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Background and Objective: Checkpoint inhibitor therapy (CPI) has demonstrated survival benefits in urothelial carcinoma (UC); however, not all patients benefit from CPI due to resistance. Combining sitravatinib, a multitargeted receptor tyrosine kinase inhibitor of TYRO3, AXL, and MERTK (TAM) receptors and VEGFR2, with CPI may improve antitumor responses. Our objective was to assess the efficacy and safety of sitravatinib plus nivolumab in patients with advanced/metastatic UC., Methods: The 516-003 trial (NCT03606174) is an open-label, multicohort phase 2 study evaluating sitravatinib plus nivolumab in patients with advanced/metastatic UC enrolled in eight cohorts depending on prior treatment with CPI, platinum-based chemotherapy (PBC), or antibody-drug conjugate (ADC). Overall, 244 patients were enrolled and treated with sitravatinib plus nivolumab (median follow-up 14.1-38.2 mo). Sitravatinib (free-base capsules 120 mg once daily [QD] or malate capsule 100 mg QD) plus nivolumab (240 mg every 2 wk/480 mg every 4 wk intravenously)., Key Findings and Limitations: The primary endpoint was objective response rate (ORR; RECIST v1.1). The secondary endpoints included progression-free survival (PFS) and safety. The Predictive probability design and confidence interval methods were used. Among patients previously treated with PBC, ORR, and median PFS were 32.1% and 3.9 mo in CPI-naïve patients (n = 53), 14.9% and 3.9 mo in CPI-refractory patients (n = 67), and 5.4% and 3.7 mo in CPI- and ADC-refractory patients (n = 56), respectively. Across all cohorts, grade 3 treatment-related adverse events (TRAEs) occurred in 51.2% patients and grade 4 in 3.3%, with one treatment-related death (cardiac failure). Immune-related adverse events occurred in 50.4% patients. TRAEs led to sitravatinib/nivolumab discontinuation in 6.1% patients., Conclusions and Clinical Implications: Sitravatinib plus nivolumab demonstrated a manageable safety profile but did not result in clinically meaningful ORRs in patients with advanced/metastatic UC in the eight cohorts studied., Patient Summary: In this study, the combination of two anticancer drugs, sitravatinib and nivolumab, resulted in manageable side effects but no meaningful responses in patients with bladder cancer., (Copyright © 2023 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: The majority of patients with metastatic castration-resistant prostate cancer (mCRPC) will have disease progression of a uniformly fatal disease. mCRPC is driven by both activated androgen receptors and elevated intratumoural androgens; however, the current standard of care is therapy that targets a single androgen signalling mechanism. We aimed to investigate the combination treatment using apalutamide plus abiraterone acetate, each of which suppresses the androgen signalling axis in a different way, versus standard care in mCRPC., Methods: ACIS was a randomised, placebo-controlled, double-blind, phase 3 study done at 167 hospitals in 17 countries in the USA, Canada, Mexico, Europe, the Asia-Pacific region, Africa, and South America. We included chemotherapy-naive men (aged ≥18 years) with mCRPC who had not been previously treated with androgen biosynthesis signalling inhibitors and were receiving ongoing androgen deprivation therapy, with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and a Brief Pain Inventory-Short Form question 3 (ie, worst pain in the past 24 h) score of 3 or lower. Patients were randomly assigned (1:1) via a centralised interactive web response system with a permuted block randomisation scheme (block size 4) to oral apalutamide 240 mg once daily plus oral abiraterone acetate 1000 mg once daily and oral prednisone 5 mg twice daily (apalutamide plus abiraterone-prednisone group) or placebo plus abiraterone acetate and prednisone (abiraterone-prednisone group), in 28-day treatment cycles. Randomisation was stratified by presence or absence of visceral metastases, ECOG performance status, and geographical region. Patients, the investigators, study team, and the sponsor were masked to group assignments. An independent data-monitoring committee continually monitored data to ensure ongoing patient safety, and reviewed efficacy data. The primary endpoint was radiographic progression-free survival assessed in the intention-to-treat population. Safety was reported for all patients who received at least one dose of study drug. This study is completed and no longer recruiting and is registered with ClinicalTrials.gov, number NCT02257736., Findings: 982 men were enrolled and randomly assigned from Dec 10, 2014 to Aug 30, 2016 (492 to apalutamide plus abiraterone-prednisone; 490 to abiraterone-prednisone). At the primary analysis (median follow-up 25·7 months [IQR 23·0-28·9]), median radiographic progression-free survival was 22·6 months (95% CI 19·4-27·4) in the apalutamide plus abiraterone-prednisone group versus 16·6 months (13·9-19·3) in the abiraterone-prednisone group (hazard ratio [HR] 0·69, 95% CI 0·58-0·83; p<0·0001). At the updated analysis (final analysis for overall survival; median follow-up 54·8 months [IQR 51·5-58·4]), median radiographic progression-free survival was 24·0 months (95% CI 19·7-27·5) versus 16·6 months (13·9-19·3; HR 0·70, 95% CI 0·60-0·83; p<0·0001). The most common grade 3-4 treatment-emergent adverse event was hypertension (82 [17%] of 490 patients receiving apalutamide plus abiraterone-prednisone and 49 [10%] of 489 receiving abiraterone-prednisone). Serious treatment-emergent adverse events occurred in 195 (40%) patients receiving apalutamide plus abiraterone-prednisone and 181 (37%) patients receiving abiraterone-prednisone. Drug-related treatment-emergent adverse events with fatal outcomes occurred in three (1%) patients in the apalutamide plus abiraterone-prednisone group (2 pulmonary embolism, 1 cardiac failure) and five (1%) patients in the abiraterone-prednisone group (1 cardiac failure and 1 cardiac arrest, 1 mesenteric arterial occlusion, 1 seizure, and 1 sudden death)., Interpretation: Despite the use of an active and established therapy as the comparator, apalutamide plus abiraterone-prednisone improved radiographic progression-free survival. Additional studies to identify subgroups of patients who might benefit the most from combination therapy are needed to further refine the treatment of mCRPC., Funding: Janssen Research & Development., Competing Interests: Declaration of interests FS received financial support from Janssen for the ACIS study; he reports grants or contracts, consulting fees, and payment or honoraria for lectures, presentations, speakers bureaus, and manuscript writing or educational events from Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Janssen, Myovant, Novartis, and Sanofi, outside of the submitted work. EE received financial support from Janssen for the ACIS study; she reports grants or contracts from Astellas, AstraZeneca, Janssen, Merck, Oric, and Sanofi; consulting fees from Astellas, AstraZeneca, Bayer, Janssen, Merck, Pfizer, and Sanofi; payment or honoraria for lectures, presentations, speakers bureaus, and manuscript writing or educational events from Bayer, Janssen, and Sanofi; financial support for attending meetings, travel, or both, from Astellas, Janssen, and Sanofi; participated in Data Safety Monitoring Board or Advisory Board meetings for AstraZeneca, Janssen, Merck, Pfizer, and Sanofi; and reports leadership or fiduciary roles in other board, society, committee, or advocacy group, paid or unpaid, for the European Society of Medical Oncology, outside of the submitted work. GA received financial support from Janssen for the ACIS study; he reports grants or contracts from Astellas and Janssen; consulting fees from Astellas, Bayer, Beigene, Clovis, Janssen, Novartis, Pfizer, and Sanofi; payment or honoraria for lectures, presentations, speakers bureaus, and manuscript writing or educational events from Astellas, Bayer, Janssen, Novartis, and Sanofi; financial support for attending meetings, travel, or both, from Astellas, Bayer, Janssen, Novartis, Pfizer, and Sanofi; participated in Data Safety Monitoring Board or Advisory Board meetings for Astellas, Bayer, Janssen, Novartis, Pfizer, and Sanofi; received royalties from Janssen paid to the Institute of Cancer Research; and is included on the list of rewards to discoverers of abiraterone, outside of the submitted work. TWF received grants or contracts from Agensys, Aragon Pharmaceuticals, Astellas Pharma, AstraZeneca–MedImmune, Bristol Myers Squibb, Bavarian Nordic, Dendreon, Exelixis, GTx, Janssen Oncology, La Roche-Posay, Lilly, Medivation, Merck, Novartis, Pfizer, Roche–Genentech, Sanofi, Sotio, Seagen, Seattle Genetics, and Tokai Pharmaceuticals; consulting fees from Aurora Oncology, Janssen Oncology, and Seattle Genetics; filed two patents (via the University of Colorado) related to early-stage bladder cancer treatment and detection for which he is an inventor, neither of which are currently commercialised or in clinical trials; and is the founder and a stockholder for Aurora Oncology, all outside of the submitted work. OBG received consulting fees from Bayer and Janssen and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, and educational events from Janssen, all outside of the submitted work. SO reports consulting fees, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events, and support for attending meetings, travel, or both from Astellas, AstraZeneca, Bayer, Bristol Myers Squibb, Ipsen, Janssen, Merck, Novartis, Pfizer, and Sanofi and participated in Data Safety Monitoring Board or Advisory Board meetings for Astellas, Janssen, Roche, and Sanofi, all outside of the submitted work. TS received consulting fees and payment or honoraria for lectures, presentations, speakers bureaus, and manuscript writing or educational events from Astellas Oncology, Bayer Health, Janssen, and Sanofi, all outside of the submitted work. HS received financial support from Janssen for the ACIS study; he reports grants or contracts from Asahi Kasei, Bayer, Daiichi Sankyo, Kissei, Nippon Kayaku, Nippon Shinyaku, Ono, Sanofi, Taiho, and Takeda; consulting fees from Astellas, AstraZeneca, Bayer, Eli Lilly, Janssen, MSD, Nihon Medi-Physics, Roche–Chugai, and Sanofi; payment or honoraria for lectures, presentations, speakers bureaus, and manuscript writing or educational events from Astellas, AstraZeneca, Bayer, Janssen, Merck Biopharma, MSD, Nippon Shinyaku, Ono, Otsuka, Pfizer, Sanofi, and Takeda; support for attending meetings, travel, or both from Astellas, Bayer, Eli Lilly, Janssen, and Sanofi; and participated in Advisory Board meetings for Astellas, Bayer, Janssen, Roche–Chugai, and Sanofi, outside of the submitted work. DER received support from Janssen for the ACIS study; she reports uncompensated participation in advisory boards for AstraZeneca, Bayer, Genentech, Janssen, and Myovant, outside of the submitted work. DW, KY, PDP, SB-M, SL, JL, ST, KBB, SDM, and SAM are employed by Janssen Research & Development, and hold stock in Johnson & Johnson. FF declares no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Overcoming taxane resistance remains a major clinical challenge in metastatic castrate-resistant prostate cancer (mCRPC). Loss of DNA repair proteins is associated with resistance to anti-microtubule agents. We propose that alterations in DNA damage response (DDR) pathway contribute to taxane resistance, and identification of these alterations may provide a potential therapeutic target to resensitize docetaxel-refractory mCRPC to taxane-based therapy., Methods: Alterations in DDR gene expression in our prostate cancer cell line model of docetaxel-resistance (DU145-DxR) derived from DU-145 cells were determined by DDR pathway-specific polymerase chain reaction array and immunoblotting. The PRKDC gene encoding DNA-PKc (DNA-dependent protein kinase catalytic unit), was noted to be overexpressed and evaluated for its role in docetaxel resistance. Cell viability and clonogenic survival of docetaxel-treated DU145-DxR cells were assessed after pharmacologic inhibition of DNA-PKc with three different inhibitors-NU7441, LTURM34, and M3814. Response to second-line cytotoxic agents, cabazitaxel and etoposide upon DNA-PKc inhibition was also tested. The impact of DNA-PKc upregulation on DNA damage repair was evaluated by comet assay and analysis of double-strand breaks marker, γH2AX and Rad51. Lastly, DNA-PKc inhibitor's effect on MDR1 activity was assessed by rhodamine 123 efflux assay., Results: DDR pathway-specific gene profiling revealed significant upregulation of PRKDC and CDK7, and downregulation of MSH3 in DU145-DxR cells. Compared to parental DU145, DU145-DxR cells sustained significantly less DNA damage when exposed to etoposide and docetaxel. Pharmacologic inhibition of DNA-PKc, a component of NHEJ repair machinery, with all three inhibitors, significantly resensitized DU145-DxR cells to docetaxel. Furthermore, DNA-PKc inhibition also resensitized DU145-DxR to cabazitaxel and etoposide, which demonstrated cross-resistance. Inhibition of DNA-PKc led to increased DNA damage in etoposide- and docetaxel-treated DU145-DxR cells. Finally, DNA-PKc inhibition did not affect MDR1 activity, indicating that DNA-PKc inhibitors resensitized taxane-resistant cells via an MDR1-independent mechanism., Conclusion: This study supports a role of DDR genes, particularly, DNA-PKc in promoting resistance to taxanes in mCRPC. Targeting prostatic DNA-PKc may provide a novel strategy to restore taxane sensitivity in taxane-refractory mCRPC., (© 2021 Wiley Periodicals LLC.)

Androgen receptor signaling is crucial for prostate cancer growth and is positively regulated in part by intratumoral CYP3A5. As African American (AA) men often carry the wild type CYP3A5 and express high levels of CYP3A5 protein, we blocked the wild type CYP3A5 in AA origin prostate cancer cells and tested its effect on androgen receptor signaling. q-PCR based profiler assay identified several AR regulated genes known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 processes several commonly prescribed drugs and many of these are CYP3A5 inducers or inhibitors. In this study, we test the effect of these commonly prescribed CYP3A5 inducers/inhibitors on AR signaling. The results show that the CYP3A5 inducers promoted AR nuclear translocation, downstream signaling, and cell growth, whereas CYP3A5 inhibitors abrogated them. The observed changes in AR activity is specific to alterations in CYP3A5 activity as the effects are reduced in the CYP3A5 knockout background. Both the inducers tested demonstrated increased cell growth of prostate cancer cells, whereas the inhibitors showed reduced cell growth. Further, characterization and utilization of the observation that CYP3A5 inducers and inhibitors alter AR signaling may provide guidance to physicians prescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT, particularly AA.

Background Aurora kinase overexpression or amplifications are associated with high proliferation, poor prognosis, and therapeutic resistance in human tumors. AMG 900 is an investigational, oral, selective pan-Aurora kinase inhibitor. Methods This first-in-human trial included dose-escalation and dose-expansion phases ( ClinicalTrials.gov : NCT00858377). Dose escalation evaluated the safety, tolerability, and pharmacokinetics of AMG 900 in advanced solid tumors and determined the maximum tolerated dose (MTD) with/without granulocyte colony-stimulating factor (G-CSF) prophylaxis. Dose expansion evaluated clinical activity in three tumor types: taxane- and platinum-resistant ovarian cancer, taxane-resistant triple-negative breast cancer (TNBC), and castration-resistant and taxane- or cisplatin/etoposide-resistant prostate cancer (CRPC). AMG 900 was administered 4 days on/10 days off at 1-50 mg/day during escalation and at the MTD with G-CSF during expansion. Results AMG 900 showed rapid absorption with fast clearance, supporting once-daily dosing. The MTD was 25 mg/day, increasing to 40 mg/day with G-CSF. Grade ≥ 3 treatment-related adverse events included neutropenia (37%), anemia (23%), leukopenia (14%), and thrombocytopenia (12%). During dose expansion, 3/29 (10.3%, 95% CI: 2.0%-28.0%) evaluable patients with ovarian cancer experienced partial response by central imaging per RECIST 1.1; median duration of response was 24.1 weeks (95% CI: 16.1-34.1). Seven patients (24.1%, 95% CI: 10.3%-43.5%) experienced partial response per Gynecologic Cancer InterGroup criteria; 5/9 patients positive for p53 expression responded to treatment. No objective responses were observed in patients with TNBC or CRPC per RECIST 1.1. Conclusions AMG 900 40 mg/day with G-CSF had manageable toxicity and demonstrated single-agent activity in patients with heavily pretreated, chemotherapy-resistant ovarian cancer.

Background: Neoplastic cells proliferate rapidly and obtain requisite building blocks by reprogramming metabolic pathways that favor growth. Previously, we observed that prostate cancer cells uptake and store lipids in the form of lipid droplets, providing building blocks for membrane synthesis, to facilitate proliferation and growth. Mechanisms of lipid uptake, lipid droplet dynamics and their contribution to cancer growth have yet to be defined. This work is focused on elucidating the prostate cancer-specific modifications in lipid storage pathways so that these modified gene products can be identified and therapeutically targeted., Methods: To identify genes that promote lipid droplet formation and storage, the expression profiles of candidate genes were assessed and compared between peripheral blood mononuclear cells and prostate cancer cells. Subsequently, differentially expressed genes were inhibited and growth assays performed to elucidate their role in the growth of the cancer cells. Cell cycle, apoptosis and autophagy assays were performed to ascertain the mechanism of growth inhibition., Results: Our results indicate that DGAT1, ABHD5, ACAT1 and ATGL are overexpressed in prostate cancer cells compared to PBMCs and of these overexpressed genes, DGAT1 and ABHD5 aid in the growth of the prostate cancer cells. Blocking the expression of both DGAT1 and ABHD5 results in inhibition of growth, cell cycle block and cell death. DGAT1 siRNA treatment inhibits lipid droplet formation and leads to autophagy where as ABHD5 siRNA treatment promotes accumulation of lipid droplets and leads to apoptosis. Both the siRNA treatments reduce AMPK phosphorylation, a key regulator of lipid metabolism. While DGAT1 siRNA reduces phosphorylation of ACC, the rate limiting enzyme in de novo fat synthesis and triggers phosphorylation of raptor and ULK-1 inducing autophagy and cell death, ABHD5 siRNA decreases P70S6 phosphorylation, leading to PARP cleavage, apoptosis and cell death. Interestingly, DGAT-1 is involved in the synthesis of triacylglycerol where as ABHD5 is a hydrolase and participates in the fatty acid oxidation process, yet inhibition of both enzymes similarly promotes prostate cancer cell death., Conclusion: Inhibition of either DGAT1 or ABHD5 leads to prostate cancer cell death. Both DGAT1 and ABHD5 can be selectively targeted to block prostate cancer cell growth.

Background: The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5., Methods: To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation., Results: CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization., Conclusion: As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy., (© 2015 Wiley Periodicals, Inc.)

Unlabelled: Tumors with BRCA germline mutations are defective in repairing DNA double-strand breaks (DSB) through homologous recombination (HR) pathways, making them sensitive to PARP inhibitors (PARPi). However, BRCA germline mutations are rare in prostate cancer limiting the ability to therapeutically target these pathways. This study investigates whether histone deacetylase (HDAC) inhibitors (HDACi), reported to modulate DSB repair pathways in sporadic cancers, can downregulate DSB repair pathways and sensitize prostate cancer cells to PARPi. Prostate cancer cells cotreated with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) and the PARPi, olaparib, demonstrated a synergistic decrease in cell viability compared with single-agent treatment (combination index < 0.9), whereas normal prostatic cells did not. Similarly, clonogenicity was significantly decreased after cotreatment. Flow cytometric cell-cycle analysis and Annexin-V staining revealed significant apoptosis upon treatment with SAHA+olaparib. This coincided with increased DNA damage observed by immunofluorescence microscopy analysis of γH2AX foci, a marker of DSBs. In addition, immunoblot analysis showed a significant and persistent increase in nuclear γH2AX levels. Both SAHA and olaparib downregulated the expression of HR-related proteins, BRCA1 and RAD51, whereas SAHA + olaparib had an additive effect on RAD51. Silencing RAD51 sensitized prostate cancer cells to SAHA and olaparib alone. Collectively, cotreatment with HDACi and PARPi downregulated HR-related protein expression and concomitantly increased DNA damage, resulting in prostate cancer cell death., Implications: These findings provide a strong rationale for supporting the use of combined HDAC and PARP inhibition in treating advanced prostate cancer., (©2014 American Association for Cancer Research.)

Background: Visceral disease, non-nodal soft-tissue metastases predominantly involving the lung and liver, is a negative prognostic factor in patients with metastatic castration-resistant prostate cancer (mCRPC). An exploratory analysis of COU-AA-301 assessed whether abiraterone acetate (AA) improved overall survival (OS) in mCRPC patients with visceral disease progressing post docetaxel., Methods: In COU-AA-301, post-docetaxel mCRPC patients were randomized 2:1 to AA 1000 mg (n=797) or placebo (n=398) once daily, each with prednisone 5 mg b.i.d. The primary end point was OS; secondary end points included radiographic progression-free survival (rPFS), PSA response rate and objective response rate (ORR). Treatment effects in visceral disease (n=352) and non-visceral disease (n=843) subsets were examined using final data (775 OS events)., Results: AA plus prednisone produced similar absolute improvement in median OS in patients with (4.6 months) and without (4.8 months) visceral disease versus prednisone; hazard ratios (HRs) were 0.79 (95% confidence interval (CI): 0.60-1.05; P=0.102) and 0.69 (95% CI: 0.58-0.83; P<0.0001), respectively. Treatment with AA plus prednisone significantly and comparably improved secondary endpoint outcomes versus prednisone in both the subsets: the HRs for rPFS were 0.60 (95% CI: 0.46-0.78; P=0.0002) and 0.68 (95% CI: 0.58-0.80; P<0.0001) in visceral and non-visceral disease subsets, respectively. PSA response rates were 28% versus 7% in the visceral disease subsets and 30% versus 5% in the non-visceral disease subsets (both P<0.0001), and ORRs were 11% versus 0% (P=0.0058) and 19% versus 5% (P=0.0010), respectively. The incidence of grade 3/4 adverse events was similar between the subsets and between the treatment arms in each subset. Adverse events related to CYP17 blockade were increased in the AA arms and were similar in patients with or without visceral disease., Conclusions: AA plus prednisone provides significant clinical benefit, including improvements in OS and secondary end points, in post-docetaxel mCRPC patients with or without baseline visceral disease. The presence of visceral disease does not preclude clinical benefit from abiraterone.

Urothelial carcinoma of the bladder with choriocarcinomatous features is a rare presentation among genitourinary cancers. In this study, the case of a 42-year-old woman who presented with menstrual irregularity and positive urine and serum beta-human chorionic gonadotropin tests is presented. Pelvic ultrasound showed no intrauterine pregnancy. Laparoscopy and hysteroscopy with dilatation and curettage were negative for evidence of trophoblastic tissue. Computed tomography of the abdomen and pelvis showed an intravesical fundal mass, with no evidence of extravesical disease. Cystoscopy and transurethral resection of the bladder tumor diagnosed an invasive high-grade urothelial carcinoma with trophoblastic differentiation and multiple foci of choriocarcinomatous morphology. The patient received 3 cycles of neoadjuvant chemotherapy with methotrexate, vinblastine, adriamycin and cisplatin and then underwent partial cystectomy, which was negative for any residual tumor. This is the first reported case of a positive urine pregnancy test leading to the diagnosis of urothelial carcinoma.

Background: Neutral endopeptidase (NEP) is a transmembrane cell surface peptidase present on prostatic epithelial cells that catalytically inactivates small peptide substrates. Neutral endopeptidase loss is associated with prostate cancer growth, progression, and increased angiogenesis. We examined whether NEP expression is regulated by hypoxia, frequently encountered in the tumor microenvironment., Methods: NEP expression was compared in prostate cancer cell lines cultured in normoxic and hypoxic conditions. The NEP activity, protein levels, and mRNA levels were determined using enzyme assay, Western blotting and q-PCR analysis, respectively. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay (ChIP) was used to confirm the negative regulation of NEP at the transcriptional level by hypoxia responsive elements (HREs)., Results: The results indicate that NEP expression was inhibited under hypoxic conditions in the NEP positive LNCaP, C4-2, and 22RV1 cells and human umbilical vascular endothelial (HUVEC) cells. NEP regulation appeared to be predominantly at the transcriptional level as NEP mRNA expression significantly reduced with hypoxia, concordant with the kinetics of protein levels, and NEP enzyme activity. A search of the NEP gene sequence revealed three putative HREs upstream of the NEP promoter. Two of the HREs demonstrated a specific reduction of shift in the presence of cobalt chloride; specificity of the binding sites was confirmed by ChIP., Conclusions: Our data indicate a novel mechanism where hypoxia negatively regulates the tumor suppressor function of NEP in prostate cancer. The negative regulation of NEP is mediated by binding of the HIF1-α protein binding to the HREs present upstream of the NEP promoter., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Abiraterone acetate improved overall survival in metastatic castration-resistant prostate cancer at a preplanned interim analysis of the COU-AA-301 double-blind, placebo-controlled phase 3 study. Here, we present the final analysis of the study before crossover from placebo to abiraterone acetate (after 775 of the prespecified 797 death events)., Methods: Between May 8, 2008, and July 28, 2009, this study enrolled 1195 patients at 147 sites in 13 countries. Patients were eligible if they had metastatic castration-resistant prostate cancer progressing after docetaxel. Patients were stratified according to baseline Eastern Cooperative Oncology Group (ECOG) performance status, worst pain over the past 24 h on the Brief Pain Inventory-Short Form, number of previous chemotherapy regimens, and type of progression. Patients were randomly assigned (ratio 2:1) to receive either abiraterone acetate (1000 mg, once daily and orally) plus prednisone (5 mg, orally twice daily) or placebo plus prednisone with a permuted block method via an interactive web response system. The primary endpoint was overall survival, analysed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00091442., Findings: Of the 1195 eligible patients, 797 were randomly assigned to receive abiraterone acetate plus prednisone (abiraterone group) and 398 to receive placebo plus prednisone (placebo group). At median follow-up of 20·2 months (IQR 18·4-22·1), median overall survival for the abiraterone group was longer than in the placebo group (15·8 months [95% CI 14·8-17·0] vs 11·2 months [10·4-13·1]; hazard ratio [HR] 0·74, 95% CI 0·64-0·86; p<0·0001). Median time to PSA progression (8·5 months, 95% CI 8·3-11·1, in the abiraterone group vs 6·6 months, 5·6-8·3, in the placebo group; HR 0·63, 0·52-0·78; p<0·0001), median radiologic progression-free survival (5·6 months, 5·6-6·5, vs 3·6 months, 2·9-5·5; HR 0·66, 0·58-0·76; p<0·0001), and proportion of patients who had a PSA response (235 [29·5%] of 797 patients vs 22 [5·5%] of 398; p<0·0001) were all improved in the abiraterone group compared with the placebo group. The most common grade 3-4 adverse events were fatigue (72 [9%] of 791 patients in the abiraterone group vs 41 [10%] of 394 in the placebo group), anaemia (62 [8%] vs 32 [8%]), back pain (56 [7%] vs 40 [10%]), and bone pain (51 [6%] vs 31 [8%])., Interpretation: This final analysis confirms that abiraterone acetate significantly prolongs overall survival in patients with metastatic castration-resistant prostate cancer who have progressed after docetaxel treatment. No new safety signals were identified with increased follow-up., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The breast cancer susceptibility genes 1 (BRCA1) and 2 (BRCA2) are cellular proteins involved in DNA repair. They are normally expressed in the breast, ovaries, prostate, and other tissues. Their germline mutation is the cause of hereditary breast-ovarian cancer syndromes. BRCA mutation carriers are also susceptible to other cancers, notably prostate cancer. In this article, we review the role of BRCA genes in the pathogenesis and clinical course of prostate cancer. We also discuss the molecular mechanisms of action and the therapeutic implications of BRCA germline mutations.

Our aim was to determine the utility of circulating micro RNA miR-141 as a potential biomarker of therapeutic response in prostate cancer (CaP) patients. We compared the values of miR-141 in plasma of 21 CaP patients to the levels of prostate specific antigen (PSA), circulating tumor cells (CTC) and lactate dehydrogenase (LDH). Data suggest a strong correlation of miR-141 values and clinical course. For prostate cancer (CaP), the measurement of prostate-specific antigen (PSA) and radiographic studies do not adequately predict response to therapy and survival, and, therefore, new relevant biomarkers are needed. We and other researchers have shown that longitudinal measurements of PSA, circulating tumor cells (CTC), and lactate dehydrogenase (LDH) may aid in predicting response to therapy. Results of recent studies have determined that circulating microRNA (miRNA) miR-141 is detected in plasma of patients with CaP. We, therefore, compared the temporal changes of miR-141 with the levels of CTC, LDH, and PSA in 21 patients with CaP, and longitudinally examined these markers alone or in combinations to determine the utility of miR-141 in the predicting a patient's clinical course and response to therapy. Levels of miR-141 in plasma of 21 patients with CaP were measured by using quantitative reverse transcription-polymerase chain reaction. A total of 35 intervals were assessed. Directional changes (increasing or decreasing) in PSA, CTC, and miR-141 had sensitivity in predicting clinical outcome (progression vs. nonprogressing) of 78.9%. Logistic regression modeling of the probability of clinical progression demonstrates that miR-141 levels predicted clinical outcomes with an odds ratio of at least 8.3. miR-141 also had the highest correlation with temporal changes of PSA with a correlation of R = 0.77 (P < .001). In this retrospective study, miR-141 demonstrated a similar ability to predict clinical progression when compared with other clinically validated biomarkers. Furthermore, miR-141 demonstrated high correlation with changes of the other biomarkers., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: Little information exists regarding the utility circulating tumor cell (CTC) enumeration in hormone sensitive prostate cancer. We enumerated CTC in 33 consecutive patients undergoing androgren deprivation therapy (ADT) at our institution. Multivariate analysis revealed baseline CTC as the only independent predictor of progression to CRPC. These data suggest that baseline CTC may identify those unlikely to benefit from ADT., Introduction: Circulating tumor cell (CTC) enumeration by using the Cellsearch platform has established prognostic and predictive value in patients with metastatic castration-resistant prostate cancer (mCRPC). Limited information exists regarding the clinical utility of CTC enumeration in metastatic hormone-sensitive prostate cancer (mHSPC). The goal of this study was to prospectively determine the relative clinical utility of CTCs in mHSPC., Patients and Methods: We analyzed serial CTC in conjunction with other classic biomarkers in 33 consecutive patients treated at the Nevada Cancer Institute with HSPC initiating androgen deprivation therapy and correlated these patients with prognostic prostate-specific antigen (PSA) endpoints and onset of CRPC., Results: Initial CTC correlated positively with lactate dehydrogenase and alkaline phosphatase, and were unrelated to PSA and testosterone. In univariate analysis, baseline CTC, alkaline phosphatase, lactate dehydrogenase, testosterone, and follow-up CTC were individual predictors of progression to CRPC. In a multivariate Cox regression, only baseline CTC retained independent predictive value. Threshold analysis revealed the cutpoint that optimized specificity and sensitivity of the test to be 3 cells per 7.5 mL whole blood. Baseline CTC also correlated well with PSA nadir benchmarks., Conclusions: Initial CTC values predict the duration and magnitude of response to hormonal therapy. CTC enumeration may identify patients at risk of progression to CRPC before initiation of androgen deprivation therapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Biosynthesis of extragonadal androgen may contribute to the progression of castration-resistant prostate cancer. We evaluated whether abiraterone acetate, an inhibitor of androgen biosynthesis, prolongs overall survival among patients with metastatic castration-resistant prostate cancer who have received chemotherapy., Methods: We randomly assigned, in a 2:1 ratio, 1195 patients who had previously received docetaxel to receive 5 mg of prednisone twice daily with either 1000 mg of abiraterone acetate (797 patients) or placebo (398 patients). The primary end point was overall survival. The secondary end points included time to prostate-specific antigen (PSA) progression (elevation in the PSA level according to prespecified criteria), progression-free survival according to radiologic findings based on prespecified criteria, and the PSA response rate., Results: After a median follow-up of 12.8 months, overall survival was longer in the abiraterone acetate-prednisone group than in the placebo-prednisone group (14.8 months vs. 10.9 months; hazard ratio, 0.65; 95% confidence interval, 0.54 to 0.77; P<0.001). Data were unblinded at the interim analysis, since these results exceeded the preplanned criteria for study termination. All secondary end points, including time to PSA progression (10.2 vs. 6.6 months; P<0.001), progression-free survival (5.6 months vs. 3.6 months; P<0.001), and PSA response rate (29% vs. 6%, P<0.001), favored the treatment group. Mineralocorticoid-related adverse events, including fluid retention, hypertension, and hypokalemia, were more frequently reported in the abiraterone acetate-prednisone group than in the placebo-prednisone group., Conclusions: The inhibition of androgen biosynthesis by abiraterone acetate prolonged overall survival among patients with metastatic castration-resistant prostate cancer who previously received chemotherapy. (Funded by Cougar Biotechnology; COU-AA-301 ClinicalTrials.gov number, NCT00638690.).

Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostate epithelial cells and lost in ~50% of primary prostate cancers. NEP directly associates with multiple proteins at the cell surface including Ezrin/Radixin/Moesin (ERM) proteins and the PTEN tumor suppressor protein. Analysis of the N-terminal sequence of the NEP cytosolic domain (N-terminal MGKSESQMDI TDINTPKPKK KQRWTR) identified a myristoylation consensus site. Mutation of Gly-2 to Arg significantly decreased (3)H-myristoylation activity, and correlated with translocation of NEP from the plasma membrane to a perinuclear domain as demonstrated by immunofluorescence staining and Western blotting with an NEP-specific antibody. Removal of this myristoylation residue did not affect NEP enzymatic specific activity. Myristoylated NEP recruited more PTEN protein to the cell membrane fraction than unmyristoylated NEP. These data demonstrate that NEP is myristoylated at Gly-2 and that this modification is an intrinsic signal for membrane targeting.

Purpose: Circulating tumor cells (CTC) have been recently accepted by the Food and Drug Administration of the United States as a prognostic tool in advanced prostate cancer. However, a number of questions remain about the use of the test. The optimal clinical cut-off has never been determined. Also, the predictive value of CTCs in the setting of low-burden advanced prostate cancer has not been evaluated. Herein we describe our experience with the CellSearch method of CTC enumeration., Experimental Design: CTCs enumerated from 100 patients with castration-resistant prostate cancer were correlated with clinicopathologic characteristics and conventional biomarkers, such as prostate-specific antigen and lactate dehydrogenase. Patients received ongoing medical oncologic follow-up for up to 26 months, and overall survival status was documented., Results: Forty-nine of the patients (49%) were alive at the end of the study. CTC counts correlate well with overall survival (P < 0.001) but are also tightly interrelated to other biomarkers. Threshold analysis identified 4 CTC/7.5 cc (compared with the approved value of 5) as an optimal cut-off value with respect to correlation with survival outcomes as well as predictive of metastatic disease. Univariate analysis confirmed a tight interrelationship between cut-off CTC values and biomarkers. Multivariate analysis with bootstrap sampling validation identified lactate dehydrogenase (P = 0.002) and CTCs (P = 0.001) as independently prognostically significant., Conclusions: Baseline CTC values provide important prognostic information and specific prediction of metastatic disease. Their presence correlates with classic biomarkers.

Neutral endopeptidase (NEP) is a cell surface peptidase that catalytically inactivates a variety of physiologically active peptides including basic fibroblast growth factor (FGF-2). We investigated the effect of using lentivirus to overexpress NEP in NEP-deficient DU145 prostate cancer cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP), catalytically inactive mutant NEP (L-NEPmu), and green fluorescent protein (L-GFP) were stably introduced into DU145 cells. FGF-2 levels in cell culture supernatants decreased by 80% in L-NEP-infected DU145 cells compared to cells infected with L-NEPmu or L-GFP (P<0.05) while levels of other angiogenic factors were not altered. In vitro tubulogenesis of human vascular endothelial cells induced by conditioned media from DU145 cells infected with L-NEP was significantly reduced compared with that from DU145 cells infected with L-GFP (P<0.05). Tumor xenografts from L-NEP-infected DU145 cells were significantly smaller compared to control cell xenografts and vascularity within these tumors was decreased (P<0.05). Our data suggest that stable expression of NEP in DU145 cells inhibits prostate cancer tumorigenicity by inhibiting angiogenesis, with a probable mechanism being proteolytic inactivation of FGF-2.

Prostate-specific membrane antigen (PSMA) is an integral membrane glycoprotein expressed in prostatic epithelia and is being evaluated as a therapeutic target in prostate cancer. It undergoes constitutive receptor-mediated endocytosis via clathrin-coated pits, which is enhanced in the presence of monoclonal antibodies directed against it. We describe distinct interactions of PSMA with clathrin and the clathrin adaptor protein-2 (AP-2) complex, two components of clathrin-coated pits. The intracellular N-terminal domain of PSMA interacts with the N-terminal globular domain of clathrin heavy chain. Deletion analysis revealed an important determinant of this interaction residing within the proximal portion of the clathrin heavy chain N-terminal domain (amino acids 1-85) distinct from the clathrin binding sites of other known clathrin-binding proteins. Furthermore, PSMA interacts with the ear domain of alpha-adaptin (an AP-2 subunit), and a glutamic acid residue at position 7 in the cytoplasmic tail of PSMA is essential for this interaction. These data indicate that PSMA exhibits a high affinity, specific association with the clathrin-based endocytic machinery by distinct interactions with both clathrin and AP-2. Thus, although PSMA is a new member of the dual AP and clathrin binding proteins, its alpha-adaptin and clathrin heavy chain binding determinants are distinct from those of other members.

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.

Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.01) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis.

The neutral endopeptidase (NEP) gene is transcriptionally regulated by androgen in prostate cancer cells. We previously identified in the NEP gene an androgen responsive element (NEP-ARE) and an androgen responsive region (NEP-ARR) that together conveyed only moderate androgen-inducibility [Mol. Cell. Endocrinol. 170 (2000) 131]. Therefore, we characterized the entire genomic structure of the NEP gene and identified ARE1 (ACTCAACAttgTGTCCTTT) and ARE2 (CAGGACAtttTGTCCC), which are located in the 3'-untranslated region and in intron 17, respectively. Steroid-dependent enhancement of transcription was assayed by transfecting the pGL-3-luciferase reporter plasmid containing three copies of ARE1 or ARE2 into PC-3 cells. Luciferase activities were increased 3.6-fold (ARE1) and 5-fold (ARE2) by androgen (AR), 4.2-fold (ARE1) and 8.2-fold (ARE2) by dexamethasone, and 3-fold (ARE1) and 4.1-fold (ARE2) by progesterone. Mutation of the ARE1 and ARE2 sequences completely abrogated androgen-inducibility. We next showed that both ARE1 and ARE2 are involved in the transcriptional regulation of the NEP gene, demonstrating in vitro and in vivo binding with AR as determined by electrophoretic mobility gel shift and chromatin immunoprecipitation (ChIP) assays, Furthermore, ARE1 and ARE2 mediate coordinated androgen-inducibility in both an SV40 promoter and the native NEP type 1 promoter. These data indicate the newly identified ARE1 and ARE2 together with the previously identified NEP-ARE function as androgen response elements, and that androgen regulation of the NEP gene is regulated by the coordinated action of multiple AREs in prostate cancer cells.

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

We have recently demonstrated that the nonvisual arrestins, beta-arrestin and arrestin3, interact with high affinity and stoichiometrically with clathrin, and we postulated that this interaction mediates internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this study, we localized the clathrin binding domain of arrestin3 using a variety of approaches. Truncation mutagenesis demonstrated that the COOH-terminal half of arrestin3 is required for clathrin interaction. Assessment of the clathrin binding properties of various glutathione S-transferase-arrestin3 fusion proteins indicated that the predominant clathrin binding domain is contained within residues 367-385. Alanine scanning mutagenesis further localized this domain to residues 371-379, and site-directed mutagenesis demonstrated the critical importance of both hydrophobic (Leu-373, Ile-374, and Phe-376) and acidic (Glu-375 and Glu-377) residues in the arrestin3/clathrin interaction. These results are complementary to the observation that hydrophobic and basic residues in clathrin are critical for its interaction with nonvisual arrestins (Goodman, O. B. , Jr., Krupnick, J. G., Gurevich, V. V., Benovic, J. L., and Keen, J. H. (1997) J. Biol. Chem. 272, 15017-15022). Lastly, an arrestin3 mutant in which Leu-373, Ile-374, and Phe-376 were mutated to Ala was significantly defective in its ability to promote beta2-adrenergic receptor internalization in COS-1 cells when compared with wild-type arrestin3. Taken together, these results implicate a discrete region of arrestin3 in high affinity binding to clathrin, an interaction critical for agonist-promoted internalization of the beta2-adrenergic receptor.

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

We have utilized a rabbit reticulocyte lysate coupled transcription-translation system to express the large subunits of the clathrin associated protein-2 (AP-2) complex so that their individual functions may be studied separately. Appropriate folding of each subunit into N-terminal core and C-terminal appendage domains was confirmed by limited proteolysis. Translated beta 2 subunit bound to both assembled clathrin cages and immobilized clathrin trimers, confirming and extending earlier studies with preparations obtained by chemical denaturation-renaturation. Translated alpha a exhibited rapid, reversible and specific binding to clathrin cages. As with native AP-2, proteolysis of alpha a bound to clathrin cages released the appendages, while cores were retained. Further digestion revealed a approximately 29-kDa alpha a clathrin-binding fragment that remained tightly cage-associated. Translated alpha a also bound to immobilized clathrin trimers, although with greater sensitivity to increasing pH than the translated beta 2 subunit. Clathrin binding by both the alpha and beta subunits is consistent with a bivalent cross-linking model for lattice assembly (Keen, J. H. (1987) Cell Biol. 105, 1989). It also raises the possibility that the alpha-clathrin interaction may have other consequences, such as modulation of lattice stability or shape, or other alpha functions.