Bladder Tumorigenesis Research Articles

Loss of Cxcr2 in Myeloid Cells Promotes Tumour Progression and T Cell Infiltration in Invasive Bladder Cancer.

CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2 flox/flox ). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status.

Nuevo modelo pronóstico de supervivencia del cáncer de vejiga basado en los genes vinculados a la ferroptosis (FRGS)

BackgroundBladder cancer (BLCA) is a malignant urothelial carcinoma with a high mortality rate. Ferroptosis is a new type of programmed cell death and functions in suppressing tumor growth and progression. However, few studies focus on ferroptosis and BLCA. Materials and methodsWe explored the potential oncogenic roles of ferroptosis-related genes in BLCA based on multiple public datasets. We then used univariate and multivariate cox regression to build a new survival model based on ferroptosis-related genes to predict the survival of BLCA. ResultsWe found that 23 ferroptosis-related genes had a strong correlation with each other in BLCA. Eight ferroptosis-related genes, CDKN1A, HSPA5, NFE2L2, MT1G, FANCD2, CISD1, TFRC, NCOA4, had a significantly different expression and heat-map. HSPA5 and CISD1 have a statistically significant difference in OS and DFS. Besides, CISD1 had an ideal nomogram to predict the 1-3-5-year OS (C-index: 0.701, P<.001). Furthermore, HSPA5 and CISD1 had a lower DNA methylation rate than normal tissue and HSPA5 had a positive connection with tumor mutational burden (TMB) (P=.02). In addition, HSPA5 participated in the DNA replication and P53 signaling pathway, and CISD1 mediated the oxidative phosphorylation and positive regulation of the intrinsic apoptotic signaling pathway. ConclusionFerroptosis-related genes had a strong correlation with BLCA, notably, HSPA5 and CISD1 may play a role in inducing ferroptosis to suppress bladder tumorigenesis and CISD1 can be a novel prognostic biomarker as well as an effective target for diagnosis and treatment in BLCA.

Persistent γ-H2AX Formation and Expression of Stem Cell Markers in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Bladder Carcinogenesis in Rats.

We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.

A new survival model based on ferroptosis-related genes (FRGS) for prognostic prediction in bladder cancer

BackgroundBladder cancer (BLCA) is a malignant urothelial carcinoma with a high mortality rate. Ferroptosis is a new type of programmed cell death and functions in suppressing tumor growth and progression. However, few studies focus on ferroptosis and BLCA. Materials and methodsWe explored the potential oncogenic roles of ferroptosis-related genes in BLCA based on multiple public datasets. We then used univariate and multivariate cox regression to build a new survival model based on ferroptosis-related genes to predict the survival of BLCA. ResultsWe found that 23 ferroptosis-related genes had a strong correlation with each other in BLCA. Eight ferroptosis-related genes, CDKN1A, HSPA5, NFE2L2, MT1G, FANCD2, CISD1, TFRC, NCOA4, had a significantly different expression and heat-map. HSPA5 and CISD1 have a statistically significant difference in OS and DFS. Besides, CISD1 had an ideal nomogram to predict the 1-3-5-year OS (C-index: 0.701, P < .001). Furthermore, HSPA5 and CISD1 had a lower DNA methylation rate than normal tissue and HSPA5 had a positive connection with TMB (P = .02). In addition, HSPA5 participated in the DNA replication and P53 signaling pathway, and CISD1 mediated the oxidative phosphorylation and positive regulation of the intrinsic apoptotic signaling pathway. ConclusionFerroptosis-related genes had a strong correlation with BLCA, notably, HSPA5 and CISD1 may play a role in inducing ferroptosis to suppress bladder tumorigenesis and CISD1 can be a novel prognostic biomarker as well as an effective target for diagnosis and treatment in BLCA.

Abstract 831: Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity

Abstract Upregulation of the non-tyrosine kinase receptor neuropilin-2 (NRP2) is described for several tumor entities and correlates with disease progression. Two membrane-anchored isoforms are transcribed from the NRP2 gene that only differ in their terminal exons to encode transmembrane stretches and short cytoplasmic tails, respectively. While NRP2a was shown to support tumor cell proliferation, NRP2b facilitates tumor cell migration, resulting in enhanced metastatic spreading. A third, soluble NRP2 isoform encoding only aminoterminal protein domains exists as dimeric protein and functions in sequestration of the VEGF-C ligand. By using a next-generation sequencing approach to enrich for NRP2 terminal exons, we identified NRP2-Mo83, a novel NRP2 isoform, from a high expressing tumor cell line, and verified secreted NRP2-Mo83 protein by mass spectrometry in cell supernatants. Preliminary mass spectrometric data give an indication of the presence of soluble NRP2-Mo83 protein in human serum samples.NRP2-Mo83 mRNA is expressed at variable quantities in vitro in a panel of bladder carcinoma cell lines, but is downregulated in a small cohort of macrodissected urothelial carcinoma samples. Transcript abundances of a NRP2 transcript classified as non-coding with high sequence similarity to the NRP2-Mo83 3’ terminus confirm the observed downregulation in the TCGA bladder carcinoma cohort.Preliminary data indicate that recombinant NRP2-Mo83 protein exerts a dose-dependent inhibitory effect on tumor cell growth in vitro in all tested bladder carcinoma cell lines in the presence of serum. However, a fraction of cell lines does not respond to NRP2-Mo83 under serum-starved conditions. Proliferation of human endothelial cells (HUVEC) is inhibited in a dose-dependent manner by recombinant NRP2-Mo83 protein in vitro. In vivo, NRP2-Mo83 reveals an anti-angiogenic effect on the chicken chorioallantoic membrane.Recombinant NRP2-Mo83 is secreted by HEK293 cells. Enrichment of endogenous and purification of recombinant NRP2-Mo83 protein is achieved by exploiting its capacity to bind heparin. In contrast to the described s9NRP2 isoform, NRP2-Mo83 is largely monomeric in solution, as verified by size exclusion chromatography. We identified N and O glycosylation as well as the capacity for polysialation as posttranslational modifications of recombinant NRP2-Mo83 protein, similar to membrane anchored NRP2 isoforms. NanoDSF experiments indicate that calcium ions stabilize NRP2-Mo83. We present NRP2-Mo83 as a novel NRP2 splice isoform, validated its expression at the mRNA and protein level. NRP2-Mo83 appears down-regulated during bladder tumorigenesis, congruent with its function in bladder tumor growth inhibition and its anti-angiogenic properties in vitro and in vivo. The identification of NRP2-Mo83 in human serum may suggest a suppressive function even under non-pathological conditions. Citation Format: Thomas Mayr, Paul L. Dix, Sayed-Mohammad Hasheminasab, Chuanpit Hampel, Marc Sylvester, Niels Schneberger, Sarah Foerster, Gregor Hagelueken, Samikshan Dutta, Glen Kristiansen, Regine Schneider-Stock, Kaustubh Datta, Michael H. Muders. Discovery of a novel soluble Neuropilin-2 isoform with antiangiogenic and antitumorigenic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 831.

Androgen Receptor Expression Is a Predictor of Poor Outcome in Urothelial Carcinoma

BackgroundSeveral preclinical and clinical studies demonstrated a strong correlation between androgen receptor (AR) signaling and bladder tumorigenesis. This study aims to evaluate the prognostic significance of AR expression in metastatic urothelial carcinoma (mUC).MethodsSamples from a phase III trial (LaMB, NCT00949455) which compared maintenance lapatinib versus placebo after completion of first-line platinum-based chemotherapy in patients with HER 1/2-positive mUC of the bladder were collected. Corresponding baseline and follow-up data included patients enrolled in the study and those who screen failed. AR expression was assessed independently by a single pathologist who was blinded to the study. Samples were grouped according to AR expression (negative vs. positive) and correlated with baseline tumor characteristics and survival.ResultsOf the 446 screened samples in the LaMB study, 90 were retrospectively analyzed for AR expression. There were no correlations between AR expression and tumor stage (r = −0.10), tumor grade (r = 0.05) at diagnosis, or subsequent treatment with lapatinib (r = −0.04). The median progression-free survival was 6 months (95% CI, 3.20–6.80) in the AR-negative group and 5 months (95% CI, 3.41–6.59) in the AR-positive group [HR 0.54 (95% CI, 0.31–0.92), p = 0.02]. Similarly, patients with AR-negative disease had more favorable overall survival (OS) with 16 months (95% CI, 6.6–25.4) and 11 months (95% CI, 7.0–15.0) in the AR-positive group [HR 0.55 (95% CI, 0.31–0.98), p = 0.04]. In the multivariate analysis, AR expression was significantly associated with worse OS (p = 0.045).ConclusionsAR expression is a predictor of poor outcome and presents a targetable alteration in patients with mUC.

PD53-08 E-CIGARETTE EXPOSURE IN MICE PROMOTES CHANGES IN DNA METHYLATION OF THE BLADDER UROTHELIUM

You have accessJournal of UrologyCME1 May 2022PD53-08 E-CIGARETTE EXPOSURE IN MICE PROMOTES CHANGES IN DNA METHYLATION OF THE BLADDER UROTHELIUM Ibardo Zambrano, Ujjawal Manocha, Weida Gong, Yi-Hsuan Tsai, Manhua Zhu, Melissa Herman, Marc Bjurlin, and William Kim Ibardo ZambranoIbardo Zambrano More articles by this author , Ujjawal ManochaUjjawal Manocha More articles by this author , Weida GongWeida Gong More articles by this author , Yi-Hsuan TsaiYi-Hsuan Tsai More articles by this author , Manhua ZhuManhua Zhu More articles by this author , Melissa HermanMelissa Herman More articles by this author , Marc BjurlinMarc Bjurlin More articles by this author , and William KimWilliam Kim More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To date, no group has studied the epigenetic effects of e-cigarettes (EC) on the urothelium. DNA methylation changes appear to be one of the earliest events in bladder tumorigenesis and can be readily found in normal urothelium adjacent to bladder tumors. EC exposure causes urothelial hyperplasia in mice but not bladder tumors. We Vtherefore asked whether EC exposure results in early field alterations of DNA methylation in the bladder urothelium. METHODS: Mice (4 females, 4 males; 35 weeks old) were exposed to 12% nicotine in 1:1 polyethylene glycol: vegetable glycerol or vehicle daily: 3 hr sessions, 3 sec vape duration, 10 min interval between vapes. Plasma nicotine and cotinine serum concentrations were measured using LC-MS. Reduced-representation bisulfite sequencing (RRBS) was performed using DNA from microdissected urothelium harvested at 4 weeks (Zymo Research Kit). Number of loci covered by each sample was determined and data was filtered to only include loci with >10 reads. Differentially methylated loci were determined (p<0.01). Gene Set Enrichment Analysis was performed to contextualize the DNA methylation changes. RESULTS: No significant adverse events were observed in the mice during the exposure period to either vehicle or nicotine. Acute and repeated exposure resulted in significantly higher levels of nicotine and cotinine serum concentration in the nicotine vs. the vehicle group (Figure 1). No bladder tumors formed. RRBS analysis demonstrated that EC exposure resulted in hypermethylation of the promoters of 665 genes and hypomethylation of 285 genes after accounting for sex (p <0.05) Pathway analysis showed enrichment of MYC target genes in hypomethylated regions and enrichment of genes associated with oxidative phosphorylation, cilium assembly, and ciliopathies in hypermethylated loci. CONCLUSIONS: Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. vehicle control. EC metabolites in urine may result in aberrant methylation patterns in the bladder urothelium. Results are suggestive of a pre-malignant phenotype driven by EC exposure via stimulation of MYC targets and silencing of genes responsible for establishing primary cilia. Loss of primary cilia in epithelial cells has been associated with loss of cell polarity and increased cell migration. Source of Funding: NCI Geographical Management of Cancer Health Disparities Program (3P30-CA016086), UNC Lineberger Innovation Award 2020 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e912 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ibardo Zambrano More articles by this author Ujjawal Manocha More articles by this author Weida Gong More articles by this author Yi-Hsuan Tsai More articles by this author Manhua Zhu More articles by this author Melissa Herman More articles by this author Marc Bjurlin More articles by this author William Kim More articles by this author Expand All Advertisement PDF DownloadLoading ...

Germline BRCA1 and BRCA2 mutations and the risk of bladder or kidney cancer in Poland

IntroductionThe role of the BRCA1 and BRCA2 genes in bladder and renal tumorigenesis is unclear. Our goal was to determine the prevalence of specific founder mutations genes BRCA1 (5328 insC, C61G and 4153 delA) and BRCA2 (C5972T) mutations in bladder and kidney cancer patients from Poland.Materials and methodsWe genotyped 1028 patients with bladder cancer and 688 cases with kidney cancer and two control groups.ResultsA BRCA1 mutation (all variants combined) was detected in peripheral blood leukocytes in 5 out of 1028 (0.5%) bladder cases and in 17 of 4000 controls (0.4%) (odds ratio [OR], (OR = 1.1; 95% CI 0.42–3.11; p = 1.0). Among 688 unselected kidney cancer cases a BRCA1 mutations was reported in three patients (0.4%) (OR = 1.0; 95% CI 0.29–3.51; p = 1.0). The mutation C5972T in BRCA2 was observed in 54 bladder cancer patients (5.2%) and in 159 of 2791 healthy controls (5.7%) (OR = 0.9; 95% CI 0.66–1.26; p = 0.6). Fifty kidney cancer cases carried a BRCA2 mutation (7.3%) (OR = 1.3; 95% CI 0.93–1.80; p = 0.1).ConclusionIn conclusion, we found no difference in the prevalence of BRCA1 and BRCA2 founder mutations between cases and healthy controls. The mutations BRCA1 and BRCA2 seem not to play a role in bladder and kidney cancer development in Polish patients.

KRT6A expedites bladder cancer progression, regulated by miR-31-5p

ABSTRACT Bladder cancer is one of the most severe life-threatening illnesses worldwide. To contribute to a solution to this public health issue, here, we sought to identify a novel biomarker for the early diagnosis of bladder tumors. We conducted RNA sequence analysis utilizing samples from tumorous tissue and adjacent healthy tissue in bladder cancer patients and found that KRT6A was upregulated in bladder tumor tissues, suggesting that it might be a candidate for involvement in bladder tumorigenesis. Accordingly, we performed a series of experiments to further verify the role of KRT6A in bladder tumor progression. Our results revealed that KRT6A promoted bladder tumor cell viability, proliferation, and adhesion, while diminishing bladder tumor cell apoptosis. We also focused on the role of epigenetics in bladder tumors and verified that KRT6A was a miR-31-5p target gene, and its positive effect on bladder tumor progression was relieved by miR-31-5p. Overall, this study sheds new light regarding a novel oncogenic regulatory axis, KRT6A/miR-31-5p, which is related to bladder tumor growth.

Identification of the gene expression changes and gene regulatory aspects in ELF3 mutant bladder cancer.

Recent genome-wide studies revealed the molecular subtypes and mutational landscape of bladder cancer, which is the 10th most common cancer causing many deaths. ELF3 is one of the frequently mutated genes in bladder cancer with 14% alteration rate. It mainly functions as an epithelial transcription factor and its proper function is critical for the urothelium development. However, the impact of ELF3 mutations in bladder cancer is currently unknown. In this study, we analysed the gene expression data available for primary bladder cancer and bladder cancer cell lines according to the mutation status of ELF3. Our results show that de-regulated genes common in cell lines and primary tissue are primarily involved in ameboidal type cell migration and cell-cell junction organization. Additionally, we identify that ELF3-mutant cases in primary samples significantly overexpress PIK3C2B and ELF3 and PIK3C2B and ELF3 are significantly co-mutated in many cancer types. Our integrative analysis with existing Hi-C data further revealed the genes proximally located to ELF3, including PIK3C2B to be upregulated in ELF3 mutant cases, potentially as a result of truncated ELF3 protein product and subsequent changes in regulatory interactions. Our results provide important insights about how ELF3 mutation contributes to bladder tumorigenesis and uncover previously unknown dependencies.

Germline HOXB13 mutation p.G84E do not confer an increased bladder or kidney cancer risk in polish population

IntroductionThe role of HOXB13 in bladder and renal tumorigenesis is unclear. Our goal was to determine the prevalence of HOXB13 p.G84E mutation in bladder and kidney cancer patients from Poland.Materials and methods1418 patients with bladder cancer and 813 cases with kidney cancer and 4497 controls were genotyped for HOXB13 p.G84E.Resultsp.G84E mutation of HOXB13 gene was detected in three of 1418 (0.2%) bladder cancer cases and in six of 4497 controls (odds ratio [OR], 1.6; 95% CI 0.39–6.36; p = 0.8). Among 813 kidney cancer cases HOXB13 mutations was reported in three patients (0,4%) (odds ratio [OR], (OR = 2,8; 95% CI 0.69–11.11; p = 0.3). In cases with mutations in the HOXB13 gene, the family history of cancer was negative.ConclusionHOXB13 mutation was not associated with bladder or kidney cancer. Mutation p.G84E in HOXB13 seem not to play a role in bladder and kidney cancer development in Polish patients.

MiRNA-182-5p promotes human bladder cancer proliferation and migration through the FOXF2/SHH axis.

Increasing evidence suggests that microRNAs (miRNAs) play critical roles in bladder tumorigenesis and development by combining with the 3' untranslated regions (3'-UTRs) of the corresponding mRNAs to negatively regulate gene expression. The role of miR-182-5p in bladder cancer (BC) remains unclear. Therefore, this study aimed to clarify the functional role of miR-182-5p in BC. We predicted candidate mRNAs for miR-182-5p via three databases (TarBase, ENCORI, and miRDB). Dual-luciferase reporter assays and target prediction confirmed FOXF2 as a potential target of miR-182-5p. Quantitative RT-PCR (qRT-PCR) showed that endogenous miR-182-5p expression was significantly upregulated in BC cell lines and clinical samples of BC patients. IHC, western blotting, and qRT-PCR assays indicated that FOXF2 expression was concurrently downregulated in BC tissues and BC cell lines. Gain- and loss-of-function studies showed that the overexpression of miR-182-5p enhanced the proliferation and migration of BC cells, while the downregulation of miR-182-5p showed the opposite results. The effects induced by miR-182-5p were attenuated with the restoration of FOXF2 expression. In BC cells, the upregulation of miR-182-5p not only decreased FOXF2 expression but also markedly increased Sonic hedgehog (SHH) pathway levels. These findings suggested that FOXF2 directly binds to miR-182-5p and that miR-182-5p acts as a tumor promoter in BC genesis and metastasis by targeting FOXF2. In addition, miR-182-5p plays a pro-cancer role by downregulating FOXF2 and activating the SHH pathway.

BUB1 drives the occurrence and development of bladder cancer by mediating the STAT3 signaling pathway

BackgroundThe incidence of bladder urothelial carcinoma (UC), a common malignancy of the urinary tract, is approximately three times higher in men than in women. High expression of the mitotic kinase BUB1 is associated with the occurrence and development of several cancers, although the relationship between BUB1 and bladder tumorigenesis remains unclear.MethodsUsing a microarray approach, we found increased BUB1 expression in human BCa. The association between BUB1 and STAT3 phosphorylation was determined through molecular and cell biological methods. We evaluated the impact of pharmacologic inhibition of BUB1 kinase activity on proliferation and BCa progression in vitro and in vivo.ResultsIn this study, we found that BUB1 expression was increased in human bladder cancer (BCa). We further identified through a series of molecular and cell biological approaches that BUB1 interacted directly with STAT3 and mediated the phosphorylation of STAT3 at Ser727. In addition, the findings that pharmacologic inhibition of BUB1 kinase activity significantly suppressed BCa cell proliferation and the progression of bladder cancer in vitro and in vivo were further verified. Finally, we found that the BUB1/STAT3 complex promoted the transcription of STAT3 target genes and that depletion of BUB1 and mutation of the BUB1 kinase domain abrogated this transcriptional activity, further highlighting the critical role of kinase activity in the activation of STAT3 target genes. A pharmacological inhibitor of BUB1 (2OH-BNPP1) was able to significantly inhibit the growth of BCa cell xenografts.ConclusionThis study showed that the BUB1 kinase drives the progression and proliferation of BCa by regulating the transcriptional activation of STAT3 signaling and may be an attractive candidate for therapeutic targeting in BCa.

RAF1 amplification: an exemplar of MAPK pathway activation in urothelial carcinoma.

Despite recent therapeutic gains in the treatment of advanced bladder cancer, the overall survival in patients with metastatic disease remains poor and further therapeutic discovery is needed. Advanced bladder cancer is a molecularly heterogeneous disease, and the identification of driver genetic alterations has led to effective targeted therapeutic agents, such as fibroblast growth factor receptor (FGFR) inhibitors. In this issue of the JCI, Bekele et al. identify a subtype of muscle-invasive bladder cancer (MIBC) that harbors RAF1 amplification. The authors showed that RAF1 inhibition, with pan-RAF inhibitors, and the combination of RAF1 inhibition with MEK inhibition were efficacious in preclinical models harboring RAF1 amplifications as well as in tumors with HRAS and NRAS mutations. This study highlights RAF1 amplification as a driver event in bladder cancer and establishes the central role of the MAPK pathway in bladder tumorigenesis.

RNF144A deficiency promotes PD-L1 protein stabilization and carcinogen-induced bladder tumorigenesis.

RNF144A is a DNA damage-induced E3 ubiquitin ligase that targets proteins involved in genome instability for degradation, e.g., DNA-PKcs and BMI1. RNF144A is frequently mutated or epigenetically silenced in cancer, providing the rationale to evaluate RNF144A loss of function in tumorigenesis. Here we report that RNF144A-deficient mice are more prone to the development of bladder tumors upon carcinogen exposure. In addition to DNA-PKcs and BMI1, we identify the immune checkpoint protein PD-L1 as a novel degradation target of RNF144A, since these proteins are expressed at higher levels in Rnf144a KO tumors. RNF144A interacts with PD-L1 in the plasma membrane and intracellular vesicles and promotes poly-ubiquitination and degradation of PD-L1. Therefore, Rnf144a KO stabilizes PD-L1 and leads to a reduction of tumor-infiltrating CD8+ T cell populations in the BBN-induced bladder tumors. The bladder tumors developed in WT and Rnf144a KO mice primarily express CK5 and CK14, markers of basal cancer subtype, as expected in BBN-induced bladder tumors. Intriguingly, the Rnf144a KO tumors also express GATA3, a marker for the luminal subtype, suggesting that RNF144A loss of function promotes features of cellular differentiation. Such differentiation features in Rnf144a KO tumors likely result from a decrease of EGFR expression, consistent with the reported role of RNF144A in maintaining EGFR expression. In summary, for the first time our study demonstrates the in vivo tumor suppressor activity of RNF144A upon carcinogenic insult. Loss of RNF144A promotes the expression of DNA-PKcs, BMI1 and PD-L1, likely contributing to the carcinogen-induced bladder tumorigenesis.

Urinary Bladder Microbiome Identification Protocol with Proteomics in Bladder Cancer Patients and Review of the Literature

Abstract Urine was conventionally thought to be sterile. However, recent evidence about the presence of microorganisms residing the urinary tract has led to an emerging field of investigation about the potential role of urinary microbiome in the pathogenesis of urinary bladder cancer. Urinary microbiota refers to the different microbe populations present in the urinary tract while a variety of genetic, environmental, and experimental parameters have been investigated as predisposing factors of microbial composition. Different methods of urine collection as well as experimental methodology on microbiome’s characterization consist of well-defined factors that may alter the microbial composition. Few preliminary data have been reported so far implicating microorganisms as causative and prognostic factors of bladder tumorigenesis, examining mostly midstream-voided urine samples while the most commonly used analyzing technique was 16S rRNA sequencing method. In the present study, a protocol of microbiome identification using proteomics is reported analyzing differences in microbial composition between bladder cancer patients and healthy controls while a review of the current evidence is presented. To the best of our knowledge, proteomics has not been described as a possible method of microbiome characterization before.

PD37-03 EXPRESSION OF MUTANT PIK3CA IN MURINE UROTHELIAL CELLS PROVIDES A NOVEL MODEL OF EARLY STAGE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-03 EXPRESSION OF MUTANT PIK3CA IN MURINE UROTHELIAL CELLS PROVIDES A NOVEL MODEL OF EARLY STAGE BLADDER CANCER Lauren Shuman, Hironobu Yamashita, Thomas Wildermuth Hershey, Xue-Ru Wu, Joshua Warrick, and David DeGraff, Hershey Lauren Shuman Lauren Shuman More articles by this author , Hironobu YamashitaHironobu Yamashita More articles by this author , Thomas Wildermuth HersheyThomas Wildermuth Hershey More articles by this author , Xue-Ru WuXue-Ru Wu More articles by this author , Joshua WarrickJoshua Warrick More articles by this author , and David DeGraff, HersheyDavid DeGraff, Hershey More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite the fact that approximately 70% of newly diagnosed bladder cancers are non-invasive tumors with high rates of recurrence, non-invasive bladder cancer is significantly understudied. In part, the general lack of appropriate models to validate the contribution of specific molecular drivers in bladder tumorigenesis is a significant issue. Activating mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) are a frequent event in early stage bladder cancer, yet an in vivo model for understanding these mutations in bladder cancer is not available. METHODS: To address this gap, we have created a novel Upk2-Cre/Pik3caH1047R mouse model which expresses one or two copies of mutant Pik3caH1047R in a urothelial-specific manner. Experimental and genetic control mice were aged for 6 or 12 months, and bladders were collected for analysis. Western blotting and immunohistochemistry was used to confirm the functionality of mutant Pik3ca as well as to characterize urothelial expression differences. RESULTS: Mutant Pik3ca activity was confirmed by immunohistochemistry for phospho-Akt(Ser473). At 6 months of age, mice carrying one or two copies of mutant Pik3ca developed urothelial hyperplasia. While mice with one copy of Pik3caH1047R failed to progress to frank malignancy, activation of two copies of mutant Pik3ca resulted in the development of papillary bladder cancer at 12 months of age. Increased proliferation of bladder urothelium was confirmed by measuring urothelial thickness as well as immunohistochemistry for Ki67. Consistent with human papillary bladder cancer, immunohistochemistry also revealed high expression of luminal markers Foxa1, Pparγ, and Gata3, as well as low expression of basal markers Krt5/6 and Krt14. CONCLUSIONS: These data provide evidence of the establishment and characterization of Upk2-Cre/Pik3caH1047R mice as a novel, clinically relevant model of non-invasive bladder cancer. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e655-e656 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lauren Shuman More articles by this author Hironobu Yamashita More articles by this author Thomas Wildermuth Hershey More articles by this author Xue-Ru Wu More articles by this author Joshua Warrick More articles by this author David DeGraff, Hershey More articles by this author Expand All Advertisement Loading ...

PD37-11 TROPOMYOSIN ISOFORM SWITCHING IN UROTHELIAL BLADDER CANCER AS A DIAGNOSTIC BIOMARKER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-11 TROPOMYOSIN ISOFORM SWITCHING IN UROTHELIAL BLADDER CANCER AS A DIAGNOSTIC BIOMARKER Nada Humayun-Zakaria, Douglas Ward, Ben Abbotts, Maurice Zeegers, KK Cheng, Nick James, Richard Bryan, and Roland Arnold Nada Humayun-ZakariaNada Humayun-Zakaria More articles by this author , Douglas WardDouglas Ward More articles by this author , Ben AbbottsBen Abbotts More articles by this author , Maurice ZeegersMaurice Zeegers More articles by this author , KK ChengKK Cheng More articles by this author , Nick JamesNick James More articles by this author , Richard BryanRichard Bryan More articles by this author , and Roland ArnoldRoland Arnold More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The urgent unmet need for biomarker discovery for the early detection and surveillance of bladder cancer in parallel with advancements in high throughput sequencing has fuelled understanding of molecular biology and pathology, paving the way for ‘precision medicine’. Transcriptomic analyses of Tropomyosin (TPM) have revealed differential expression of isoforms with ‘isoform switching’ in the presence of urothelial bladder cancer (UBC) making them promising candidates as diagnostic biomarkers. METHODS: RNA-sequence quantification was performed on 3 cancer datasets (Cancer Cell Line Encyclopaedia (CCLE), The Cancer Genome Atlas (TCGA) and West Midland’s Bladder Cancer Prognosis Program (BCPP)) and one non-cancer dataset (GEO Accession number GSE133624) using a bespoke bioinformatics pipeline – ‘IsoTPM’. Wilcoxon rank sum determined p-values for pairwise-comparison of isoform expression in cancer vs non-cancer. Subsequent qPCR analysis with isoform-specific primers was performed on in-house CCLE cell lines followed by Sanger sequencing. Evaluation of TPM isoform expression at protein level was achieved using mass spectrometry (MS) on CCLE and BCPP samples to delineate peptides of interest. RESULTS: The differential expression (DE) of two TPM1 isoforms, TPM1-201 and TPM1-206, appeared as dominant transcripts in non-cancer and cancer datasets, respectively. TPM4 analyses revealed high abundance of TPM4-218 in cancer datasets in comparison to TPM4-221 found exclusively in non-cancer. Additionally, TPM4-205 was expressed in non-muscle-invasive tumours (BCPP). qPCR analysis showed the relative expressions of TPM1-206 and TPM4-218 in a range of cell lines. MS revealed ∼20 peptides, including splice-junction exons from TPM4-218 and TPM1-206, from both cancer datasets (BCPP and CCLE). CONCLUSIONS: Comparative analysis of non-cancer and cancer datasets demonstrates that TPM1-201 isoform switching to TPM1-206 accompanies early bladder tumorigenesis and/or epithelial-mesenchymal-transition (EMT). TPM4-218 is upregulated in all cancer datasets accompanied by downregulation of TPM4-221 also suggesting an association with bladder tumorigenesis. Source of Funding: QEHB charities funded NH-Z's research © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e659-e660 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nada Humayun-Zakaria More articles by this author Douglas Ward More articles by this author Ben Abbotts More articles by this author Maurice Zeegers More articles by this author KK Cheng More articles by this author Nick James More articles by this author Richard Bryan More articles by this author Roland Arnold More articles by this author Expand All Advertisement Loading ...

Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

To study the progression of bladder cancer from non-muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. SIGNIFICANCE: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

High CD39 expression is associated with the non-muscle-invasive phenotype of human bladder cancer.

Background: An accurate prediction of progression is critical to define the management of bladder cancer (BC). The ectonucleotidases CD39 and CD73 play strategic roles in calibrating purinergic signals via an extracellular balance between ATP and adenosine. The altered expression of these enzymes plays a potential role in tumor invasion and metastasis, therefore, has been proposed to be used for prognosis of solid tumor. In BC this is not yet clear.Objective: This study aimed to evaluate CD39 and CD73 expression in a cohort of patients with non-muscle-invasive (NMI) and muscle-invasive (MI) BC regard to its association with clinicopathological features.Materials and Methods: Retrospective clinical follow-up data and primary urothelial BC specimens of 162 patients were used (87 from patients who underwent transurethral resection and 75 from cystectomized patients). Tissue microarrays were constructed, and immunohistochemistry for CD39 and CD73 was performed to make associations with clinicopathological data.Results: Overall, 96 were NMI (59.3%) and 66 MI (40.7%). CD39 immunoreactivity in BC cells was found in 72% of the cases, while CD73 was found in 97%. High CD39 expression alone was more frequent in NMI BC (p < 0.001), while CD73 expression was not powerful to predict the stage of BC. The association of both markers confirmed that only CD39 has potential in BC prognosis.Conclusions: The altered expression of CD39 presented herein supports the idea that this ectonucleotidase may be involved in bladder tumorigenesis. High expression of CD39 in tumor cells is correlated with the early stage of BC.