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During progression, luminal muscle-invasive bladder cancer (MIBC) can transition to the aggressive basal-squamous (Ba/Sq) subtype. Reduced expression of forkhead box A1 (FOXA1) in the urothelium is a hallmark and driver of the Ba/Sq transcriptional state and squamous differentiation. Ba/Sq tumors are highly inflamed; however, the specific inflammatory pathways contributing to the Ba/Sq state are unknown. In this study, transcriptomic analyses of The Cancer Genome Atlas MIBC cohort were performed to determine whether immune response gene signatures were associated with MIBC molecular states. Results showed that Ba/Sq MIBCs were enriched for the interferon-γ (IFN-γ)-dominant signature. Ba/Sq MIBCs exhibited increased IFN-γ/Janus kinase (JAK)/STAT pathway activity, corresponding to reduced FOXA1 regulon activity. Immunohistochemistry of MIBC specimens demonstrated that JAK1 expression was significantly increased in tumor areas with squamous differentiation. IFN-γ treatment of luminal MIBC cell lines significantly decreased the expression of luminal transcriptional drivers, including FOXA1, and increased the expression of Ba/Sq markers in a STAT1-dependent manner. RNA-sequencing analyses identified IFN-γ as a driver of the Ba/Sq state. The ability of IFN-γ to repress FOXA1 in luminal cells was abrogated by ruxolitinib inhibition of JAK1/2 activity. Additionally, pharmacologic inhibition or genetic ablation of JAK1 restored FOXA1 expression in Ba/Sq MIBC cells. These findings are the first to identify IFN-γ as an epithelial cell-extrinsic mechanism to repress FOXA1 and drive the Ba/Sq state in MIBC., Competing Interests: Disclosure Statement D.J.D. received a research grant from Bristol Myers Squibb; and is currently employed at Johnson & Johnson. M.J. is on an advisory board at Seattle Genetics, Gilead; received travel/accommodation from Dava Oncology, Caris; teaches at Curio; is a grant mentor for Bristol Myers Squibb Foundation; and received a research grant from AstraZeneca., (Copyright © 2025. Published by Elsevier Inc.)

Upper tract urothelial carcinoma (UTUC) is a relatively rare yet aggressive malignancy. While radical nephroureterectomy (RNU) remains the cornerstone treatment, UTUC has high local and metastatic relapse rates, leading to a dismal prognosis. To identify the clinicopathological factors associated with an increased risk of local and metastatic relapse in UTUC, we conducted a retrospective analysis of 133 consecutive UTUC patients who underwent RNU from 1998 to 2018. Patients lost to follow-up or with a history of bladder cancer were excluded from the study. The remaining 87 patients were categorized into two subgroups: those with tumor recurrence/relapse (40 cases) and those without recurrence/relapse (47 cases). Clinical and pathological characteristics were compared across the two groups. Multiple factors are associated with UTUC recurrence/relapse including larger tumor size, histology divergent differentiations/subtypes, high tumor grade, advanced pathologic T stage, positive margin, lymphovascular invasion (LVI), positive lymph node status, and preoperative hydronephrosis. Multivariate Cox regression analysis revealed that squamous differentiation predicted recurrence/relapse (p = 0.012), independent of tumor stage. Moreover, compared to the conventional histology type, UTUC with squamous differentiation had a significantly higher relapse rate (p = 0.0001) and poorer survival (p = 0.0039). This observation was further validated in invasive high-grade UTUC cases. Our findings suggest that many pathological factors contribute to UTUC recurrence/relapse, particularly, squamous differentiation may serve as an independent risk predictor for relapse and a potent prognosticator for adverse cancer-specific survival in UTUC patients. Recognizing and thoroughly assessing the pathological factors is essential for better oncologic management of UTUC., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sex-determining region Y-box 2 (SOX2) is a transcription factor with a central role in embryologic development. SOX2 is also an oncogene in several cancer types. Prior work by our group has shown SOX2 activity associates with cell cycle dysregulation in early-stage bladder cancer. The present study was thus undertaken to broadly investigate SOX2 in bladder cancer, with emphasis on associations with tumor stage, clinical outcomes, and tumorigenicity. Gene expression was quantified by immunohistochemistry in an established tissue microarray (n=303 cystectomy specimens, all stages) and whole tissue sections of noninvasive papillary urothelial carcinoma (n=25). Gene expression by RNA sequencing was evaluated in non-muscle invasive and muscle-invasive cohorts from publicly available repositories. By immunohistochemistry, SOX2 was expressed in 40% of whole tissue sections of noninvasive papillary carcinoma, which correlated with SOX2 expression by RNA sequencing (r=0.6, P=0.001, Spearman correlation). Expression tended to be focal (median H-score =6). SOX2 was expressed in only 9% of TMA cases, consistent with focal expression. SOX2 expression was substantially higher in muscle-invasive compared with noninvasive papillary urothelial carcinoma by RNA sequencing (P<0.001, Wilcoxon rank sum test). SOX2 expression associated with stage progression in lamina-propria invasive cancers (hazard ratio =2, P=0.05, Cox model, binary, RNA sequencing) but not noninvasive papillary cancers (P=0.5, Cox model, binary, RNA sequencing). SOX2 expression did not associate with overall survival in muscle-invasive carcinoma. Activity of SOX2 in bladder cancer was tested in vivo using murine allografts created with MB49 cells that express human SOX2 (MB49-SOX). MB49-SOX allografts expressed this protein focally by immunohistochemistry, much like human tumors. Compared with controls, MB49 allografts demonstrated larger tumor size (P=0.03, Wilcoxon rank sum test) and higher tumor burden in mesenteric metastases (P=0.009, Wilcoxon rank sum test). Though SOX2 expression is focal within tumors, it may drive tumorigenesis, increase growth rate, and promote aggressive features of bladder cancer, particularly stage progression of early-stage disease., Competing Interests: None., (AJCEU Copyright © 2024.)

Competing Interests: Scott Delacroix, Jared Foster, Francesca Khani, Joseph Liao, Abdul Tawab-Amiri Pamela West, Alex Wyatt: No conflicts. David McConkey: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Grants and contracts – Department of Defense, Mark Foundation, Commonwealth Foundation. Brian Baumann: Consulting fees – Boston Scientific, Varian, Novartis, Blue Earth; leadership or fiduciary role – Board of Directors, National Association for Proton Therapy. Stephanie Cooper Greenberg: Patient advocate, Johns Hopkins Greenberg Bladder Cancer Institute and NCI’s Bladder Cancer Task Force. David DeGraff: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review Grants and contracts – Bristol Myers Squibb, Department of Defense; receipt of material – Astra-Zeneca. Jason Efstathiou: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review Consulting fees – Blue Earth Diagnostics, Boston Scientific, AstraZeneca, Genentech, Astellas/Pfizer, Lantheus; payment or honoraria – IBA, Elekta, UpToDate; advisory boards – Merck, Roivant Pharma, Myovant Sciences, Janssen, Bayer Healthcare, Progenics Pharmaceuticals, Pfizer, Astellas, Gilead, Lantheus, Blue Earth Diagnostics, Angiodynamics; committee leadership – Vice-Chair, GU Committee, NRG; Board member (unpaid) – Massachusetts Prostate Cancer Coalition (MPCC) American College of Radiation Oncology (ACRO), Radiation Oncology Institute (ROI). Susan Groshen: Financial support from NCI/GUSC for attending a 2-day workshop that led to the CTPM. Edward Kadel: Patent, “PD-L1 PROMOTER METHYLATION IN CANCER”; stock or stock options, 89bio, Apple, Amazon, Alphabet, Eli Lilly & Co, Inspire Medical Systems, Merck & Co, Roche, SPDR S&P Biotech ETF, Teladoc; employee of Genentech/Roche. William Kim: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Grants or contracts – NIH R01-CA241810, Merck; consulting fees – Focal Medical (spouse); advisory board – OncoRev; stock or stock options – Abbvie, Amgen, Apellis, BeiGene, BMS, Lilly, ImmunityBio, Moderna, Natera, NovoNordisk, Revolution Medicines, Tango Therapeutics, Zentalis; other financial or non-financial interests – Focal Medical, ownership interest (spouse). Seth Lerner: Co-Editor-in-Chief of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Advisory board – FerGene, Pfizer. BMS, Incyte; consultant – Vaxiion, Verity, Protara, C2i Genomics, Ferring, Aura Bioscience, Surge Therapeutics; clinical trials – Genentech (SWOG), Vaxiion, Endo, FKD, JBL (SWOG), Merck (Alliance), Aura Bioscience, Surge Therapeutics, QED Therapeutics; honoraria – UroToday, co-editor bladder cancer section UpToDate, Grand Rounds Urology; compensated editorial responsibilities – IOS Press. Trevor Levin: Grants or contracts – NCI SBIR 5R44CA200174-05; patents issued or pending – WO/2017/056451, WO/2020/023630, WO/2020/243587, WO/2022/216865A1; leadership or fiduciary role – Convergent Genomics (Executive and Board roles); stock or stock options – Convergent Genomics. Matthew Milowski: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Consulting: Loxo/Lilly; payments or honoraria, Medscape, Research to Practice; other financial or non-financial interests: Co-Editor in Chief, Clinical Genitourinary Cancer, Elsevier. Joshua Meeks: grants or contracts, VA, NIH, Department of Defense; consulting fees, Merck, AstraZeneca, Incyte, Janssen, BMS, UroGen, Prokarium, Imvax, Pfizer, Seagen/Astellas, Ferring; payments, Olympus, AUA, UroToday, OncLive; advisory boards, Astra-Zeneca, Medscape. David Miyamoto: grant support from NIH and the Radiation Oncology Institute. Kent Mouw: consulting fees, EMD Serono, Pfizer, UroGen, Riva Therapeutics. Eugene Pietzak: participation in a Data Safety Monitoring Committee or Advisory Board, QED Pharmaceuticals, Merck, Janssen, Urogen; society leadership, Chair of the Program Committtee, 2024 BCAN Think Tank. David Solit: consulting fees from Pfizer, Fog Pharma, PaigeAI, BridgeBio, Scorpion Therapeutics, FORE Therapeutics, Function Oncology, Pyramid, and Elsie Biotechnologies, Inc.; participation on a Data Safety Monitoring Board for Rain Therapeutics; stock or stock options from Scorpion Therapeutics, FORE Therapeutics, Function Oncology, Pyramid, Elsie Biotechnologies, Inc. Debashish Sundi: grants or contracts from Janssen, consulting fees from Janssen. Sara Wobker: royalties – Wolters Kluwer; honoraria – College of American Pathologists, American Society of Clinical Pathology, USCAP; travel reimbursement – Kidney Cancer Society. Andrea Apolo: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Peter Black: Editorial Board member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review. Consulting fees – AbbVie, AstraZeneca, Astellas, Bayer, BMS, Combat, EMD-Serono, Ferring, Janssen, Merck, Nonagen, Nanobot, NanOlogy, Pfizer, Photocure, Prokarium, Sumitomo, TerSera, Tolmar, Verity; payments – Janssen, TerSera, Bayer, Pfizer; patents issued or pending – Veracyte.

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an in vivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3ca H1047R mouse model expressing one or two R26-Pik3ca H1047R alleles in a urothelium-specific manner was generated. Pik3ca H1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3ca H1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3ca H1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3ca H1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Patients with bladder cancer (BC) who are cisplatin ineligible or have unresectable disease have limited treatment options. Previously, we showed targeting programmed death-ligand 1 (PD-L1) with durvalumab (durva) and radiation therapy (RT) combination was safe in BC. We now report results from a phase II study evaluating the toxicity and efficacy of durva and RT in localized BC., Methods: This is a single-arm, multi-institutional phase II study; N=26. Enrolled patients had pure or mixed urothelial BC (T2-4 N0-2 M0) with unresectable tumors and were unfit for surgery or cisplatin ineligible. Patients received durva concurrently with RT ×7 weeks, followed by adjuvant durva × 1 year., Primary Endpoints: (A) progression-free survival (PFS) at 1 year and (B) disease control rate (DCR) post adjuvant durva. Key secondary endpoints: (A) complete response (CR) post durvaRT (8 weeks), (B) overall survival (OS), (C) PFS and (D) toxicity. Correlative studies included evaluation of baseline tumor and blood (baseline, post durvaRT) for biomarkers., Results: Median follow-up was 27 months. Evaluable patients: 24/26 post durvaRT, 22/26 for DCR post adjuvant durva, all patients for PFS and OS. Post adjuvant durva, DCR was seen in 72.7%, CR of 54.5%. 1-year PFS was 71.5%, median PFS was 21.8 months. 1-year OS was 83.8%, median OS was 30.8 months. CR at 8 weeks post durvaRT was 62.5%. Node positive (N+) patients had similar median PFS and OS. DurvaRT was well tolerated. Grade ≥3 treatment-related adverse events: anemia, high lipase/amylase, immune-nephritis, transaminitis, dyspnea (grade 4-COPD/immune), fatigue, rash, diarrhea and scleritis. No difference in outcome was observed with PD-L1 status of baseline tumor. Patients with CR/PR or SD had an increase in naïve CD4 T cells, a decrease in PD-1+CD4 T cells at baseline and an increase in cytokine-producing CD8 T cells, including interferon gamma (IFNγ) producing cells, in the peripheral blood., Conclusion: Durva with RT followed by adjuvant durva was safe with promising efficacy in localized BC patients with comorbidities, including N+ patients. Larger randomized studies, like S1806 and EA8185, are needed to evaluate the efficacy of combining immunotherapy and RT in BC., Trial Registration Number: NCT02891161., Competing Interests: Competing interests: MJ: Advisory board for Seagen (personal fees). Research funds—AstraZeneca, Pfizer, Eisai (drug only), BMS LT: None. JZ: None. VW: None. TS: None. MK: None. DK: None. JL: None. SH: Research grant—Eli Lilly, BMSHE: None. AS: None. SM: None. HZ: Pfizer, personal fees from BeiGene, personal fees from BMS, outside the submitted work. JW: None. RH: None. BG: None. MS: None. JD: Personal fees from Sanofi outside the submitted work. DJD: Research funds-BMSYZ: Advisory Board: Amgen, Roche Diagnostics, Novartis, Janssen, Eisai, Exelixis, Castle Bioscience, Array, Bayer, Pfizer, Clovis, EMD serono. Grant/research support from: Institution clinical trial support from NewLink Genetics, Pfizer, Exelixis, Eisai. DSMC: Janssen Research and Development. Consultant honorarium: Pfizer, Novartis., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Despite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9. Furthermore, using primary cortical neurons, we observed aberrant BMP signaling and dysregulated expression of neuronal genes upon manipulating the AWS complex, indicating that the AWS-CUL4-BMP axis plays a role in regulating neuronal lineage specification in vivo. Thus, our findings uncover a sophisticated cellular signaling network mobilized by a prominent NDD risk factor, presenting multiple potential therapeutic targets for NDD., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To determine if there are histologic differences relative to tobacco exposure in buccal mucosa. Substitution urethroplasty outcomes may be worse in tobacco users and we investigate if the buccal graft is inherently damaged due to chronic tobacco exposure., Methods: Subjects undergoing substitution urethroplasty with buccal graft harvest were prospectively consented in this IRB approved study. Subjects with poor dentition were excluded. A detailed tobacco use history was obtained. Cotinine testing was performed day of surgery to confirm or exclude active tobacco use. Trimmed portions of harvested graft were sent for tissue processing. Standard hematoxylin and eosin staining was performed. A single blinded pathologist performed analysis of the slides. Using a scale of none, mild, moderate, or severe slides were analyzed for cytologic atypia, architectural complexity, inflammation, and keratinization. Evidence of vascular damage was noted and the type of inflammation if present was classified., Results: Twenty-five buccal grafts were analyzed. No evidence of vascular damage or cytologic atypia were noted in any grafts. While mild architectural complexity and mild inflammation, typically lymphocytic, were noted in several of the buccal mucosa sections, this did not appear to correlate with tobacco exposure. The majority of grafts demonstrating increased keratinization correlated with significant tobacco exposure, but this was not consistently noted in all those with tobacco use., Conclusions: Buccal mucosa in patients with tobacco exposure did not show significant histologic alterations. Outcomes of substitution urethroplasty may be more impacted by persistent systemic exposure causing local ischemia as opposed to the graft tissue itself., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors., (© 2022. The Author(s).)

Human bladder cancer (BCa) exhibits morphological and molecular heterogeneity which can complicate treatment. Morphologically, more than 90% of BCa is classified as urothelial cell carcinoma (UCC). Among other histological variants, UCC with squamous differentiation (SqD) shows a worse prognosis than pure UCC. In addition, basal-squamous BCa is enriched for SqD, and these tumors have a poor prognosis. Therefore, it is critical to elucidate the mechanisms to drive the basal-squamous phenotype of human BCa. Laminin-332 is a major glycoprotein of the epithelial basement membrane. It is well known that laminin-332 is a favorable target for extracellular matrix proteases such as matrix metalloproteinases (MMPs) in various diseases. Accumulating evidence indicates the significant role of laminin-332 in tumorigenesis. Here, we analyzed the expression of laminin-332 genes ( LAMA3, LAMB3, LAMC2 ) in molecular subtypes of human BCa using publicly available data from The Cancer Genome Atlas (TCGA). Additionally, we also used q-RT-PCR to characterize laminin-332 gene expression between distinct molecular subtypes of human BCa cell lines. Our analysis of publicly available data show that laminin-332 genes are highly expressed in the basal-squamous molecular subtype of human BCa. In addition, we show laminin-332 genes are highly expressed in basal-squamous human BCa cell lines. Moreover, the expression of both LAMA3 and LAMC2 are negatively correlated with expression of the luminal transcription factor (TF) FOXA1 in the TCGA data. We also demonstrate that laminin-332 genes are downregulated by the overexpression of FOXA1 in a human basal-squamous BCa cell line (5637). Taken together, these results suggest that laminin-332 gene expression may be a biomarker of BCa patients with basal-squamous disease., Competing Interests: None., (AJCEU Copyright © 2022.)

Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor's transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma., (© 2022. The Author(s).)

Background: Patients with metastatic urothelial carcinoma (mUC) have poor prognosis, so further development of novel combinations for these patients is needed., Objective: To assess the safety and efficacy of eribulin mesylate (eribulin) with avelumab in mUC., Design, Setting, and Participants: This was an open-label, phase 1b study in which patients with mUC who were cisplatin-ineligible and treatment-naïve or platinum-resistant were treated with eribulin and avelumab. A 3 + 3 design was used. The study was prematurely terminated because the free study drug became unavailable, but we performed extended follow-up for patients enrolled in the study., Intervention: Patients received eribulin 1.1 mg/m 2 plus avelumab 10 mg/kg on days 1 and 15 in every 28-d cycle in cohort 0, or eribulin 1.4 mg/m 2 plus avelumab 10 mg/kg on days 1 and 15 in every 28-d cycle in cohort +1., Outcome Measurements and Statistical Analysis: The primary objectives were to determine the maximum tolerated dose (MTD) of eribulin with avelumab and assess the objective response rate. A key secondary endpoint was to assess efficacy by evaluating the disease control rate. Exploratory endpoints included PD-1 expression on T cells in peripheral blood and in tumor cells, and tumor DNA sequencing., Results and Limitations: A total of six patients were enrolled in the MTD group (n = 3 in cohort 0 and n = 3 in cohort +1). No dose-limiting toxicity (DLT) was observed in cohort 0, whereas two DLT events were observed in cohort +1. Two patients in cohort 0 had a partial response that was durable, with one patient having a durable response for 7.8 mo. Disease control was observed in 4/6 patients (66.7%). Owing to the early termination, MTD could not be determined., Conclusions: While early termination of this trial precludes any definitive conclusions, the combination of eribulin and avelumab shows promise in mUC. We observed that treatment was better tolerated and efficacious at lower doses of eribulin. Further research is warranted for this combination in mUC., Patient Summary: We evaluated different doses of eribulin (a chemotherapy drug) in combination with a fixed dose of avelumab (an antibody used to treat several different cancers) in a small group of patients with metastatic cancer of the urinary tract. The lower dose of eribulin was easier to tolerate and the combination had an anti-cancer effect. This trial is registered at ClinicalTrials.gov as NCT03502681., (Copyright © 2021 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Although cancer is highly heterogeneous, all metastatic cancer is considered American Joint Committee on Cancer (AJCC) Stage IV disease. The purpose of this project was to redefine staging of metastatic cancer. Internal validation of nationally representative patient data from the National Cancer Database (n = 461 357; 2010-2013), and external validation using the Surveillance, Epidemiology and End Results database (n = 106 595; 2014-2015) were assessed using the concordance index for evaluation of survival prediction. A Cox proportional hazards model was used for overall survival by considering identified phenotypes (latent classes) and other confounding variables. Latent class analysis was performed for phenotype identification, where Bayesian information criterion (BIC) and sample-size-adjusted BIC were used to select the optimal number of distinct clusters. Kappa coefficients assessed external cluster validation. Latent class analysis identified five metastatic phenotypes with differences in overall survival (P < .0001): (Stage IVA) nearly exclusive bone-only metastases (n = 59 049, 12.8%; median survival 12.7 months; common in lung, breast and prostate cancers); (IVB) predominant lung metastases (n = 62 491, 13.5%; 11.4 months; common in breast, stomach, kidney, ovary, uterus, thyroid, cervix and soft tissue cancers); (IVC) predominant liver/lung metastases (n = 130 014, 28.2%; 7.0 months; common in colorectum, pancreatic, lung, esophagus and stomach cancers); (IVD) bone/liver/lung metastases predominant over brain (n = 61 004, 13.2%; 5.9 months; common in lung and breast cancers); and (IVE) brain/lung metastases predominant over bone/liver (n = 148 799, 32.3%; 5.7 months; lung cancer and melanoma). Long-term survivors were identified, particularly in Stages IVA-B. A pan-cancer nomogram model to predict survival (STARS: site, tumor, age, race, sex) was created, validated and provides 13% better prognostication than AJCC: 1-month concordance index of 0.67 (95% confidence interval [CI]: 0.66-0.67) vs 0.61 (95% CI: 0.60-0.61). STARS is simple, uses easily accessible variables, better prognosticates survival outcomes and provides a platform to develop novel metastasis-directed clinical trials., (© 2021 UICC.)

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

Deciphering the mechanisms that drive transdifferentiation to neuroendocrine prostate cancer (NEPC) is crucial to identifying novel therapeutic strategies against this lethal and aggressive subtype of advanced prostate cancer (PCa). Further, the role played by exosomal microRNAs (miRs) in mediating signaling mechanisms that propagate the NEPC phenotype remains largely elusive. The unbiased differential miR expression profiling of human PCa cells genetically modulated for TBX2 expression led to the identification of miR-200c-3p. Our findings have unraveled the TBX2/miR-200c-3p/SOX2/N-MYC signaling axis in NEPC transdifferentiation. Mechanistically, we found that: (1) TBX2 binds to the promoter and represses the expression of miR-200c-3p, a miR reported to be lost in castrate resistant prostate cancer (CRPC), and (2) the repression of miR-200c-3p results in the increased expression of its targets SOX2 and N-MYC. In addition, the rescue of mir-200c-3p in the context of TBX2 blockade revealed that miR-200c-3p is the critical intermediary effector in TBX2 regulation of SOX2 and N-MYC. Further, our studies show that in addition to the intracellular mode, TBX2/miR-200c-3p/SOX2/N-MYC signaling can promote NEPC transdifferentiation via exosome-mediated intercellular mechanism, an increasingly recognized and key mode of propagation of the NEPC phenotype.

The molecular landscape in non-muscle-invasive bladder cancer (NMIBC) is characterized by large biological heterogeneity with variable clinical outcomes. Here, we perform an integrative multi-omics analysis of patients diagnosed with NMIBC (n = 834). Transcriptomic analysis identifies four classes (1, 2a, 2b and 3) reflecting tumor biology and disease aggressiveness. Both transcriptome-based subtyping and the level of chromosomal instability provide independent prognostic value beyond established prognostic clinicopathological parameters. High chromosomal instability, p53-pathway disruption and APOBEC-related mutations are significantly associated with transcriptomic class 2a and poor outcome. RNA-derived immune cell infiltration is associated with chromosomally unstable tumors and enriched in class 2b. Spatial proteomics analysis confirms the higher infiltration of class 2b tumors and demonstrates an association between higher immune cell infiltration and lower recurrence rates. Finally, the independent prognostic value of the transcriptomic classes is documented in 1228 validation samples using a single sample classification tool. The classifier provides a framework for biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials.

Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown. RESULT: We determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. CONCLUSION: In summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.

Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15 INK4B , p14 ARF and p16 INK4A ), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15 INK4B is markedly stronger than p16 INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15 INK4B , but not those expressing HRas and lacking p16 INK4A , develop early-onset bladder tumors. The potency of CDKN2B/p15 INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15 INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15 INK4B on cell proliferation, and unveil mechanisms whereby p15 INK4B aberrations may underpin cancer and non-cancer conditions.

Bone metastasis frequently occurs in advanced-stage prostate cancer (PCa) patients. Understanding the mechanisms that promote PCa-mediated bone destruction is important for the identification of therapeutic targets against this lethal disease. We found that forkhead box A2 (FOXA2) is expressed in a subset of PCa bone metastasis specimens. To determine the functional role of FOXA2 in PCa metastasis, we knocked down the expression of FOXA2 in PCa PC3 cells, which can grow in bones and elicit an osteolytic reaction. The PC3/FOXA2-knockdown cells generated fewer bone lesions following intra-tibial injection compared to control cells. Further, we found that FOXA2 knockdown decreased the expression of PTHLH , which encodes PTHrP, a well-established factor that regulates bone remodeling. These results indicate that FOXA2 is involved in PCa bone metastasis., Competing Interests: None., (AJTR Copyright © 2020.)

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

Histological and molecular analyses of urothelial carcinoma often reveal intratumoural and intertumoural heterogeneity at the genomic, transcriptional and cellular levels. Despite the clonal initiation of the tumour, progression and metastasis often arise from subclones that can develop naturally or during therapy, resulting in molecular alterations with a heterogeneous distribution. Variant histologies in tumour tissues that have developed distinct morphological characteristics divergent from urothelial carcinoma are extreme examples of tumour heterogeneity. Ultimately, heterogeneity contributes to drug resistance and relapse after therapy, resulting in poor survival outcomes. Mutation profile differences between patients with muscle-invasive and metastatic urothelial cancer (interpatient heterogeneity) probably contribute to variability in response to chemotherapy and immunotherapy as first-line treatments. Heterogeneity can occur on multiple levels and averaging or normalizing these alterations is crucial for clinical trial and drug design to enable appropriate therapeutic targeting. Identification of the extent of heterogeneity might shape the choice of monotherapy or additional combination treatments to target different drivers and genetic events. Identification of the lethal tumour cell clones is required to improve survival of patients with urothelial carcinoma.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Although advanced bladder cancer overall has a poor prognosis, a subset of patients demonstrate durable response to immune checkpoint inhibitors. Evidence shows that the response to checkpoint inhibitors may be associated with type and degree of immune infiltration in the tumor microenvironment. Here, we evaluated immune markers stratified by molecular subtypes and histologic variants. The study utilized a series of urothelial carcinomas (UCs) by tissue microarray, on which histologic variants and molecular subtypes had previously been established. PD1, CD3, CD8 and CD68 expression was evaluated by immunohistochemistry in tumor infiltrating immune cells, while PD-L1 expression in the tumor microenvironment was assessed. Each marker was scored semi-quantitatively (score 0-3). Tumors were clustered by marker scores using agglomerative methods, and associations among markers, histologies, and molecular subtypes were analyzed. PD-L1 expression in the tumor microenvironment significantly correlated with presence of CD3, CD8 and chronic inflammation. Urothelial carcinoma may be classified as either immune high or low based on marker expression. The immune high group is enriched in higher CD3, PD-L1, and genomically-unstable molecular subtype, suggesting it may respond to checkpoint inhibitors. We also identified a degree of intratumoral heterogeneity in immune markers in bladder cancer.

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to regional differences in molecular subtype within the tumor, as well as during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPARɣ has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPARɣ-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of three human bladder cancer cells with a PPARɣ agonist identified a number of TFs regulated by PPARɣ activation, several of which are implicated in urothelial and squamous differentiation. One PPARɣ-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPARɣ activation to repress TFAP2A, while PPARɣ antagonist and PPARɣ siRNA knockdown studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPARɣ inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition during tumor progression.

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Creation of genetically engineered mouse models of bladder cancer often involves the use of several background strains in conjunction with the carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). However, carcinogen susceptibility in commonly used strains, as well as phenotypic differences is not well characterized., Objectives: To determine differences in susceptibility and phenotypic outcome following BBN exposure of C57BL/6 and FVB, two strains commonly used for model development., Methods: Male C57BL/6 and FVB mice were exposed to BBN (0.05%) in drinking water for 12 and 16 weeks. Dissected bladders were characterized by histological and immunohistochemical analyses. Gene Ontology analysis was performed to identify differences in gene expression across strains following BBN exposure., Results: While the C57BL/6 strain developed non-invasive tumors, FVB mice developed muscle invasive bladder cancer with squamous and/or glandular differentiation. Glandular differentiation was exclusively observed in the FVB strain. FVB tumors were highly immunogenic and inflamed by the presence of high expression of Cd274 (Pdl-1), murine histocompatibility complex (H2) and pro-inflammatory cytokines (Il-5 and Il-17)., Conclusions: Following BBN exposure, FVB mice undergo rapid tumorigenesis and disease progression characterized by Pdl-1 expression and development of glandular differentiation. These studies identify a degree of tumor heterogeneity in the FVB tumors previously undescribed, and identify FVB mice as a potentially useful model for the study of bladder adenocarcinoma and the inflammatory tumor microenvironment., Competing Interests: None.

Epigenetic aberrations are prominent in bladder cancer (BC) and contribute to disease pathogenesis. We characterized histone deacetylase (HDAC) expression, a family of deacetylation enzymes, in both in vitro and in vivo BC model systems and analyzed expression data from The Cancer Genome Atlas (TCGA). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis was used to determine the expression status of Class I and II HDACs in ten human BC cell lines, while qRT-PCR was used to determine HDAC expression in 24 human tumor specimens. The TCGA cohort consists of 408 muscle invasive BC (MIBC) clinical samples and analysis of this data set identified expression of HDAC4 and - 9 as being associated with basal-squamous disease. These findings agree with qRT-PCR results identifying increased expression of HDAC4 , - 7 , and - 9 in basal BC cell lines ( p < 0.05; Kruskal-Wallis test) and in clinical specimens with invasive bladder cancer (not statistically significant). We also observed increased expression in Hdac4, -7, and -9 in commonly used BC mouse models. Here, we identify suitable preclinical model systems for the study of HDACs, and show increased expression of Class IIa HDACs, specifically HDAC4 and HDAC9 , in basal BC cell lines and in invasive clinical specimens. These results suggest this class of HDACs may be best suited for targeted inhibition in patients with basal BC.

Measures of mitotic activity predict behavior of noninvasive papillary urothelial carcinoma of the urinary bladder, but it is unclear what role these should have in tumor grading. In this article, we compare measures of mitotic activity to contemporary tumor grading, specifically in their association with recurrence of noninvasive papillary urothelial carcinoma. The study uses a retrospective cohort of 199 tumors from 124 patients. Mitotic activity was treated as a categorical variable (mitotic-inert, mitotic-low, or mitotic-high). Evaluating only first-occurrence tumors, recurrence was more frequent in mitotic-high (hazard ratio [HR], 8.8; P < .0001, Cox model) and mitotic-low tumors (HR, 3.7; P = .017) compared with mitotic-inert tumors, when controlling for treatment with intravesical bacillus Calmette-Guerin, age, and sex. Recurrence was likewise more frequent in high-grade tumors (HR, 3.1; P = .00019, Cox model) compared with low-grade tumors, controlling for these factors. However, mitotic group, but not tumor grade, was significantly associated with recurrence in a multivariate Cox model including mitotic group, tumor grade, and treatment status (HR, 6.5 [P = .0025] for mitotic-high versus reference; HR, 3.7 [P = .018] for mitotic-low versus reference). Frailty models including both first-occurrence and recurrent tumors showed similar results. Isolating the analysis to first occurrence, low-grade tumors, recurrence was more frequent in mitotic-high (HR, 6.8; P = .0044, Cox model) and mitotic-low (HR, 3.4; P = .027) tumors compared with mitotic-inert tumors. The findings indicate that mitotic activity is associated with behavior of noninvasive papillary urothelial carcinoma and may be valuable as an adjunct to the contemporary grading system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tumorigenesis requires accumulation of genetic and epigenetic alterations, some of which drive tumor initiation. "Oncogene addiction" describes the phenomenon that (1) well-established cancers are dependent on one mutated oncogene or pathway for the maintenance of a malignant phenotype and that (2) withdrawal of the single oncogenic event leads to growth arrest and/or cancer regression. While oncogene addiction has been experimentally validated in advanced tumor models, its role in tumor precursors has not been investigated. We utilized the requirement of Forkhead box A1 (Foxa1) for transcriptional activation of the Upk2-promoter to temporally control the expression of Upk2-HRAS* oncogene, an inducer of urothelial hyperplasia in transgenic mice. Inducible homozygous knockout of Foxa1 in Upk2-HRAS*/UBC-Cre ERT2 /Foxa1 loxp/loxp mice results in reduced HRAS* levels. This led to a marked reduction of urothelial proliferation as evidenced by urothelial thinning, degenerative changes such as intracellular vacuole formation, and reduced Ki67 expression. Reduced proliferation did not affect basal, Krt14-positive cells, supporting the fact that Foxa1-regulated Upk2-HRAS* expression occurs primarily in supra-basal cells. Our results indicate that maintenance of urothelial hyperplasia in Upk2-HRAS* mice depends on continuous expression of Foxa1 and activated HRAS, and that mutated receptor tyrosine kinases, FOXA1 and/or other downstream effectors may mediate oncogene addiction in urothelial hyperplasia.

Molecular subtyping may inform on prognosis and treatment response in bladder cancer. However, intratumoral molecular heterogeneity is not well studied in this disease and could complicate efforts to use molecular subtyping to guide patient management. To investigate intratumoral heterogeneity in bladder cancer, we examined molecular subtypes in a consecutive, retrospective cystectomy series of histologic variant bladder cancers and conventional urothelial carcinomas co-occurring with them. Molecular subtypes were assigned as per the approach reported by Lund University, an approach that incorporates cell cycle alterations and markers of differentiation, to give the urothelial-like, genomically unstable, basal-squamous, mesenchymal-like, and neuroendocrine-like subtypes. The majority (93%) of tumors were classified as urothelial like, genomically unstable, or basal squamous. Among patients with more than one tumor histology, 39% demonstrated molecular heterogeneity among the different tumor histologies. This was greatest for the basal-squamous subtype, 78% of which co-occurred with either urothelial-like or genomically unstable carcinoma (among cases with multiple histologies). In contrast, there was no co-occurrence of urothelial-like and genomically unstable carcinoma in the same patient. The findings indicate that bladder cancer is often molecularly heterogeneous, particularly in the basal-squamous subtype. This raises the concern for sampling error in laboratory tests that guide therapy based on molecular subtyping. Patient summary: In this report, we investigated molecular diversity among different areas from the same tumor in patients with bladder cancer. We found that different areas from the same tumor are often molecularly different. We conclude that this biological diversity must be taken into account when interpreting clinical molecular tests performed on bladder cancer samples., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Opioid receptors are G protein-coupled receptors that bind opioid ligands including endorphins and enkephalins. The existence of a number of opioid receptors, including the mu-opioid receptor (OPRM1), delta-opioid receptor (OPRD1), kappa-opioid receptor (OPRK1) and zeta-opioid receptor (OGFR) have been reported. However, the potential expression and role of these receptors on human prostate carcinogenesis is unknown. In the present study, we examined opioid receptor expression in human prostate cancer cell lines and in prostate cancer tissue. We observed using quantitative real-time PCR analysis that OGFR and OGFRL1 mRNA is expressed in all examined prostate cancer cell lines as well as in an immortalized, non-tumorigenic prostate epithelial cell line (RWPE-1). Conversely, OPRK1 mRNA expression was detected in a more limited number of cell lines (LNCaP and VCaP), while OPRD1 and OPRM1 mRNA expression was undetectable in all examined prostate cell lines. Interestingly, androgen sensitive LNCaP cells expressed high amounts of OPRK1, OGFR and OGFRL1 compared to other cell lines. Therefore, we investigated the effect of androgen on the mRNA expression of OPRK1, OGFR, OGFRL1 in the LNCaP cell line. Our results demonstrated that the synthetic androgen (R1881) represses mRNA of OPRK1, OGFR and OGFRL1 in a time-dependent manner. Furthermore, immunohistochemistry demonstrated OGFR is expressed at high levels in prostate cancer tissue compared to benign tissue, and that OGFR expression is high in undifferentiated and aggressive prostate cancer tissue. This is the first study showing OGFR and OGFRL1 are androgen repressed genes, and these results suggest a role for the opioid signaling axis in prostate cancer., Competing Interests: None.

Micropapillary morphology in invasive urothelial carcinoma is an established predictor of aggressive disease. It is unknown, however, if prominent retraction is associated with more aggressive disease in the absence of classic micropapillary morphology. We reviewed a retrospective series of 309 radical cystectomy specimens with clinical follow-up data and documented the presence or absence of invasive urothelial carcinoma with prominent retraction clefts, defined as invasive carcinoma with retraction involving the majority of invasive tumor nests in at least one 100× field but without classic micropapillary morphology. Invasive carcinomas with plasmacytoid, sarcomatoid, nested, and small cell morphology were excluded, as were cases without lymph node sampling. In invasive conventional urothelial carcinoma, the presence of prominent retraction clefts was associated lymph node metastasis (odds ratio 4.7, P = .0015, Fisher exact test) but not pathologic tumor stage or several other oncologic parameters (all Ps > .10). Similarly, invasive urothelial carcinoma with micropapillary morphology had lymph node metastasis more frequently than conventional urothelial carcinoma without prominent retraction clefts (P < .001, Fisher exact test), but there was no difference in pathologic tumor stage or oncologic parameters (all Ps > .10). There was no statistically significant difference in rates of lymph node metastasis between invasive urothelial carcinoma with micropapillary morphology and conventional urothelial carcinoma with prominent retraction clefts (P = .54, Fisher exact test). The findings suggest that prominent retraction in invasive urothelial carcinoma may be associated with more aggressive disease, even in the absence of classic micropapillary morphology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Genomic and transcriptional studies have identified discrete molecular subtypes of bladder cancer. These observations could be the starting point to identify new treatments. Several members of the forkhead box (FOX) superfamily of transcription factors have been found to be differentially expressed in the different bladder cancer subtypes. In addition, the FOXA protein family are key regulators of embryonic bladder development and patterning. Both experimental and clinical data support a role for FOXA1 and FOXA2 in urothelial carcinoma. FOXA1 is expressed in embryonic and adult urothelium and its expression is altered in urothelial carcinomas and across disparate molecular bladder cancer subtypes. FOXA2 is normally absent from the adult urothelium, but developmental studies identified FOXA2 as a marker of a transient urothelial progenitor cell population during bladder development. Studies also implicate FOXA2 in bladder cancer and several other FOX proteins might be involved in development and/or progression of this disease; for example, FOXA1 and FOXO3A have been associated with clinical patient outcomes. Future studies should investigate to what extent and by which mechanisms FOX proteins might be directly involved in bladder cancer pathogenesis and treatment responses.

Discrete bladder cancer molecular subtypes exhibit differential clinical aggressiveness and therapeutic response, which may have significant implications for identifying novel treatments for this common malignancy. However, research is hindered by the lack of suitable models to study each subtype. To address this limitation, we classified bladder cancer cell lines into molecular subtypes using publically available data in the Cancer Cell Line Encyclopedia (CCLE), guided by genomic characterization of bladder cancer by The Cancer Genome Atlas (TCGA). This identified a panel of bladder cancer cell lines which exhibit genetic alterations and gene expression patterns consistent with luminal and basal molecular subtypes of human disease. A subset of bladder cancer cell lines exhibit in vivo histomorphologic patterns consistent with luminal and basal subtypes, including papillary architecture and squamous differentiation. Using the molecular subtype assignments, and our own RNA-seq analysis, we found overexpression of GATA3 and FOXA1 cooperate with PPARɣ activation to drive transdifferentiation of a basal bladder cancer cells to a luminial phenotype. In summary, our analysis identified a set of human cell lines suitable for the study of molecular subtypes in bladder cancer, and furthermore indicates a cooperative regulatory network consisting of GATA3, FOXA1, and PPARɣ drive luminal cell fate.

Objective: To determine the prognostic significance of Forkhead Box A1 (FOXA1) expression in patients with upper tract urothelial carcinoma (UTUC) undergoing radical nephroureterectomy (RNU)., Materials and Methods: A retrospective analysis of 566 patients undergoing RNU at seven academic medical centers was performed. Tissue microarrays were subjected to immunohistochemistry using a commercially available polyclonal FOXA1 antibody. Logistic regression determined the association of FOXA1 expression with pathologic features and survival outcomes., Results: Three hundred twenty-two men and 244 women were included. The pathologic distribution of specimens included 53% muscle-invasive or greater (≥pT2), 74% high-grade, 16% with flat architecture, 13% with necrosis, 21% with lymphovascular invasion, 18% with concomitant carcinoma in situ, and 8% with positive lymph nodes. The median FOXA1 score was 5.0 (range: 0-8). Lower FOXA1 expression was significantly correlated with advanced pathologic stage (≥pT3) (P = .02), concomitant carcinoma in situ (P = .006), and renal pelvis (vs ureter) location (P < .0001). At a median follow-up of 27.0 months (range: 3-196), 139 patients (25%) experienced disease recurrence and 121 (21%) died from the disease. In a multivariate model, lower FOXA1 expression was independently associated with disease recurrence (hazard ratio [HR]: 1.11, 95% confidence interval [CI]: 1.05-1.62, P = .04), cancer-specific mortality (HR: 1.17, 95% CI: 1.03-1.92, P = .04), and all-cause mortality (HR: 1.08, 95% CI: 1.02-1.18, P = .05)., Conclusion: Lower FOXA1 expression is associated with adverse pathologic features and inferior survival outcomes for UTUC patients undergoing RNU. These data indicate lower FOXA1 expression may be a marker of aggressive disease in UTUC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Studies evaluating enhancer of zeste homolog 2 (EZH2) expression and oncologic outcomes in bladder cancer have been discrepant. EZH2 expression in noninvasive bladder cancer is not well studied. We thus set out to address the discrepancy in previous reports, and to study expression of EZH2 in noninvasive bladder cancer and its associations, in a large cystectomy cohort., Materials and Methods: EZH2 expression was evaluated in tissue microarray material (invasive and noninvasive cancer). Associations between EZH2 expression and oncologic outcomes, tumor stage, and disease type were determined. Receiver operating characteristic analysis was performed for EZH2 expression in the diagnosis of invasive carcinoma and flat carcinoma in situ (CIS) compared to benign urothelium., Results: EZH2 expression was most common in CIS, followed by invasive carcinoma, noninvasive papillary urothelial carcinoma, and benign urothelium, in decreasing order (P<0.05 all comparisons, linear model). The receiver operating characteristic analysis demonstrated an area under the curve of 0.92 for CIS and 0.83 for invasive carcinoma, both compared to benign urothelium. At a cutoff of≥4, this corresponded to sensitivities of 89% and 73%, and specificities of 82% and 82%, for CIS and invasive carcinoma, respectively. The EZH2 expression was not associated with oncologic outcomes, including recurrence-free survival and death from bladder cancer. The EZH2 expression status (positive or negative) of noninvasive and invasive carcinomas taken from the same bladder correlated (P = 0.05, Fisher exact)., Conclusion: That EZH2 status of noninvasive and invasive cancer correlated in individual patients suggests that EZH2 may be a marker of lineage. EZH2 may offer diagnostic utility, particularly in flat urothelial CIS vs. benign urothelium. The present study supports that EZH2 expression in bladder cancer is not predictive of oncologic outcome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.

Background: Recent molecular characterization studies focusing on bladder cancer have provided a wealth of information, including the identification of specific molecular subtypes of this disease. Interestingly, a particular molecular subtype identified by several different groups is characterized, at least in part, by the presence of squamous differentiation (SqD) in a significant fraction of primary tumors. Tumors that exhibit SqD are extremely aggressive. Moreover, conflicting reports exist relative to the sensitivity of bladder tumors exhibiting SqD to multimodal treatment. Bladder cancers that exhibit SqD appear to be distinct clinical entities and are often associated with a specific molecular subtype; therefore, it is important to understand the molecular drivers of this process., Purpose: Because presence of SqD is closely associated with a basal molecular phenotype, we review the evidence for specific pathways in SqD. In addition, we pose key areas for future exploration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We previously found loss of forkhead box A1 (FOXA1) expression to be associated with aggressive urothelial carcinoma of the bladder, as well as increased tumor proliferation and invasion. These initial findings were substantiated by The Cancer Genome Atlas, which identified FOXA1 mutations in a subset of bladder cancers. However, the prognostic significance of FOXA1 inactivation and the effect of FOXA1 loss on urothelial differentiation remain unknown. Application of a univariate analysis (log-rank) and a multivariate Cox proportional hazards regression model revealed that loss of FOXA1 expression is an independent predictor of decreased overall survival. An ubiquitin Cre-driven system ablating Foxa1 expression in urothelium of adult mice resulted in sex-specific histologic alterations, with male mice developing urothelial hyperplasia and female mice developing keratinizing squamous metaplasia. Microarray analysis confirmed these findings and revealed a significant increase in cytokeratin 14 expression in the urothelium of the female Foxa1 knockout mouse and an increase in the expression of a number of genes normally associated with keratinocyte differentiation. IHC confirmed increased cytokeratin 14 expression in female bladders and additionally revealed enrichment of cytokeratin 14-positive basal cells in the hyperplastic urothelial mucosa in male Foxa1 knockout mice. Analysis of human tumor specimens confirmed a significant relationship between loss of FOXA1 and increased cytokeratin 14 expression., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: SOX2 is a member of SOX (SRY-related high mobility group box) family of transcription factors., Methods: In this study, we examined the expression of SOX2 in murine and human prostatic specimens by immunohistochemistry., Results: We found that SOX2 was expressed in murine prostates during budding morphogenesis and in neuroendocrine (NE) prostate cancer (PCa) murine models. Expression of SOX2 was also examined in human prostatic tissue. We found that SOX2 was expressed in 26 of the 30 BPH specimens. In these BPH samples, expression of SOX2 was limited to basal epithelial cells. In contrast, 24 of the 25 primary PCa specimens were negative for SOX2. The only positive primary PCa was the prostatic NE tumor, which also showed co-expression of synaptophysin. Additionally, the expression of SOX2 was detected in all prostatic NE tumor xenograft lines. Furthermore, we have examined the expression of SOX2 on a set of tissue microarrays consisting of metastatic PCa tissues. Expression of SOX2 was detected in at least one metastatic site in 15 of the 24 patients with metastatic castration-resistant PCa; and the expression of SOX2 was correlated with synaptophysin., Conclusions: SOX2 was expressed in developing prostates, basal cells of BPH, as well as prostatic NE tumors.

Purpose of Review: High-risk, nonmuscle invasive bladder cancer (HR-NMIBC) represents a costly and difficult-to-treat disease, the molecular pathogenesis of which has a limited understanding. Most preclinical models for the study of bladder cancer are more appropriate for the study of advanced disease. However, recent key advances in preclinical animal models places us at an opportune position to better understand HR-NMIBC., Recent Findings: Discoveries in the basic sciences allow us to better understand tumor biology when building models of bladder cancer. Of note, a key study on urothelial progenitor cells recently highlighted an important role for Sonic hedgehog-positive cells and retinoid signaling that is essential for urothelial development and regeneration. In the translational realm, transgenic mouse models continue to be used, with a recent interest in the role of Wnt/beta-catenin in urothelial carcinomas. Tissue recombination models are also being increasingly utilized to better recreate the tissue microenvironment and better understand stromal-epithelial interactions and the impact of genetic alterations on tissue differentiation. Lastly, the avatar mouse systems, which involve direct xenotransplantation of human tumor specimens into immunocompromised mice, represent an additional approach to study cancer characteristics in a preserved tissue context., Summary: With molecular alterations remaining an unclear area of our understanding of HR-NMIBC, preclinical models of bladder cancer serve as essential tools to discover specific genetic compromises in disease pathogenesis and the therapeutics to treat them.

When the National Institutes of Health Mouse Models of Human Cancer Consortium initiated the Prostate Steering Committee 15 years ago, there were no genetically engineered mouse (GEM) models of prostate cancer (PCa). Today, a PubMed search for "prostate cancer mouse model" yields 3,200 publications and this list continues to grow. The first generation of GEM utilized the newly discovered and characterized probasin promoter driving viral oncogenes such as Simian virus 40 large T antigen to yield the LADY and TRAMP models. As the PCa research field has matured, the second generation of models has incorporated the single and multiple molecular changes observed in human disease, such as loss of PTEN and overexpression of Myc. Application of these models has revealed that mice are particularly resistant to developing invasive PCa, and once they achieve invasive disease, the PCa rarely resembles human disease. Nevertheless, these models and their application have provided vital information on human PCa progression. The aim of this review is to provide a brief primer on mouse and human prostate histology and pathology, provide descriptions of mouse models, as well as attempt to answer the age old question: Which GEM model of PCa is the best for my research question?

Protein-protein interactions (PPI) are a hallmark of cellular signaling. Such interactions occur abundantly within the cellular milieu and encompass interactions involved in vital cellular processes. Understanding the various types, mechanisms, and consequences of PPIs with respect to cellular signaling and function is vital for targeted drug therapy. Various types of small-molecule drugs and targeted approaches to drug design have been developed to modulate PPIs. Peptidomimetics offer an exciting class of therapeutics as they can be designed to target specific PPIs by mimicking key recognition motifs found at critical points in the interface of PPIs (e.g., hotspots). In contrast to peptides, peptidomimetics do not possess a natural peptide backbone structure but present essential functional groups in a required three-dimensional pattern complimentary to the protein-binding pocket. This design feature overcomes many limitations of peptide therapeutics including limited stability toward peptidases, poor transport across biologic membranes, and poor target specificity. Equally important is deciphering the structural requirements and amino acid residues critical to PPIs. This review provides an up-to-date perspective of the complexity of cellular signaling and strategies for targeting PPIs in disease states, particularly in cancer, using peptidomimetics, and highlights that the rational design of agents that target PPIs is not only feasible but is of the utmost clinical importance., (©2014 American Association for Cancer Research.)

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.

Purpose: Metastasis, the main cause of death from cancer, remains poorly understood at the molecular level., Experimental Design: Based on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo., Results: SPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models., Conclusions: We present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer., (Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.)

Macrophage migratory inhibitory factor (MIF) is a proinflammatory cytokine shown to promote tumorigenesis. Using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model of bladder cancer, we previously showed that MIF knockout mice display decreased angiogenesis and invasion compared with wild-type. This study examines the role of MIF in bladder cancer via use of oral inhibitors of MIF. In vitro, high-grade bladder cancer cells were treated with recombinant human MIF +/- (rhMIF+/-) inhibitor. Measurements included cell counts, proliferation by (3)H-thymidine incorporation (TdR), extracellular signal-regulated kinase (ERK) phosphorylation by western blot analysis, messenger RNA (mRNA) expression by quantitative PCR and protein secretion by enzyme-linked immunosorbent assay. Treatment with rhMIF increased ERK phosphorylation, cell counts, TdR and mRNA expression and protein secretion of vascular endothelial growth factor, which were blocked by specific inhibitors of ERK and MIF. In vivo, 3-month-old male C57Bl/6 mice were given BBN for 22 and 16 weeks in study 1 and study 2, respectively. Mice (n = 8-10 per group) were gavaged with vehicle or doses of MIF inhibitors daily from weeks 16-22 in both studies. Average bladder weights, reflecting tumor mass, tumor stage/burden, mitotic rate and proliferation indices, and microvessel densities were reduced in inhibitor groups versus controls. In summary, MIF promotes bladder cancer via increasing cell proliferation and angiogenesis and oral inhibitors of MIF may prove useful in treatment of this disease.

Differentiation is defined as the ability of a cell to acquire full functional behavior. For instance, the function of bladder urothelium is to act as a barrier to the diffusion of solutes into or out of the urine after excretion by the kidney. The urothelium also serves to protect the detrusor muscle from toxins present in stored urine. A major event in the initiation and progression of bladder cancer is loss of urothelial differentiation. This is important because less differentiated urothelial tumors (higher histologic tumor grade) are typically associated with increased biologic and clinical aggressiveness. The differentiation status of urothelial carcinomas can be assessed by histopathologic examination and is reflected in the assignment of a histologic grade (low-grade or high-grade). Although typically limited to morphologic evaluation in most routine diagnostic practices, tumor grade can also be assessed using biochemical markers. Indeed, current pathological analysis of tumor specimens is increasingly reliant on molecular phenotyping. Thus, high priorities for bladder cancer research include identification of (1) biomarkers that will enable the identification of high grade T1 tumors that pose the most threat and require the most aggressive treatment; (2) biomarkers that predict the likelihood that a low grade, American Joint Committee on Cancer stage pTa bladder tumor will progress into an invasive carcinoma with metastatic potential; (3) biomarkers that indicate which pTa tumors are most likely to recur, thus enabling clinicians to prospectively identify patients who require aggressive treatment; and (4) how these markers might contribute to biological processes that underlie tumor progression and metastasis, potentially through loss of terminal differentiation. This review will discuss the proteins associated with urothelial cell differentiation, with a focus on those implicated in bladder cancer, and other proteins that may be involved in neoplastic progression. It is hoped that ongoing discoveries associated with the study of these differentiation-promoting proteins can be translated into the clinic to positively impact patient care., (Copyright © 2013 Elsevier Inc. All rights reserved.)