expression and merit further evaluation as predictive biomarkers of response for PSMA-targeted therapies in larger, prospective cohorts. PATIENT SUMMARY: Through analysis of prostate cancer samples, we report that the presence of prostate-specific membrane antigen (PSMA) is extremely variable both within one patient and between different patients. This may limit the usefulness of PSMA scans and PSMA-targeted therapies. We show for the first time that prostate cancers with defective DNA repair produce more PSMA and so may respond better to PSMA-targeting treatments..

BACKGROUND: Detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. OBJECTIVE: To determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). DESIGN, SETTING, AND PARTICIPANTS: CTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: CTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. RESULTS AND LIMITATIONS: Of the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19-184 vs 9, 2-64; Mann-Whitney test p<0.001) and biopsy AR-V7 protein expression (median H-score, IQR: 100, 63-148 vs 15, 0-113; Mann-Whitney test p=0.004) than CTC+/AR-V7- samples. However, both CTC- (63%) and CTC+/AR-V7- (62%) patients had detectable AR-V7 protein in contemporaneous biopsies. After accounting for baseline characteristics, there was shorter OS in CTC+/AR-V7+ patients than in CTC- patients (hazard ratio [HR] 2.13; 95% confidence interval [CI] 1.23-3.71; p=0.02); surprisingly, there was no evidence that CTC+/AR-V7+ patients had worse OS than CTC+/AR-V7- patients (HR 1.26; 95% CI 0.73-2.17; p=0.4). A limitation of this study was the heterogeneity of treatment received. CONCLUSIONS: Studies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. PATIENT SUMMARY: Liquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use..

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is a lethal but clinically heterogeneous disease, with patients having variable benefit from endocrine and cytotoxic treatments. Intrapatient genomic heterogeneity could be a contributing factor to this clinical heterogeneity. Here, we used whole-genome sequencing (WGS) to investigate genomic heterogeneity in 21 previously treated CRPC metastases from 10 patients to investigate intrapatient molecular heterogeneity (IPMH).Experimental Design: WGS was performed on topographically separate metastases from patients with advanced metastatic prostate cancer. IPMH of the RB1 gene was identified and further evaluated by FISH and IHC assays. RESULTS: WGS identified limited IPMH for putative driver events. However, heterogeneous genomic aberrations of RB1 were detected. We confirmed the presence of these RB1 somatic copy-number aberrations, initially identified by WGS, with FISH, and identified novel structural variants involving RB1 in 6 samples from 3 of these 10 patients (30%; 3/10). WGS uncovered a novel deleterious RB1 structural lesion constituted of an intragenic tandem duplication involving multiple exons and associating with protein loss. Using RB1 IHC in a large series of mCRPC biopsies, we identified heterogeneous expression in approximately 28% of mCRPCs. CONCLUSIONS: mCRPCs have a high prevalence of RB1 genomic aberrations, with structural variants, including rearrangements, being common. Intrapatient genomic and expression heterogeneity favors RB1 aberrations as late, subclonal events that increase in prevalence due to treatment-selective pressures..

Castration-resistant prostate cancer (CRPC) that has developed resistance to the new-generation androgen receptor (AR) antagonist enzalutamide is a lethal disease. Transcriptome analysis of multiple prostate cancer models identified CXCR7, an atypical chemokine receptor, as one of the most upregulated genes in enzalutamide-resistant cells. AR directly repressed CXCR7 by binding to an enhancer 110 kb downstream of the gene and expression was restored upon androgen deprivation. We demonstrate that CXCR7 is a critical regulator of prostate cancer sensitivity to enzalutamide and is required for CRPC growth in vitro and in vivo. Elevated CXCR7 activated MAPK/ERK signaling through ligand-independent, but β-arrestin 2-dependent mechanisms. Examination of patient specimens showed that CXCR7 and pERK levels increased significantly from localized prostate cancer to CRPC and further upon enzalutamide resistance. Preclinical studies revealed remarkable efficacies of MAPK/ERK inhibitors in suppressing enzalutamide-resistant prostate cancer. Overall, these results indicate that CXCR7 may serve as a biomarker of resistant disease in patients with prostate cancer and that disruption of CXCR7 signaling may be an effective strategy to overcome resistance. SIGNIFICANCE: These findings identify CXCR7-mediated MAPK activation as a mechanism of resistance to second-generation antiandrogen therapy, highlighting the therapeutic potential of MAPK/ERK inhibitors in CRPC..

BACKGROUND: Understanding the integrated immunogenomic landscape of advanced prostate cancer (APC) could impact stratified treatment selection. METHODS: Defective mismatch repair (dMMR) status was determined by either loss of mismatch repair protein expression on IHC or microsatellite instability (MSI) by PCR in 127 APC biopsies from 124 patients (Royal Marsden [RMH] cohort); MSI by targeted panel next-generation sequencing (MSINGS) was then evaluated in the same cohort and in 254 APC samples from the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF). Whole exome sequencing (WES) data from this latter cohort were analyzed for pathogenic MMR gene variants, mutational load, and mutational signatures. Transcriptomic data, available for 168 samples, was also performed. RESULTS: Overall, 8.1% of patients in the RMH cohort had some evidence of dMMR, which associated with decreased overall survival. Higher MSINGS scores associated with dMMR, and these APCs were enriched for higher T cell infiltration and PD-L1 protein expression. Exome MSINGS scores strongly correlated with targeted panel MSINGS scores (r = 0.73, P < 0.0001), and higher MSINGS scores associated with dMMR mutational signatures in APC exomes. dMMR mutational signatures also associated with MMR gene mutations and increased immune cell, immune checkpoint, and T cell-associated transcripts. APC with dMMR mutational signatures overexpressed a variety of immune transcripts, including CD200R1, BTLA, PD-L1, PD-L2, ADORA2A, PIK3CG, and TIGIT. CONCLUSION: These data could impact immune target selection, combination therapeutic strategy selection, and selection of predictive biomarkers for immunotherapy in APC. FUNDING: We acknowledge funding support from Movember, Prostate Cancer UK, The Prostate Cancer Foundation, SU2C, and Cancer Research UK..

Purpose: Persistent androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC) and confers resistance to AR-targeting therapies. Novel therapeutic strategies to overcome this are urgently required. We evaluated how bromodomain and extra-terminal (BET) protein inhibitors (BETi) abrogate aberrant AR signaling in CRPC.Experimental Design: We determined associations between BET expression, AR-driven transcription, and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer models.Results: Nuclear BRD4 protein expression increases significantly (P ≤ 0.01) with castration resistance in same patient treatment-naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR, 3.25; 95% CI, 1.50-7.01; P ≤ 0.001). BRD2, BRD3, and BRD4 RNA expression in CRPC biopsies correlates with AR-driven transcription (all P ≤ 0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of prostate cancer cells and patient-derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (P ≤ 0.001) of a patient-derived xenograft model of CRPC with AR amplification and AR-V7 expression.Conclusions: BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof-of-mechanism pharmacodynamic studies. Clin Cancer Res; 24(13); 3149-62. ©2018 AACR..

BACKGROUND: Loss of PTEN is a common genomic aberration in castration-resistant prostate cancer (CRPC) and is frequently concurrent with ERG rearrangements, causing resistance to next-generation hormonal treatment (NGHT) including abiraterone. The relationship between PTEN loss and docetaxel sensitivity remains uncertain. OBJECTIVE: To study the antitumor activity of docetaxel in metastatic CRPC in relation to PTEN and ERG aberrations. DESIGN SETTING AND PARTICIPANTS: Single-centre, retrospective analysis of PTEN loss and ERG expression using a previously described immunohistochemistry (IHC) binary classification system. Patients received docetaxel between January 1, 2006 and July 31, 2016. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Response correlations were analyzed using Pearson's χ2 tests and independent-sample t tests. Overall (OS) and progression-free survival (PFS) were analyzed using univariate and multivariate (MVA) Cox regression and Kaplan-Meier methods. RESULTS AND LIMITATIONS: Overall, 215 patients were eligible. Established metastatic CRPC prognostic factors were well balanced between PTEN loss (39%) and normal patients (61%). PTEN loss was associated with shorter median OS (25.4 vs 34.7 mo; hazard ratio [HR] 1.66, 95% confidence interval [CI] 1.18-2.13; p = 0.001). There were no differences in median PFS (8.0 vs 9.1 mo; univariate HR 1.20, 95% CI 0.86-1.68; p = 0.28) and PSA response (53.4% vs 50.6%; p = 0.74). PTEN loss was an independent prognostics factor in MVA. ERG status was available for 100 patients. ERG positivity was not associated with OS or PFS. Limitations include the retrospective nature and the single-centre analysis. CONCLUSIONS: Our findings suggest that metastatic CRPC with PTEN loss might benefit more from docetaxel than from NGHT. PATIENT SUMMARY: In this study we found that metastatic prostate cancer with loss of the PTEN switch may benefit more from docetaxel than from abiraterone..

BACKGROUND: Androgen receptor splice variant 7 (AR-V7) has been implicated in resistance to abiraterone and enzalutamide treatment in men with metastatic castration-resistant prostate cancer (mCRPC). Tissue- or cell-based in situ detection of AR-V7, however, has been limited by lack of specificity. OBJECTIVE: To address current limitations in precision measurement of AR-V7 by developing a novel junction-specific AR-V7 RNA in situ hybridization (RISH) assay compatible with automated quantification. DESIGN, SETTING, AND PARTICIPANTS: We designed a RISH method to visualize single splice junctions in cells and tissue. Using the validated assay for junction-specific detection of the full-length AR (AR-FL) and AR-V7, we generated quantitative data, blinded to clinical data, for 63 prostate tumor biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We evaluated clinical correlates of AR-FL/AR-V7 measurements, including association with prostate-specific antigen progression-free survival (PSA-PFS) and clinical and radiographic progression-free survival (PFS), in a subset of patients starting treatment with abiraterone or enzalutamide following biopsy. RESULTS AND LIMITATIONS: Quantitative AR-FL/AR-V7 data were generated from 56 of the 63 (88.9%) biopsy specimens examined, of which 44 were mCRPC biopsies. Positive AR-V7 signals were detected in 34.1% (15/44) mCRPC specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 11.9% (range 2.7-30.3%). Positive detection of AR-V7 was correlated with indicators of high disease burden at baseline. Among the 25 CRPC biopsies collected before treatment with abiraterone or enzalutamide, positive AR-V7 detection, but not higher AR-FL, was significantly associated with shorter PSA-PFS (hazard ratio 2.789, 95% confidence interval 1.12-6.95; p=0.0081). CONCLUSIONS: We report for the first time a RISH method for highly specific and quantifiable detection of splice junctions, allowing further characterization of AR-V7 and its clinical significance. PATIENT SUMMARY: Higher AR-V7 levels detected and quantified using a novel method were associated with poorer response to abiraterone or enzalutamide in prostate cancer..

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

Androgen receptor (AR) splice variants (AR-Vs) have been implicated in the development and progression of metastatic prostate cancer. AR-Vs are truncated isoforms of the AR, a subset of which lack a ligand-binding domain and remain constitutively active in the absence of circulating androgens, thus promoting cancer cell proliferation. Consequently, AR-Vs have been proposed to contribute not only to resistance to anti-androgen therapies but also to resistance to radiotherapy in patients receiving combination therapy by promoting DNA repair. AR-Vs, such as AR-V7, have been associated with unfavourable clinical outcomes in patients; however, attempts to specifically inhibit or prevent the formation of AR-Vs have, to date, been unsuccessful. Thus, novel therapeutic strategies are desperately needed to address the oncogenic effects of AR-Vs, which can drive lethal forms of prostate cancer. Disruption of alternative splicing through modulation of the spliceosome is one such potential therapeutic avenue; however, our understanding of the biology of the spliceosome and how it contributes to prostate cancer remains incomplete, as reflected in the dearth of spliceosome-targeted therapeutic agents. In this Review, the authors outline the current understanding of the role of the spliceosome in the progression of prostate cancer and explore the therapeutic utility of manipulating alternative splicing to improve patient care..

Purpose: CHD1 deletions and SPOP mutations frequently cooccur in prostate cancer with lower frequencies reported in castration-resistant prostate cancer (CRPC). We monitored CHD1 expression during disease progression and assessed the molecular and clinical characteristics of CHD1-deleted/SPOP-mutated metastatic CRPC (mCRPC).Experimental Design: We identified 89 patients with mCRPC who had hormone-naive and castration-resistant tumor samples available: These were analyzed for CHD1, PTEN, and ERG expression by IHC. SPOP status was determined by targeted next-generation sequencing (NGS). We studied the correlations between these biomarkers and (i) overall survival from diagnosis; (ii) overall survival from CRPC; (iii) duration of abiraterone treatment; and (iv) response to abiraterone. Relationship with outcome was analyzed using Cox regression and log-rank analyses.Results: CHD1 protein loss was detected in 11 (15%) and 13 (17%) of hormone-sensitive prostate cancer (HSPC) and CRPC biopsies, respectively. Comparison of CHD1 expression was feasible in 56 matched, same patient HSPC and CRPC biopsies. CHD1 protein status in HSPC and CRPC correlated in 55 of 56 cases (98%). We identified 22 patients with somatic SPOP mutations, with six of these mutations not reported previously in prostate cancer. SPOP mutations and/or CHD1 loss was associated with a higher response rate to abiraterone (SPOP: OR, 14.50 P = 0.001; CHD1: OR, 7.30, P = 0.08) and a longer time on abiraterone (SPOP: HR, 0.37, P = 0.002, CHD1: HR, 0.50, P = 0.06).Conclusions: SPOP-mutated mCRPCs are strongly enriched for CHD1 loss. These tumors appear highly sensitive to abiraterone treatment. Clin Cancer Res; 24(22); 5585-93. ©2018 AACR..

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency.Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity.Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses.Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635-44. ©2018 AACR..

BRD4 belongs to the bromodomain and extraterminal (BET) family of chromatin reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi) has indicated antitumor activity against multiple cancer types. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs) and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ) pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. In support of this, we also show that, in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa) radiation therapy. Thus, in addition to regulating gene expression, BRD4 is also a central player in the repair of DNA DSBs, with significant implications for cancer therapy..

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Biomarkers for more precise patient care are needed in metastatic prostate cancer. We have reported a phase II trial (TOPARP-A) of the PARP inhibitor olaparib in metastatic prostate cancer, demonstrating antitumor activity associating with homologous recombination DNA repair defects. We now report targeted and whole-exome sequencing of serial circulating cell-free DNA (cfDNA) samples collected during this trial. Decreases in cfDNA concentration independently associated with outcome in multivariable analyses (HR for overall survival at week 8: 0.19; 95% CI, 0.06-0.56; P = 0.003). All tumor tissue somatic DNA repair mutations were detectable in cfDNA; allele frequency of somatic mutations decreased selectively in responding patients (χ2P < 0.001). At disease progression, following response to olaparib, multiple subclonal aberrations reverting germline and somatic DNA repair mutations (BRCA2, PALB2) back in frame emerged as mechanisms of resistance. These data support the role of liquid biopsies as a predictive, prognostic, response, and resistance biomarker in metastatic prostate cancer.Significance: We report prospectively planned, serial, cfDNA analyses from patients with metastatic prostate cancer treated on an investigator-initiated phase II trial of olaparib. These analyses provide predictive, prognostic, response, and resistance data with "second hit" mutations first detectable at disease progression, suggesting clonal evolution from treatment-selective pressure and platinum resistance. Cancer Discov; 7(9); 1006-17. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Kondrashova et al., p. 984See related article by Quigley et al., p. 999This article is highlighted in the In This Issue feature, p. 920..

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth. Clinically, Bag-1L protein levels increase with progression to castration-resistant PCa (CRPC) and high levels of Bag-1L in primary PCa associate with a reduced clinical benefit from abiraterone when these tumors progress. Intriguingly, residues in Bag-1L important for its interaction with the AR AF-1 are within a potentially druggable pocket, implicating Bag-1L as a potential therapeutic target in PCa..

BACKGROUND: The neutrophil to lymphocyte ratio (NLR) has been shown to be highly prognostic across many tumor types, and predictive of treatment outcome in advanced prostate cancer, and has been postulated to be an indirect measure of tumor inflammation. We evaluated the effect of low-dose steroids on NLR in men suffering from castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: The NLR was evaluated in a prospective randomized phase II trial that compared prednisolone 5 mg twice daily and dexamethasone 0.5 mg daily administered to 75 chemotherapy and abiraterone/enzalutamide-naive CRPC patients. NLR was examined at baseline (BL), after 6 and 12 weeks of corticosteroid treatment; associations with >50% prostate-specific antigen (PSA) response, duration of response (PSA progression-free interval), and overall survival (OS) were tested using logistic regression and Cox regression analysis. RESULTS: The median NLR for all evaluable patients was 2.6 at BL; 2.9 at 6 weeks; and 4.0 at 12 weeks. After low-dose corticosteroid initiation, 46 patients had a decline in PSA with 24 confirmed responders. BL NLR (log10) associated with a PSA response (odds ratio, .029, 95% confidence interval [CI], .002-.493; P = .014), and with the extent of the PSA decline (P = .009). A favorable BL NLR (less than median) associated with a 5.5-fold higher odds of a PSA >50% response (95% CI, 1.3-23.9; P = .02). Higher BL NLR (log10) associated with a shorter time to PSA progression (hazard ratio [HR], 9.5; 95% CI, 2.3-39.9; P = .002). In multivariate analysis BL NLR as a discrete variable was independently associated with PSA progression (HR, 3.5; 95% CI, 1.5-8.1; P = .003). NLR at 6 weeks was also associated with duration of benefit; in the favorable NLR category time to PSA progression was 10.8 months, for those who converted to an unfavorable (greater than median) category 4.5 months, and for those remaining in a unfavorable category only 1.5 months (95% CI, 0.5-2.5; P = .003). OS was 33.1 months (95% CI, 24.2-42.0) and 21.9 months (95% CI, 19.3-24.4) for those with an favorable and unfavorable BL NLR, respectively. CONCLUSION: Treatment-naive CRPC patients with a high BL or during-treatment NLR appear not to benefit from low-dose corticosteroids. The immunological implications of an unfavorable NLR, and whether corticosteroids might drive prostate cancer progression in patients harboring a high NLR, warrant further study..

Androgen receptor (AR) splice variants (SV) have been implicated in the development of metastatic castration-resistant prostate cancer and resistance to AR targeting therapies, including abiraterone and enzalutamide. Agents targeting AR-SV are urgently needed to test this hypothesis and further improve the outcome of patients suffering from this lethal disease. Clin Cancer Res; 22(17); 4280-2. ©2016 AACRSee related article by Yang et al., p. 4466..

PURPOSE: Erlotinib is active in advanced non-small cell lung cancer (aNSCLC) particularly in patients with EGFR-sensitizing mutations. The thymidylate synthase inhibitors are active in NSCLC, but capecitabine is not well studied. This study explored the safety and activity of this oral combination. METHODS: This phase Ib trial used a 3 + 3 escalation design with a combination of erlotinib (100 mg daily) with increasing doses of capecitabine (500, 750 and 1000 mg/m(2) BD, 14/21 days), in first- and second-line aNSCLC of adenocarcinoma histology. The DLT was any drug-induced toxicity ≥grade (G)2 causing dose interruption or dose delay during the first 2 cycles. RESULTS: Forty patients were recruited, and 1 patient had an EGFR mutation. Dose escalation stopped at capecitabine 1000 mg/m(2) with expansion to 6 patients due to unpredicted DLTs in 2/6 patients: G2 creatinine rise, G2 anaemia, G3 atrial fibrillation and G3 pneumonia. MTD was capecitabine 750 mg/m(2). First-line dose escalation at the MTD led to unpredicted DLTs in 3/4 patients (G3 troponin rise, G2 rash and G2 hyperbilirubinaemia). MTD expansion in the second-line setting was well tolerated. The most common drug toxicities were gastrointestinal (35 %), followed by skin disorders (28 %). The response rate was 3 % with a disease control rate of 34 %. Median progressive-free survival was 1.6 months (95 % CI 1.4-3.5), and median overall survival was 6.1 months (95 % CI 5.1-10.1). CONCLUSION: The MTD for the combination of capecitabine and erlotinib is 750 mg/m(2) BD, 14/21 days, and 100 mg daily, respectively, which is lower than predicted. Capecitabine did not improve the efficacy of erlotinib in aNSCLC unselected for EGFR mutation..

In recent years, the therapeutic options for treating men with metastatic castration-resistant prostate cancer have increased substantially. The hormonal treatments abiraterone acetate and enzalutamide, the chemotherapeutics docetaxel and cabazitaxel, the radiopharmaceutical alpharadin and the immunotherapeutic Sipuleucel-T have entered the field. Additionally, corticosteroids, which are used extensively, have documented activity but no documented survival benefit. Physicians treating patients with metastatic prostate cancer immediately after castration resistance develops currently have at least four different options to choose from for the first treatment. These therapeutic choices and their several possible ways of sequential use have not yet been compared to each other head-to-head and may never be. Therefore, there is an unmet need to inform their use with prospective clinical data. Additionally, the new indications of docetaxel for hormone naïve prostate cancer is changing the landscape of prostate cancer treatment and questions the traditional classifications 'pre-chemotherapy' and 'post-chemotherapy'. In this work we attempt to address these challenges in the treatment of metastatic castration-resistant prostate cancer with the focus mainly on the non-cytotoxic agents. We try to integrate available clinical and preclinical information to suggest optimal ways of treatment..

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Small cell lung cancer (SCLC) is a smoking-induced malignancy with multiple toxin-associated mutations, which accounts for 15% of all lung cancers. It remains a clinical challenge with a rapid doubling time, early dissemination and poor prognosis. Despite multiple clinical trials in SCLC, platinum-based chemotherapy remains the mainstay of treatment in the first line advanced disease setting; good initial responses are nevertheless inevitably followed by disease relapse and survival ultimately remains poor. There are currently no molecularly targeted agents licenced for use in SCLC. Advances in sequencing the cancer genome and other high-throughput profiling technologies have identified aberrant pathways and mechanisms implicated in SCLC development and progression. Novel anti-tumour therapeutics that impact these putative targets are now being developed and investigated in SCLC. In this review, we discuss novel anti-tumour agents assessed in SCLC with reference to the complex molecular mechanisms implicated in SCLC development and progression. We focus on novel DNA damage response inhibitors, immune checkpoint modulators and antibody-drug conjugates that have shown promise in SCLC, and which may potentially transform treatment strategies in this disease. Finally, we envision the future management of SCLC and propose a biomarker-driven translational treatment paradigm for SCLC that incorporates next generation sequencing studies with patient tumours, circulating plasma DNA and functional imaging. Such modern strategies have the potential to transform the management and improve patient outcomes in SCLC..

AIMS: Studies suggest worse outcomes in obese women with breast cancer than in non-obese women. One potential reason may be that oncologists 'dose cap' adjuvant chemotherapy in obese patients in order to avoid excessive toxicity. Reductions from standard dosing may compromise survival outcomes in the curative setting. Here we describe the body mass index (BMI) distribution of patients in a non-trial population, the frequency with which oncologists dose cap and its effect on febrile neutropenia chemotherapy toxicity. MATERIALS AND METHODS: In this non-randomised study, electronic patient records retrospectively identified patients with early breast cancer who initiated neoadjuvant or adjuvant chemotherapy at the Royal Marsden Hospital between 1 January and 31 December 2013. Baseline data included age, BMI, performance status, tumour characteristics, granulocyte colony-stimulating factor and comorbidities. Chemotherapy doses, rates of dose capping across BMI groups and rates of febrile neutropenia were reported. RESULTS: In total, 325 patients were eligible: 79 (24.5%) were obese (BMI ≥ 30), 109 (33.5%) were overweight (BMI ≥25 - <30) and 137 (42%) were normal bodyweight (BMI < 25). Sixteen patients (20.5%) in the obese group received dose-capped chemotherapy. Overall, 62 patients (19%) had an episode of febrile neutropenia. Obese patients receiving uncapped chemotherapy did not experience a significant difference in febrile neutropenia rates when compared with overweight or normal bodyweight groups (P = 0.5798). The febrile neutropenia rate in obese patients receiving capped chemotherapy was 6.5%, compared with 24% in obese patients receiving uncapped chemotherapy (P = 0.1216). CONCLUSION: In a non-trial population of obese patients, dose capping is frequently used. Obese patients receiving uncapped chemotherapy do not experience increased febrile neutropenia rates when compared with uncapped overweight or normal bodyweight patients. Furthermore, dose capping was associated with a trend towards lower rates of febrile neutropenia than in other groups and may indicate relative under-dosing of chemotherapy. This supports international guidelines that state that obese patients should not be dose capped..

BACKGROUND: The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. OBJECTIVE: To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. DESIGN, SETTING, AND PARTICIPANTS: Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. RESULTS AND LIMITATIONS: Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30-136.3) compared to after (HS 140, IQR 105-167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004-1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. CONCLUSIONS: We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. PATIENT SUMMARY: In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time..

OBJECTIVES: To determine the incidence and risk factors for thromboembolic events (TE) and febrile neutropenia (FN) in patients receiving systemic chemotherapy for early breast cancer (EBC). METHODS: 325 patients received FEC75, FEC100-T or ECaP for EBC in 2013. RESULTS: TE occurred in 7.4% and FN in 19.1% of patients. Risk factors for TE were: central venous catheter (p = 0.011). Risk factors for FN were: FEC100-T treatment versus FEC75 and ECaP (p ≤ 0.001); lower pre-treatment neutrophil count (p = 0.009) and poorer performance status (p = 0.012). Two patients died from treatment-related toxicities. CONCLUSION: In real-world experience, the majority of patients completed adequate treatment, despite significant complications..

BACKGROUND: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC. METHODS: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas(®)), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently. RESULTS: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features. CONCLUSION: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC..

Breast cancer is the most common cancer in women worldwide. The majority of deaths attributed to breast cancer are a result of metastatic disease, and 30% of early breast cancers (EBC) will develop distant disease. The 5-year survival of patients with metastatic disease is estimated at 23%. Breast cancer subtypes continue to be stratified histologically on oestrogen, progesterone and human epidermal growth factor-2 (HER2) receptor expression. HER2-positive breast cancers represent 25% of all breast cancer diagnoses. The therapies available for metastatic breast cancer (MBC) are expanding, in particular within the field of HER2-positive disease, with the approval of trastuzumab, pertuzumab, lapatinib and trastuzumab emtansine (TDM-1). Recently, TDM-1 has been shown to improve progression-free survival in HER2 MBC when compared to capecitabine and lapatinib in clinical studies. Its main toxicities are deranged liver function tests and thrombocytopenia. There have also been cases of acute liver failure. Therefore, its use in acute hepatic dysfunction, to our knowledge, has been neither studied nor reported. We report a patient with progressive HER2-positive MBC who had previously responded to multiple HER2-targeted therapies that presented with acute hepatic dysfunction. She was treated with dose-reduced TDM-1 safely, with clear evidence of rapid biochemical, clinical and radiological response. This allowed dose escalation of TDM-1, and the patient maintains an ongoing response..

BACKGROUND: Serum total human chorionic gonadotrophin β subunit (hCGβ) level might have prognostic value in urothelial transitional cell carcinoma (TCC) but has not been investigated for independence from other prognostic variables. METHODS: We utilised a clinical database of patients receiving chemotherapy between 2005 and 2011 for urothelial TCC and an independent cohort of radical cystectomy patients for validation purposes. Prognostic variables were tested by univariate Kaplan-Meier analyses and log-rank tests. Statistically significant variables were then assessed by multivariate Cox regression. Total hCGβ level was dichotomised at < vs ≥2 IU l(-1). RESULTS: A total of 235 chemotherapy patients were eligible. For neoadjuvant chemotherapy, established prognostic factors including low ECOG performance status, normal haemoglobin, lower T stage and suitability for cisplatin-based chemotherapy were associated with favourable survival in univariate analyses. In addition, low hCGβ level was favourable when assessed either before (median survival not reached vs 1.86 years, P=0.001) or on completion of chemotherapy (4.27 vs 0.42 years, P=0.000002). This was confirmed in multivariate analyses and in patients receiving first- and second-line palliative chemotherapy, and in a radical cystectomy validation set. CONCLUSIONS: Serum total hCGβ level is an independent prognostic factor in patients receiving chemotherapy for urothelial TCC in both curative and palliative settings..

BAG-1, a multifunctional protein, interacts with a plethora of cellular targets where the interaction with HSC70 and HSP70, is considered vital. Structural studies have demonstrated the C-terminal of BAG-1 forms a bundle of three alpha-helices of which helices 2 and 3 are directly involved in binding to the chaperones. Here we found peptides derived from helices 2 and 3 of BAG-1 interfered with BAG-1:HSC70 binding. We confirmed that a 12 amino-acid peptide from helix 2 directly interacted with HSC70 and when introduced into MCF-7 and ZR-75-1 cells, these peptides inhibited their growth. In conclusion, we have identified a small domain within BAG-1 which appears to play a critical role in the interaction with HSC70..

The C-terminal BAG domain is thought to play a key role in BAG-1-induced survival and proliferation by mediating protein-protein interactions, for example, with heat shock proteins HSC70 and HSP70, and with RAF-1 kinase. Here, we have identified thioflavin S (NSC71948) as a potential small-molecule chemical inhibitor of these interactions. NSC71948 inhibited the interaction of BAG-1 and HSC70 in vitro and decreased BAG-1:HSC70 and BAG-1:HSP70 binding in intact cells. NSC71948 also reduced binding between BAG-1 and RAF-1, but had no effect on the interaction between two unrelated proteins, BIM and MCL-1. NSC71948 functionally reversed the ability of BAG-1 to promote vitamin D3 receptor-mediated transactivation, an activity of BAG-1 that depends on HSC70/HSP70 binding, and reduced phosphorylation of p44/42 mitogen-activate protein kinase. NSC71948 can be used to stain amyloid fibrils; however, structurally related compounds, thioflavin T and BTA-1, had no effect on BAG-1:HSC70 binding, suggesting that structural features important for amyloid fibril binding and inhibition of BAG-1:HSC70 binding may be separable. We demonstrated that NSC71948 inhibited the growth of BAG-1 expressing human ZR-75-1 breast cancer cells and wild-type, but not BAG-1-deficient, mouse embryo fibroblasts. Taken together, these data suggest that NSC71948 may be a useful molecule to investigate the functional significance of BAG-1 C-terminal protein interactions. However, it is important to recognize that NSC71948 may exert additional "off-target" effects. Inhibition of BAG-1 function may be an attractive strategy to inhibit the growth of BAG-1-overexpressing cancers, and further screens of additional compound collections may be warranted..

BAG-1 is an anti-apoptotic protein that is frequently deregulated in a variety of malignancies including colorectal cancer. There are three isoforms: BAG-1L is located in the nucleus, BAG-1M and BAG-1S are located both in the nucleus and the cytoplasm. In colon cancer, the expression of nuclear BAG-1 is associated with poorer prognosis and is potentially a useful predictive factor for distant metastasis. However, the function of BAG-1 in colonic epithelial cells has not been studied. Having previously shown a predominant nuclear localisation of BAG-1 in adenoma-derived cell lines, we wanted to determine the function of nuclear BAG-1 in these non-tumourigenic cells, to identify whether nuclear BAG-1 was implicated in tumour progression in the colon. In the current report we established that nuclear BAG-1 inhibits apoptosis in a colorectal adenoma-derived cell line. We demonstrate that apoptosis induced by gamma-radiation or the vitamin D analogue EB1089 in the non-tumourigenic human colorectal adenoma-derived S/RG/C2 cell line, was preceded by a decrease in nuclear and an increase in cytoplasmic BAG-1 expression. This change in subcellular localisation of BAG-1 was due to the redistribution of the BAG-1M isoform. In addition, we have shown that the maintenance of high nuclear BAG-1 through enforced expression of the nuclear localised BAG-1L isoform enhanced cellular survival after gamma-radiation or exposure to EB1089. Furthermore the expression of cytoplasmic BAG-1S isoform fused with a nuclear localisation signal protected against gamma-radiation induced apoptosis. This demonstrates that nuclear localisation of the BAG-1 protein confers a survival advantage in colorectal adenoma-derived cells and that nuclear BAG-1 could potentially be an important survival factor in colorectal carcinogenesis..

BAG-1 is a multifunctional protein that exists as several differentially localised and functionally distinct isoforms. BAG-1 isoforms interact with a diverse array of molecular targets and regulate a wide range of cellular processes, including proliferation, survival, transcription, apoptosis, metastasis and motility. The BAG domain of BAG-1 interacts with chaperone molecules and this is considered important for many BAG-1 functions. The ability of BAG-1 to regulate such a wide variety of cellular processes suggests it might play an important role in many cancer types. For example, regulation of nuclear hormone receptor function and susceptibility to apoptosis might have a major impact on cancer development, progression and response to therapy. There is also increasing evidence that BAG-1 expression is altered in a variety of human malignancies relative to normal cells, and with further understanding of BAG-1 function it might become a powerful prognostic/predictive marker in human cancer. This review describes the structure and function of BAG-1 isoforms and the potential clinical implications of their expression in tumour cells..

Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein. In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag-1, an apoptotic regulator, in human colonic adenoma- and carcinoma-derived cell lines. Coimmunoprecipitation demonstrated that endogenous Rb and Bag-1 interact in both adenoma- and carcinoma-derived cell lines. The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag-1 complex. We report that Bag-1 is predominantly localised in the nucleus of colorectal adenoma- and carcinoma-derived epithelial cells. Disruption of the Rb/Bag-1 complex through expression of E7 changes the subcellular distribution of Bag-1, decreasing nuclear localised Bag-1. Our work establishes that the Rb protein interacts with the Bag-1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag-1 in human colonic epithelial cells..

BAG-1 is a multifunctional protein that interacts with a wide range of cellular targets including heat-shock proteins and some nuclear hormone receptors. BAG-1 exists as three major isoforms, BAG-1L, BAG-1M and BAG-1S. BAG-1L contains a nuclear localization signal, which is not present in the other isoforms, and is predominantly localized in the cell nucleus. Here we have investigated the effects of BAG-1 on function of the oestrogen receptor (ER), a key growth control molecule and target for hormonal therapy in breast cancer. We demonstrate that BAG-1L, but not BAG-1S or BAG-1M, increased oestrogen-dependent transcription in breast cancer cells. BAG-1L interacted with and stimulated the activity of both ER alpha and beta. Although BAG-1L and ERs colocalize to the nucleus, fusing BAG-1S to an heterologous nuclear localization sequence was not sufficient to stimulate transcription. Consistent with an important effect on receptor function, nuclear BAG-1 expression in breast cancers was associated with expression of the progesterone receptor, a transcriptional target of ERalpha, and was associated with improved survival in patients treated with hormonal therapy. These data suggest that BAG-1L is an important determinant of ER function in vitro and in human breast cancer..

BAG-1 is a multifunctional protein that interacts with a wide range of cellular targets. There is accumulating evidence that overexpression of BAG-1 may play an important role in breast cancer; however, the functional consequences of BAG-1 expression and its mechanism of action in breast cancer cells have not been studied in detail. Here we demonstrate that BAG-1 overexpression completely protected breast cancer cells from apoptosis and long-term growth inhibition induced by heat shock and also partially protected cells from other stresses, including hypoxia, radiation, and chemotoxic drugs. BAG-1 exists as three protein isoforms, and all isoforms prevented stress-induced growth inhibition. This required a conserved lysine in the BAG-1S ubiquitin-like domain thought to be important for proteasome binding and COOH-terminal amino acids required for interaction with the chaperone molecules, Hsc70 and Hsp70. Although expression of BAG-1 was unaltered by heat shock, endogenous and overexpressed BAG-1S relocalized from the cytoplasm to the nucleus after heat shock. The endogenous BAG-1S.Hsc70/Hsp70 complex dissociated after heat shock but was maintained at a detectable level in cells overexpressing BAG-1S. BAG-1-mediated resistance to stress-induced growth inhibition is likely to have a major impact on the development and response to therapy of breast cancer. Targeting the interaction of BAG-1 with chaperones is an attractive strategy to counter the biological effects of BAG-1..

BAG-1 is multifunctional protein which interacts with a wide range of cellular targets to regulate growth control pathways important for normal and malignant cells, including apoptosis, signaling, proliferation, transcription and cell motility. Of particular relevance to tumour cells, BAG-1 interacts with the anti-apoptotic BCL-2 protein, various nuclear hormone receptors and the 70 kDa heat shock proteins, Hsc70 and Hsp70. Interaction with chaperones may account for many of the pleiotropic effects associated with BAG-1 overexpression. Recent studies have shown that BAG-1 expression is frequently altered in malignant cells, and BAG-1 expression may have clinical value as a prognostic/predictive marker. This review summarises current understanding of molecular mechanisms of BAG-1 expression and function..