Association of Routine Preoperative Frailty Assessment With 1-Year Postoperative Mortality.

Importance: Patient frailty is a known risk factor for adverse outcomes following surgery, but data are limited regarding whether systemwide interventions related to frailty are associated with improved patient outcomes. Objective: To evaluate whether a frailty screening initiative (FSI) is associated with reduced late-term mortality after elective surgery. Design, Setting, and Participants: This quality improvement study with an interrupted time series analysis used data from a longitudinal cohort of patients in a multihospital, integrated health care system in the US. Beginning in July 2016, surgeons were incentivized to measure frailty with the Risk Analysis Index (RAI) for all patients considering elective surgery. Implementation of the BPA occurred in February 2018. The cutoff for data collection was May 31, 2019. Analyses were conducted between January and September 2022. Exposures: The exposure of interest was an Epic Best Practice Alert (BPA) used to identify patients with frailty (RAI ≥42) and prompt surgeons to document a frailty-informed shared decision-making process and consider additional evaluation by a multidisciplinary presurgical care clinic or the primary care physician. Main Outcomes and Measures: The primary outcome was 365-day mortality after the elective surgical procedure. Secondary outcomes included 30-day and 180-day mortality as well as the proportion of patients referred for additional evaluation based on documented frailty. Results: A total of 50 463 patients with at least 1 year of postsurgical follow-up (22 722 before intervention implementation and 27 741 after) were included (mean [SD] age, 56.7 [16.0] y; 57.6% women). Demographic characteristics, RAI score, and operative case mix, as defined by Operative Stress Score, were similar between time periods. After BPA implementation, the proportion of frail patients referred to a primary care physician and presurgical care clinic increased significantly (9.8% vs 24.6% and 1.3% vs 11.4%, respectively; both P < .001). Multivariable regression analysis demonstrated an 18% reduction in the odds of 1-year mortality (0.82; 95% CI, 0.72-0.92; P < .001). Interrupted time series models demonstrated a significant slope change in the rate of 365-day mortality from 0.12% in the preintervention period to -0.04% in the postintervention period. Among patients triggering the BPA, estimated 1-year mortality changed by -4.2% (95% CI, -6.0% to -2.4%). Conclusions and Relevance: This quality improvement study found that implementation of an RAI-based FSI was associated with increased referrals of frail patients for enhanced presurgical evaluation. These referrals translated to a survival advantage among frail patients of similar magnitude to those observed in a Veterans Affairs health care setting, providing further evidence for both the effectiveness and generalizability of FSIs incorporating the RAI.

Fusion Gene Detection in Prostate Cancer Samples Enhances the Prediction of Prostate Cancer Clinical Outcomes from Radical Prostatectomy through Machine Learning in a Multi-Institutional Analysis.

Prostate cancer remains one of the most fatal malignancies in men in the United States. Predicting the course of prostate cancer is challenging given that only a fraction of prostate cancer patients experience cancer recurrence after radical prostatectomy or radiation therapy. This study examined the expressions of 14 fusion genes in 607 prostate cancer samples from the University of Pittsburgh, Stanford University, and the University of Wisconsin-Madison. The profiling of 14 fusion genes was integrated with Gleason score of the primary prostate cancer and serum prostate-specific antigen level to develop machine-learning models to predict the recurrence of prostate cancer after radical prostatectomy. Machine-learning algorithms were developed by analysis of the data from the University of Pittsburgh cohort as a training set using the leave-one-out cross-validation method. These algorithms were then applied to the data set from the combined Stanford/Wisconsin cohort (testing set). The results showed that the addition of fusion gene profiling consistently improved the prediction accuracy rate of prostate cancer recurrence by Gleason score, serum prostate-specific antigen level, or a combination of both. These improvements occurred in both the training and testing cohorts and were corroborated by multiple models.

(+)-JJ-74-138 is a Novel Noncompetitive Androgen Receptor Antagonist.

Identification of novel androgen receptor (AR) antagonists may lead to urgently needed new treatments for patients with prostate cancer resistant to current AR antagonists. AR is presently the main target for treating prostate cancer. Clinically approved AR antagonists compete with dihydrotestosterone (DHT) for binding to the ligand-binding domain (LBD) of AR, and patients eventually develop resistance to these treatments. One approach to overcoming resistance is to discover compounds that inhibit AR in alternative ways. Our lab previously identified a small molecule, JJ-450, that is capable of inhibiting AR lacking LBD. To optimize the efficacy of this class of inhibitors, we developed structural analogues of JJ-450 and identified (+)-JJ-74-138 as a promising candidate. Here, we show that (+)-JJ-74-138 is more potent than JJ-450 in the inhibition of androgen-independent AR activity in enzalutamide-resistant LN95 cells. Further studies showed (+)-JJ-74-138 inhibition of castration-resistant PSA expression in all tested castration-resistant prostate cancer (CRPC) cells. (+)-JJ-74-138 inhibited mRNA expression of AR and ARv7 target genes and reduced AR level in the nucleus in the absence of androgens. Also, this analogue noncompetitively inhibited androgen-stimulated AR activity in C4-2, LN95, and 22Rv1 CRPC cells. At low dosages, (+)-JJ-74-138 inhibited the proliferation of enzalutamide-resistant AR-positive LN95 and 22Rv1 cells, but not AR-negative PC3 and DU145 cells. A surface plasmon resonance assay detected (+)-JJ-74-138 binding to AR and a chromatin immunoprecipitation assay indicated (+)-JJ-74-138 inhibited AR binding to androgen response elements. In addition, (+)-JJ-74-138 inhibited 22Rv1 xenograft tumor growth. Our observations suggest that (+)-JJ-74-138 is a novel noncompetitive AR antagonist capable of inhibiting enzalutamide-resistant CRPC.

Oncogenic Activity of Solute Carrier Family 45 Member 2 and Alpha-Methylacyl-Coenzyme A Racemase Gene Fusion Is Mediated by Mitogen-Activated Protein Kinase.

Chromosome rearrangement is one of the hallmarks of human malignancies. Gene fusion is one of the consequences of chromosome rearrangements. In this report, we show that gene fusion between solute carrier family 45 member 2 (SLC45A2) and alpha-methylacyl-coenzyme A racemase (AMACR) occurs in eight different types of human malignancies, with frequencies ranging from 45% to 97%. The chimeric protein is translocated to the lysosomal membrane and activates the extracellular signal-regulated kinase signaling cascade. The fusion protein promotes cell growth, accelerates migration, resists serum starvation-induced cell death, and is essential for cancer growth in mouse xenograft cancer models. Introduction of SLC45A2-AMACR into the mouse liver using a sleeping beauty transposon system and somatic knockout of phosphatase and TENsin homolog (Pten) generated spontaneous liver cancers within a short period. Conclusion: The gene fusion between SLC45A2 and AMACR may be a driving event for human liver cancer development.

Detection of fusion gene transcripts in the blood samples of prostate cancer patients.

Prostate cancer remains one of the most lethal cancers for men in the United States. The study aims to detect fusion transcripts in the blood samples of prostate cancer patients. We analyzed nine fusion transcripts including MAN2A1-FER, SLC45A2-AMACR, TRMT11-GRIK2, CCNH-C5orf30, mTOR-TP53BP1, KDM4-AC011523.2, TMEM135-CCDC67, LRRC59-FLJ60017 and Pten-NOLC1147 in the blood samples from 147 prostate cancer patients and 14 healthy individuals, using Taqman RT-PCR and Sanger's sequencing. Similar analyses were also performed on 25 matched prostate cancer samples for matched-sample evaluation. Eighty-two percent blood samples from the prostate cancer patients were positive for MAN2A1-FER transcript, while 41.5% and 38.8% blood samples from the prostate cancer patients were positive for SLC45A2-AMACR and Pten-NOLC1, respectively. CCNH-c5orf30 and mTOR-TP53BP1 had low detection rates, positive in only 5.4% and 4% of the blood samples from the prostate cancer patients. Only 2 blood samples were positive for KDM4B-AC011523.2 transcript. Overall, 89.8% patients were positive for at least one fusion transcript in their blood samples. The statistical analysis showed varied sensitivity of fusion transcript detection in the blood based on the types of fusions. In contrast, the blood samples from all healthy individuals were negative for the fusion transcripts. Detection of fusion transcripts in the blood samples of the prostate cancer patients may be a fast and cost-effective way to detect prostate cancer.

Clinical Utility of the Risk Analysis Index as a Prospective Frailty Screening Tool within a Multi-practice, Multi-hospital Integrated Healthcare System.

OBJECTIVE: The goal of this project was to first address barriers to implementation of the Risk Analysis Index (RAI) within a large, multi-hospital, integrated healthcare delivery system, and to subsequently demonstrate its utility for identifying at-risk surgical patients. BACKGROUND: Prior studies demonstrate the validity of the RAI for evaluating preoperative frailty, but they have not demonstrated the feasibility of its implementation within routine clinical practice. METHODS: Implementation of the RAI as a frailty screening instrument began as a quality improvement initiative at the University of Pittsburgh Medical Center in July 2016. RAI scores were collected within a REDCap survey instrument integrated into the outpatient electronic health record and then linked to information from additional clinical datasets. NSQIP-eligible procedures were queried within 90 days following the RAI, and the association between RAI and postoperative mortality was evaluated using logistic regression and Cox proportional hazards models. Secondary outcomes such as inpatient length of stay and readmissions were also assessed. RESULTS: RAI assessments were completed on 36,261 unique patients presenting to surgical clinics across five hospitals from July 1 to December 31, 2016, and 8,172 of these underwent NSQIP-eligible surgical procedures. The mean RAI score was 23.6 (SD 11.2), the overall 30-day and 180-day mortality after surgery was 0.7% and 2.6%, respectively, and the median time required to collect the RAI was 33 [IQR 23-53] seconds. Overall clinic compliance with the recommendation for RAI assessment increased from 58% in the first month of the study period to 84% in the sixth and final month. RAI score was significantly associated with risk of death (HR=1.099 [95% C.I.: 1.091 - 1.106], p < 0.001). At an RAI cutoff of ≥37, the positive predictive values for 30- and 90-day readmission were 14.8% and 26.2%, respectively, and negative predictive values were 91.6% and 86.4%, respectively. CONCLUSIONS: The RAI frailty screening tool can be efficiently implemented within multi-specialty, multi-hospital healthcare systems. In the context of our findings and given the value of the RAI in predicting adverse postoperative outcomes, health systems should consider implementing frailty screening within surgical clinics.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer.

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

Pten-NOLC1 fusion promotes cancers involving MET and EGFR signalings.

Inactivation of Pten gene through deletions and mutations leading to excessive pro-growth signaling pathway activations frequently occurs in cancers. Here, we report a Pten derived pro-cancer growth gene fusion Pten-NOLC1 originated from a chr10 genome rearrangement and identified through a transcriptome sequencing analysis of human cancers. Pten-NOLC1 fusion is present in primary human cancer samples and cancer cell lines from different organs. The product of Pten-NOLC1 is a nuclear protein that interacts and activates promoters of EGFR, c-MET, and their signaling molecules. Pten-NOLC1 promotes cancer proliferation, growth, invasion, and metastasis, and reduces the survival of animals xenografted with Pten-NOLC1-expressing cancer cells. Genomic disruption of Pten-NOLC1 induces cancer cell death, while genomic integration of this fusion gene into the liver coupled with somatic Pten deletion produces spontaneous liver cancers in mice. Our studies indicate that Pten-NOLC1 gene fusion is a driver for human cancers.

Rotavirus induces intercellular calcium waves through ADP signaling.

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Transforming growth factor beta 1 impairs benign prostatic luminal epithelial cell monolayer barrier function.

Our recent studies identifying the presence of luminal secretory protein PSA in the stroma, decreased E-cadherin expression, and reduced number of tight junction kiss points in benign prostatic hyperplasia (BPH) tissues suggest that epithelial barrier permeability is increased in BPH. However, the cause of increased epithelial permeability in BPH is unclear. Transforming growth factor beta 1 (TGF-ß1) has been reported to be up-regulated in clinical BPH specimens and TGF-ß1 overexpression induced fibrosis and inflammation in a murine model. TGF-ß1 was reported to repress the expression of E-cadherin in benign prostatic cells. However, whether and how TGF-ß1 up-regulation affects epithelial barrier permeability is unknown. Here, in vitro benign prostatic epithelial cell lines BHPrE1 and BPH-1 were utilized to determine the impact of TGF-ß1 treatment on epithelial barrier, tight junctions, and expression of E-cadherin and claudin 1 by transepithelial electrical resistance (TEER) measurement, FITC-dextran trans-well diffusion assays, qPCR, as well as transmission electron microscopy (TEM) observation. Laser capture micro-dissection (LCM) combined with reverse transcription-polymerase chain reaction (qPCR) were utilized to determine the expression of E-cadherin and claudin 1 in BPH patient specimens. TGF-ß1 treatment decreased TEER, increased FITC-dextran diffusion, and reduced the mRNA expression of junction protein claudin 1 in cultured cell monolayers. Claudin 1 mRNA but not E-cadherin mRNA was down-regulated in the luminal epithelial cells in BPH nodules compared to normal prostate tissues. Our studies suggest that TGF-ß1 could increase the permeability through decreasing the expression of claudin 1 and inhibiting the formation of tight junctions in BHPrE1 and BPH-1 monolayers. These results suggest that TGF-ß1 might play an important role in BPH pathogenesis through increasing the permeability of luminal epithelial barrier in the prostate.

EAF2 loss induces prostatic intraepithelial neoplasia from luminal epithelial cells in mice.

Defining the cell of origin for prostatic carcinogenesis is fundamentally important for understanding the mechanisms leading to prostate cancer. Lineage tracing studies have demonstrated that luminal epithelial cells are capable of self-replication in multiple organs, including the adult murine prostate, and cell of prostate cancer origin studies have shown that while both the luminal and basal murine prostate epithelial cells are capable of neoplastic transformation, luminal cells are more efficient as the origin of prostate cancer. ELL-associated factor 2 (EAF2) is an androgen responsive tumor suppressive protein expressed by prostate luminal epithelial cells that is frequently down-regulated in primary prostate tumors. EAF2 knockdown induces prostate cancer cell proliferation and invasion in vitro and mice with Eaf2 deficiency develop epithelial hyperplasia and murine prostatic intraepithelial neoplasia (mPIN) lesions. Here, we utilized an Eaf2 knockout, PSA-CreERT2 transgenic model crossed with a fluorescent reporter line to show that Eaf2 deficiency induces mPIN lesions derived from the luminal cell lineage. These results suggest that PIN lesions in the Eaf2 knockout mouse were derived from prostate luminal epithelial cells, further suggesting that the prostatic luminal epithelial cell is the major origin of prostate carcinogenesis.

The Development and Validation of Prostate Cancer-specific Physician-Hospital Networks.

OBJECTIVE: To develop prostate cancer-specific physician-hospital networks to define hospital-based units that more accurately group hospitals, providers, and the patients they serve. METHODS: Using Surveillance, Epidemiology, and End Results-Medicare, we identified men diagnosed with localized prostate cancer between 2007 and 2011. We created physician-hospital networks by assigning each patient to a physician and each physician to a hospital based on treatment patterns. We assessed content validity by examining characteristics of hospitals anchoring the physician-hospital networks and of the patients associated with these hospitals. RESULTS: We identified 42,963 patients associated with 344 physician-hospital networks. Networks anchored by a teaching hospital (compared to a nonteaching hospital) had higher median numbers of prostate cancer patients (117 [interquartile range {71-189} vs 82 {50-126}]) and treating physicians (7 [4-11] vs 4 [3-6]) (both P <0.001). On average, patients traveled farther to networks anchored by a teaching hospital (49 miles [standard deviation] [207] vs 41 [183]; P <.001). Hospitals known as high-volume centers for robotic prostatectomies, proton-beam therapy, and active surveillance had network rates for these procedures well above the mean. Hospitals known as safety net providers served higher proportions of minorities. CONCLUSION: We empirically developed prostate-cancer specific physician-hospital networks that exhibit content validity and are relevant from a clinical and policy perspective. They have the potential to become targets for policy interventions focused on improving the delivery of prostate cancer care.

A Novel Small Molecule Targets Androgen Receptor and Its Splice Variants in Castration-Resistant Prostate Cancer.

Reactivation of androgen receptor (AR) appears to be the major mechanism driving the resistance of castration-resistant prostate cancer (CRPC) to second-generation antiandrogens and involves AR overexpression, AR mutation, and/or expression of AR splice variants lacking ligand-binding domain. There is a need for novel small molecules targeting AR, particularly those also targeting AR splice variants such as ARv7. A high-throughput/high-content screen was previously reported that led to the discovery of a novel lead compound, 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2,3-dimethylphenyl)piperazin-1-yl)ethan-1-one (IMTPPE), capable of inhibiting nuclear AR level and activity in CRPC cells, including those resistant to enzalutamide. A novel analogue of IMTPPE, JJ-450, has been investigated with evidence for its direct and specific inhibition of AR transcriptional activity via a pulldown assay and RNA-sequencing analysis, PSA-based luciferase, qPCR, and chromatin immunoprecipitation assays, and xenograft tumor model 22Rv1. JJ-450 blocks AR recruitment to androgen-responsive elements and suppresses AR target gene expression. JJ-450 also inhibits ARv7 transcriptional activity and its target gene expression. Importantly, JJ-450 suppresses the growth of CRPC tumor xenografts, including ARv7-expressing 22Rv1. Collectively, these findings suggest JJ-450 represents a new class of AR antagonists with therapeutic potential for CRPC, including those resistant to enzalutamide.

The Influence of Stereotactic Body Radiation Therapy Adoption on Prostate Cancer Treatment Patterns.

PURPOSE: To our knowledge it is unknown whether stereotactic body radiation therapy of prostate cancer is a substitute for other radiation treatments or surgery, or for expanding the pool of patients who undergo treatment instead of active surveillance. MATERIALS AND METHODS: Using SEER (Surveillance, Epidemiology, and End Results)-Medicare we identified men diagnosed with prostate cancer between 2007 and 2011. We developed physician-hospital networks by identifying the treating physician of each patient based on the primary treatment received and subsequently assigning each physician to a hospital. We examined the relative distribution of prostate cancer treatments stratified by whether stereotactic body radiation therapy was performed in a network by fitting logistic regression models with robust SEs to account for patient clustering in networks. RESULTS: We identified 344 physician-hospital networks, including 30 (8.7%) and 314 (91.3%) in which stereotactic body radiation therapy was and was not performed, respectively. Networks in which that therapy was and was not done did not differ with time in the performance of robotic and radical prostatectomy, and active surveillance (all p >0.05). The relationship with intensity modulated radiation therapy did not show any consistent temporal pattern. In networks in which it was performed less intensity modulated radiation therapy was initially done but there were similar rates in later years. Brachytherapy trends differed among networks in which stereotactic body radiation therapy was vs was not performed with a lower brachytherapy rate in networks in which stereotactic body radiation therapy was done (p=0.03). CONCLUSIONS: Surgery and active surveillance rates did not differ in networks in which stereotactic body radiation therapy was vs was not performed but when that therapy was done there was a lower brachytherapy rate. Stereotactic body radiation therapy may represent more of an alternative to brachytherapy than to active surveillance.

Multifocal Extraprostatic Extension of Prostate Cancer.

OBJECTIVES: To investigate the prognostic utility of multifocal extraprostatic extension (EPE) on biochemical recurrence after radical prostatectomy. METHODS: We conducted retrospective analysis of biochemical recurrence and prognostic pathologic variables in 673 men with stage pT3a/pT3b prostate cancer from 2000 to 2012. Extent of EPE on radical prostatectomy was divided into three groups: focal EPE (tumor dimension <0.8 mm), established (≥ 0.8 mm), and multifocal (more than one focus of EPE <0.8 mm). RESULTS: Type of EPE had significant effect on recurrence with progressively lower progression-free probability and higher recurrence probability from focal to established to multifocal. Multifocal and established tumors exhibited worse prognostic features and higher hazard ratio than focal. In multivariate analysis, established and multifocal were independent prognostic factors with the greatest adverse prognostic significance associated with multifocal. CONCLUSIONS: Identification of multifocal EPE provides important prognostic information associated with increased likelihood of recurrence compared to focal and established tumors.

Patterns of stereotactic body radiation therapy: The influence of lung cancer treatment on prostate cancer treatment.

INTRODUCTION: Technology availability and prior experience with novel cancer treatments may partially drive their use. We sought to examine this issue in the context of stereotactic body radiation therapy (SBRT) by studying how its use for an established indication (lung cancer) impacts its use for an emerging indication (prostate cancer). METHODS: Using SEER-Medicare from 2007 to 2011, we developed prostate cancer-specific physician-hospital networks. Our primary dependent variable was SBRT use for prostate cancer and our primary independent variable was SBRT use for lung cancer, both at the network level. To assess the influence of SBRT availability and experiential use, we generated predicted probabilities of SBRT use for prostate cancer stratified by a network's use of lung cancer SBRT, adjusting for network characteristics. To assess intensity of use, we examined the correlation between the proportion of prostate cancer patients and lung cancer patients receiving SBRT within a network. RESULTS: We identified 316 networks that served 41,034 prostate cancer and 83,433 lung cancer patients. A network was significantly more likely to use SBRT for prostate cancer if that network used SBRT for lung cancer (e.g., in 2011, odds ratio [OR] 12.7; 95% confidence interval [CI] 3.9-41.8). The Pearson's correlation between the proportion of prostate cancer patients and lung cancer patients receiving SBRT in a network was 0.34, which was not statistically significant (P = 0.12). CONCLUSIONS: SBRT availability and experiential use for lung cancer influences its use for prostate cancer, but intensity of use for one does not relate to intensity of use for the other.

A novel androgen receptor antagonist JJ-450 inhibits enzalutamide-resistant mutant ARF876L nuclear import and function.

BACKGROUND: Castration-resistant prostate cancer can develop resistance to enzalutamide because of androgen receptor (AR) point mutations, AR overexpression, constitutively active AR splice variants, and/or elevated intratumoral androgen synthesis. The point mutation ARF876L was reported to be stimulated, instead of inhibited, by enzalutamide, thus contributing to enzalutamide resistance. We have recently developed JJ-450 as a novel AR antagonist with the potential to treat enzalutamide-resistant castration-resistant prostate cancer (CRPC). METHODS: We employed several assays to determine the impact of JJ-450 and enzalutamide on prostate cancer cell lines expressing green fluorescent protein (GFP)-ARF876L . These assays include a prostate-specific antigen enhancer/promoter-based luciferase assay to determine AR transcriptional activity, a quantitative real-time polymerase chain reaction assay, and Western blot analysis to detect expression of AR-target genes at the messenger RNA and protein level, fluorescence microscopy to show AR subcellular localization, and a 5-bromo-2'-deoxyuridine assay to measure prostate cancer cell proliferation. RESULTS: As expected, enzalutamide inhibited wild-type (WT) AR but not ARF876L transcriptional activity in the luciferase assay. In contrast, JJ-450 inhibited both WT-AR and ARF876L transcriptional activity to a similar extent. Also, enzalutamide retarded androgen-induced nuclear import of GFP-AR, but not GFP-ARF876L , whereas JJ-450 retarded nuclear import of both GFP-AR and GFP-ARF876L . To further evaluate JJ-450 inhibition of ARF876L , we stably transfected C4-2 cells separately with GFP-AR or GFP-ARF876L . Enzalutamide inhibited endogenous AR-target gene expression in C4-2-GFP-ARWT , but not in the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited AR-target gene expression in both C4-2 sublines. More importantly, enzalutamide inhibited proliferation of C4-2-GFP-ARWT , but not of the C4-2-GFP-ARF876L subline, whereas JJ-450 inhibited proliferation of both C4-2 sublines. CONCLUSION: JJ-450 inhibits enzalutamide-resistant ARF876L mutant nuclear translocation and function. Our findings suggest that JJ-450 and its analogs should be further developed to provide a potential new approach for the treatment of enzalutamide-resistant CRPC.

Relation between activity-induced intracellular sodium transients and ATP dynamics in mouse hippocampal neurons.

KEY POINTS: Employing quantitative Na+ -imaging and Förster resonance energy transfer-based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity-induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1-0.6 mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+ /K+ -ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. ABSTRACT: Excitatory neuronal activity results in the influx of Na+ through voltage- and ligand-gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+ ]i ) is mainly mediated by the Na+ /K+ -ATPase (NKA) and is one of the major energy-consuming processes in the brain. Here, we analysed the relation between different patterns of activity-induced [Na+ ]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium-binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i ) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+ ]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3-0.4 mm in both compartments. Global [Na+ ]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i . Our results suggest that ATP consumption by the NKA following global [Na+ ]i transients temporarily overrides its availability, causing a decrease in [ATP]i . Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i , suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.

E-cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer.

BACKGROUND: We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. METHODS: The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. RESULTS: The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. CONCLUSIONS: Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

Change in Functional Status After Prostate Cancer Treatment Among Medicare Advantage Beneficiaries.

OBJECTIVE: To examine the relationship between treatment and subsequent functional status among prostate cancer patients. METHODS: Using Surveillance, Epidemiology, and End Results-Medicare Health Outcomes Survey data, we identified men 65 years or older diagnosed with prostate cancer between 1998 and 2009 (follow-up through 2010) who were treated with conservative management, surgery, or radiation. Our primary outcome was functional status as measured by activities of daily living. Secondary outcomes included physical component summary and mental component summary scores, which are both calculated from the Short Form 36 (SF-36) and the Veterans RAND 12-item health survey (VR-12) questionnaires. We included patients who completed 2 surveys and performed propensity score analyses to match patients 1:5 with noncancer controls. We used generalized linear mixed effects models, accounting for clustering due to insurance plan. RESULTS: We identified 408 patients of whom 143 (35%) underwent conservative management, 59 (14%) underwent surgery, and 206 (51%) underwent radiation. Among conservative management and radiation patients, changes in functional status mirrored that of their noncancer controls (all P > .05). Among surgery patients, changes in activities of daily living scores were not significant, but physical component summary (mean difference = 4.5, P < .001) and mental component summary (mean difference = 3.3, P = .01) scores declined slightly more than for their noncancer peers. CONCLUSION: Surgery patients had a slight decline in their general functional status whereas conservative management and radiation patients had no differences in functional status compared with their noncancer peers. Although the functional status of surgery patients declined more than that of their noncancer peers, this difference may not be clinically significant.