Bladder Carcinogenesis Research Articles

An integrative approach for identification of smoking-related genes involving bladder cancer.

Tobacco smoking is one of the most important environmental risk factors involving bladder tumorigenesis. However, smoking-related genes in bladder carcinogenesis and corresponding genetic effects on bladder cancer risk remain unclear. Weighted correlation network analysis (WGCNA) underlying transcriptome of bladder cancer tissues was applied to identify smoking-related genes. The logistic regression model was utilized to estimate genetic effects of single nucleotide polymorphisms (SNPs) in smoking-related genes on bladder cancer risk in the Chinese and European populations with a total of 6510 cases and 6569 controls, as well as the interaction with smoking status. Transcriptome of cells and tissues was used to profile the expression pattern of candidate genes and their genetic variants. Our results demonstrated that a total of 24 SNPs in 14 smoking-related genes were associated with the risk of bladder cancer, of which rs9348451 in CDKAL1 exhibited an interaction with smoking status (ORinteraction = 1.38, Pinteraction = 1.08 × 10-2) and tobaccosmoking might combine with CDKAL1 rs9348451 to increase the risk of bladder cancer (Ptrend = 4.27 × 10-4). Moreover, rs9348451 was associated with CDKAL1 expression in bladder cancer, especially in smokers (P < 0.001). Besides, CDKAL1 was upregulated in bladder cancer compared to normal adjacent tissues, as well as upregulated via treatment of cigarette smoke extracts. This study highlights the important role of nurture and nature, as well as their interaction on tumorigenesis, which provides a new way to decipher the etiology of bladder cancer with smoking status.

AhRR methylation contributes to disease progression in urothelial bladder cancer.

Bladder Cancer (BCa) is the tenth most incident malignancy worldwide. BCa is mostly attributed to environmental exposure and lifestyle, particularly tobacco smoking. The Aryl Hydrocarbon Receptor Repressor (AhRR) participates in the induction of many enzymes involved in metabolizing carcinogens, including tobacco smoke components. Additionally, studies have shown that smoking demethylates the (AhRR) gene in blood, suggesting AhRR demethylation as a specific serum smoking biomarker. This study aimed to validate AhRR demethylation as a smoking biomarker in the target tissue and investigate its contribution to bladder carcinogenesis. AhRR percent methylation was tested for its association with patient smoking status and oncogenic outcome indicators, particularly p53, RB1, and FGFR3 activating mutations, muscle-invasiveness, and tumor grade, in 180 BCa tissue-based DNA. Results showed significantly higher AhRR percent methylation in muscle-invasive compared to non-muscle invasive tumors (42.86% vs. 33.98%; p= 0.011), while lower AhRR methylation was significantly associated with FGFR3 Codon 248 mutant genotype compared to wild-type (28.11% ± 9.44 vs. 37.87% ± 22.53; p= 0.036). All other tested associations were non-statistically significant. Although AhRR methylation did not predict smoking status in BCa tumors, it may be a contributor to carcinogenesis and disease progression. Our findings constitute the basis for further research.

The Impact of Meat Intake on Bladder Cancer Incidence: Is It Really a Relevant Risk?

Simple SummaryPublic health is severely challenged by bladder cancer (BC). There are few known risk factors for BC, but among these, diet seems to have a potential role in its etiology. However, the effect of meat is yet to be determined. In this study, we reviewed the influence of meat consumption on BC incidence. Our results showed different associations between meat consumption and BC depending on the type of meat consumed, the cooking method and the temperature used. Consequently, the promotion of healthy lifestyle interventions based on dietary factors can halt BC onset and can ameliorate the treatment efficacy of BC patients.Bladder cancer (BC) represents the second most common genitourinary malignancy. The major risk factors for BC include age, gender, smoking, occupational exposure, and infections. The BC etiology and pathogenesis have not been fully defined yet. Since catabolites are excreted through the urinary tract, the diet may play a pivotal role in bladder carcinogenesis. Meat, conventionally classified as “red”, “white” or “processed”, represents a significant risk factor for chronic diseases like cardiovascular disease, obesity, type 2 diabetes, and cancer. In particular, red and processed meat consumption seems to increase the risk of BC onset. The most accepted mechanism proposed for explaining the correlation between meat intake and BC involves the generation of carcinogens, such as heterocyclic amines and polycyclic aromatic hydrocarbons by high-temperature cooking. This evidence claims the consumption limitation of meat. We reviewed the current literature on potential biological mechanisms underlying the impact of meat (red, white, and processed) intake on the increased risk of BC development and progression. Toward this purpose, we performed an online search on PubMed using the term “bladder cancer” in combination with “meat”, “red meat”, “white meat” or “processed meat”. Although some studies did not report any association between BC and meat intake, several reports highlighted a positive correlation between red or processed meat intake, especially salami, pastrami, corned beef and bacon, and BC risk. We speculate that a reduction or rather a weighting of the consumption of red and processed meat can reduce the risk of developing BC. Obviously, this remark claims future indications regarding food education (type of meat to be preferred, quantity of red meat to be eaten and how to cook it) to reduce the risk of developing BC. Further well-designed prospective studies are needed to corroborate these findings.

Biomarkers to assess the risk of bladder cancer in patients presenting with haematuria are gender-specific.

IntroductionHaematuria is a common red flag symptom of urinary tract cancer. Bladder cancer (BC) is the most common cancer to present with haematuria. Women presenting with haematuria are often underdiagnosed. Currently, no gender-specific tests are utilized in clinical practice. Considerable healthcare resources are needed to investigate causes of haematuria and this study was set up to help identify markers of BC. The aim of the study was to define biomarker algorithms in haematuria patients using an expanded panel of biomarkers to diagnose BC and investigate if the algorithms are gender-specific.Materials and MethodsA total of n=675 patients with a history of haematuria were recruited from Northern Ireland hospitals. Patients were collected on a 2:1 ratio, non-BC (control) n=474: BC n=201. A detailed clinical history, urine and blood samples were collected. Biomarkers, known to be involved in the pathobiology underlying bladder carcinogenesis were investigated. Biomarkers differentially expressed between groups were investigated using Wilcoxon rank sum and linear regression.ResultsBiomarkers were gender specific. Two biomarker-algorithms were identified to triage haematuria patients; male – u_NSE, s_PAI-1/tPA, u_midkine, u_NGAL, u_MMP-9/TIMP-1 and s_prolactin (u=urine; s=serum); sensitivity 71.8%, specificity 72.8%; AUROC 0.795; and female urine biomarkers - IL-12p70, IL-13, midkine and clusterin; sensitivity 83.7%, specificity 79.7%; AUROC 0.865. Addition of the clinical variable infection to both algorithms increased both AUROC to 0.822 (DeLong p=0.014) and to 0.923 (DeLong p=0.004) for males and females, respectively. Combining clinical risk factors with biomarker algorithms would enable application of the algorithms to triage haematuria patients.ConclusionUsing gender-specific biomarker algorithms in combination with clinical risks that are associated with BC would allow clinicians to better manage haematuria patients and potentially reduce underdiagnosis in females. In this study, we demonstrate, for the first time, that blood and urine biomarkers are gender-specific when assessing risk of BC in patients who present with blood in their urine. Combining biomarker data with clinical factors could improve triage when referring patients for further investigations.

BK and JC polyomaviruses and risk of urothelial bladder carcinoma: a preliminary study in the northern shores of Persian Gulf, Iran

BackgroundBladder cancer is a challenging public health concern in South of Iran because of its high prevalence and the related medical expenses. Although the exact etiology of bladder cancer remains unknown, given the cell transforming ability and oncogenic potential of the members of Polyomaviridae families, this study was conducted to evaluate the magnitude of BK polyomavirus (BKPyV) and John Cunningham polyomavirus (JCPyV) among patients with bladder cancer residents in the northern shores of the Persian Gulf, South of Iran.MethodsTotally 211 patients with bladder cancer were enrolled in this study. Bladder biopsy samples of these patients and patients with interstitial cystitis as well as autoptic samples of healthy bladder were tested for detection of BKPyV and JCPyV by semi-nested PCR–RFLP followed by sequencing.ResultsBKPyV and JCPyV were detected in 1.7% and 6.1% of bladder cancer samples, respectively. These samples were infected with JCPyV genotypes 2, 3 and 6 and BKPyV genotypes I and IV. BKPyV and JCPyV coinfection was detected in 2 samples. Moreover, one of the healthy bladder samples was positive for BKPyV, and one of the interstitial cystitis samples was positive for JCPyV. Although the majority of infected patients were in the age group 70–79 years, male, residents in Tangestan, stage Ta–T1, and low-grade and high-grade papillary urothelial carcinoma, the prevalence of BKPyV and JCPyV among patients with bladder cancer was not statistically associated with age, gender, place of residency, and stage and grade of the tumor.ConclusionDespite identifying BKPyV and JCPyV in a number of bladder cancer biopsy specimens and the high prevalence of bladder cancer among people resident in South of Iran, it is suggested that these viruses are unlikely to be effective causative factors in bladder carcinogenesis in this region. Therefore, environmental risk factors and genetic backgrounds may have a more prominent role than human polyomaviruses in the development of bladder cancer in South of Iran.

Chemopreventive Efficacy of Thymoquinone in Chemically Induced Urinary Bladder Carcinogenesis in Rat.

The effects of thymoquinone (TQ) in a carcinogen-based models of urinary bladder cancer were evaluated, using 45 male rats in five groups. In negative control (n = 10), only tap water was given. In positive control (n = 10), the rats received 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in drinking water for 9 weeks. In preventive groups with 25 mg/kg (n = 10) and 50 mg/kg (n = 10), oral TQ was concurrently given with 0.05% BBN for 9 weeks and continued for one more week after cessation of BBN. Preventive-treatment group (n = 5) received 50 mg/kg TQ orally for 20 weeks. Five rats from each group were sequentially sacrificed in two phases: the induction phase at 12th week (except the last group) and the rest in postinduction phase at 20th week. The bladders were examined macroscopically for lesion formation, and the masses were submitted for histopathological evaluation. Markers for total oxidant status (TOS), inflammation (nuclear factor kappa B (NF-κB)), and angiogenesis (vascular endothelial growth factor (VEGF)) were also assessed. There was a reduced number of bladder lesions in the TQ groups versus the carcinogen group at both phases. Histopathological findings demonstrated a significant improvement in the abnormal morphological changes in the urothelium of the TQ-treated groups. Thymoquinone exerted a significant antioxidant and anti-inflammatory effect by a decrease in serum level of TOS and NF-κB at week 12 which was maintained low in phase two at week 20. The serum level of VEGF was also alleviated in the induction phase at week 12 and maintained low in postinduction period. In TQ preventive-treatment approach, a nonsignificant elevation of serum level of TOS and NF-κB and slight reduction in VEGF were observed at the end of the experiment. These data suggest that TQ may be effective in preventing bladder carcinogenesis, and the suggested mechanisms might be related to antioxidant, prooxidant, and anti-inflammatory properties of TQ.

Environmental exposure to microcystin-LR increases the risks of urinary bladder proliferation and carcinogenesis: Evidence from case control, animal, and in vitro studies

To date, no reported studies have explored the impacts of microcystin-LR (MC-LR) on bladder tissues, and even the occurrence of bladder cancer. The current study explores the role of MC-LR in the development of bladder cancer through human observation and experimental research. In the population study, the odds ratio of bladder cancer for MC-LR was 6.073 (95 % CI, 2.117–17.422) after adjusting interference confounders. MC-LR is mainly located in the nucleus of epithelial cells in bladder cancer tissues instead of normal tissues. A positive association was observed between MC-LR and advanced tumor stage in serum and tissues. The animal study confirmed that prolonged MC-LR treatment promoted the bladder cancer phenotype accompanied by urinary bladder proliferation. In vitro, we indicated that MC-LR activated the PI3K/AKT/GSK3β/Cyclin D1 and JAK2/STAT3/Bcl2 signaling pathways to induce the growth of SV-HUC-1 cells. Moreover, MC-LR promoted the angiogenesis of SV-HUC-1 cells through PI3K/AKT/mTOR/HIF-1α/VEGF pathway. Our study provided the first evidence that prolonged MC-LR treatment increases the incidence of bladder cancer from human investigations, mice models, and in vitro studies, implying the profound importance of the investigation of MC-LR for public health.

Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 μM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

A Comprehensive Transcriptomic Analysis of Arsenic-Induced Bladder Carcinogenesis.

Arsenic (sodium arsenite: NaAsO2) is a potent carcinogen and a known risk factor for the onset of bladder carcinogenesis. The molecular mechanisms that govern arsenic-induced bladder carcinogenesis remain unclear. We used a physiological concentration of NaAsO2 (250 nM: 33 µg/L) for the malignant transformation of normal bladder epithelial cells (TRT-HU1), exposed for over 12 months. The increased proliferation and colony-forming abilities of arsenic-exposed cells were seen after arsenic exposure from 4 months onwards. Differential gene expression (DEG) analysis revealed that a total of 1558 and 1943 (padj < 0.05) genes were deregulated in 6-month and 12-month arsenic-exposed TRT-HU1 cells. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that cell proliferation and survival pathways, such as the MAPK, PI3K/AKT, and Hippo signaling pathways, were significantly altered. Pathway analysis revealed that the enrichment of stem cell activators such as ALDH1A1, HNF1b, MAL, NR1H4, and CDH1 (p < 0.001) was significantly induced during the transformation compared to respective vehicle controls. Further, these results were validated by qPCR analysis, which corroborated the transcriptomic analysis. Overall, the results suggested that stem cell activators may play a significant role in facilitating the arsenic-exposed cells to gain a survival advantage, enabling the healthy epithelial cells to reprogram into a cancer stem cell phenotype, leading to malignant transformation.

Exploring urine sediments as a non-invasive method for DNA methylation detection in bladder cancer

BackgroundThe main epigenetic event occurring during the bladder carcinogenesis process is DNA methylation, affecting genes involved in various metabolic pathways and cell regulation. The use of biological fluids such as urine sediments could be used as a non-invasive approach to enhance bladder cancer management. In this study, we aim to determine the promoter methylation status of a panel of genes in bladder cancer on tumor biopsies and urine sediments to evaluate the usefulness of urine samples as a non-invasive approach for methylation status assessment.MethodsUsing the methylation-specific PCR technique, we explored the promoter methylation status of hTERT, TWIST1, VIM and NID2 genes in 40 tumor biopsies and their paired urine samples from Moroccan bladder cancer patients.ResultsIn this study, bladder tumors showed promoter hypermethylation frequency of individual genes as 90%, 85%, 62.5% and 72.5% in TWIST1, hTERT, NID2 and VIM genes, respectively.Interestingly, the specificity of methylation detection in urine samples was 100% and the sensitivity to detect hypermethylation of TWIST1, hTERT, NID2 and VIM genes reached 91.7%; 97.1%; 84% and 82.8%, respectively.ConclusionsOur results clearly show that the assessment of promoter hypermethylation in urine samples is highly specific and has high sensitivity. Furthermore, urine sediments would be a useful approach to detect the DNA methylation status of genes and its potential association with bladder cancer development.

The carbonic anhydrase inhibitor acetazolamide inhibits urinary bladder cancers via suppression of β-catenin signaling.

Carbonic anhydrases (CAs) play an important role in maintaining pH homeostasis. We previously demonstrated that overexpression of CA2 was associated with invasion and progression of urothelial carcinoma (UC) in humans. The purpose of the present study was to evaluate the effects of the CA inhibitor acetazolamide (Ace) on N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced bladder carcinogenesis in mice and explore the function of CA2 in muscle invasion by UC. Male mice were treated with 0.025% (experiment 1) or 0.05% BBN (experiment 2) in their drinking water for 10 weeks, then treated with cisplatin (Cis), Ace, or Cis plus Ace for 12 weeks. In experiment 1, the overall incidence of BBN‐induced UCs was significantly decreased in the BBN→Ace and BBN→Cis+Ace groups. In experiment 2, the overall incidence of BBN‐induced UCs was significantly decreased in the BBN→Cis+Ace group, and the incidence of muscle invasive UC was significantly decreased in both the BBN→Ace and the BBN→Cis+Ace groups. We also show that overexpression of CA2 by human UC cells T24 and UMUC3 significantly increased their migration and invasion capabilities, and that Ace significantly inhibited migration and invasion by CA2‐overexpressing T24 and UMUC3 cells. These data demonstrate a functional association of CA2 with UC development and progression, confirming the association of CA2 with UC that we had shown previously by immunohistochemical analysis of human UC specimens and proteome analysis of BBN‐induced UC in rats. Our finding that inhibition of CA2 inhibits UC development and muscle invasion also directly confirms that CA2 is a potential therapeutic target for bladder cancers.

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.

Abstract 745: Molecular landscape of arsenic-induced bladder carcinogenesis

Abstract Introduction: Epidemiological, pre-clinical, and clinical studies have linked chronic arsenic (sodium arsenite: NaAsO2) exposure to a myriad of adverse health dysfunctions including the development of bladder cancer (BCa). However, most of the studies on arsenic-induced BCa have been performed on either unsuitable cell lines or using a higher concentration of arsenic (mM), which does not reflect exposure levels in a real-world setting. We used the telomerase reverse transcriptase (HU-hTERT1) immortalized normal bladder epithelial and a median physiological concentration of NaAsO2 (250 nM: 33 mg/L), to determine the transcriptomic and phenotypic changes during malignant transformation of HU-hTERT1 cells. Methods: HU-hTERT1 cells were exposed to 250nM arsenic for 12 months. To analyze the effect of arsenic exposure we performed cell viability, cellular proliferation, and clonogenic assays. RNA Seq data was used to perform pathway enrichment analysis, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) studies. Results: BCa patients had significantly higher (4-5 fold) levels of arsenic (20-50 mg/L) in their urine when compared to their healthy counterparts (&amp;lt;8 mg/L). Acute exposure to high concentration of (&amp;gt; 400nM) inhibited the HU-hTERT1 growth, whereas concentrations less than 400nM did not adversely affect the viability of these cells. HU-hTER1 cells began forming colonies after six months of chronic NaAsO2 exposure, with the number of colonies increasing concurrently with the exposure time. GO and KEGG analysis showed a total of 2174 genes that are differentially expressed in arsenic-treated HU-hTER1 transforming cells (0 vs 12 months). The upregulated genes (1392) were involved in cellular response to unfolded protein, response to endoplasmic reticulum stress, proteasome-mediated ubiquitin-dependent protein degradation, autophagy signaling, and tumor angiogenesis(P&amp;lt;0.05). Interestingly, the PPI analysis demonstrated a significant upregulation in histone modification of genes. These genes were enriched for the binding sites of transcription factors that are key regulators of carcinogenic pathways including ataxia-telangiectasia mutated (ATM) kinase and cell cycle regulatory pathways demonstrating a novel link between NaAsO2 exposure and BCa. Conclusion: An in vitro model of NaAsO2-induced malignant transformation was developed. Identifying precise molecular events that lead to arsenic-induced malignant transformation of normal urothelial cells would be an important step in defining therapy for BCa. Citation Format: Ajit Kumar Navin, Vaibhav Shukla, Balaji Chandrasekaran, Ashish Tyagi, Uttara Saran, Murali K. Ankem, Chendil Damodaran. Molecular landscape of arsenic-induced bladder carcinogenesis [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 745.

Abstract 6032: Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice

Abstract Despite recent advances in molecular profiling of bladder cancer, molecular mechanisms for urothelial carcinogenesis have not fully understood. It is partly because the lack of faithful disease models that accurately recapitulate human bladder cancer. In our previous study, we have analyzed mutation landscape of chemical-induced mouse bladder cancer and human bladder cancer by exome-sequence and found that some components of histone methyltransferase complex like Kmt2c, Kmt2d, Kdm6a were highly prevalent as common mutations and associated with Trp53. Additionally, the downregulation of tumor suppressor gene PTEN is known in human muscle invasive bladder cancer (MIBC). We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have tried to make MIBC mouse model using genetically engineered mouse in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system, combined with CRISPR/Cas9 gene editing technique and 3D organoid culture system. Eight-week-old mice harboring Krt5CreERT2/+; LSL-Cas9; LSL-Trp53+/R172H were used in this study. After introducing gene recombination by administration of tamoxifen, we generated organoids from bladder epithelial cells. For further gene editing, we infected adeno-associated virus with sgPten and sgMll3 to the organoids, which successfully edited the target genes. There seemed to be no significant differences in growth of the organoids in culture between the wild-type and gene-edited organoids. However, gene-edited organoids showed tumorigenesis in vivo when inoculated in subcutaneous space, renal subcapsular space or bladder submucosa of athymic mice, whereas wild-type organoids did not show tumorigenesis in vivo. Histologically, the organoid-derived tumors showed morphological similarity to human urothelial carcinoma with squamous differentiation and intra-tumoral heterogeneity. Furthermore, tumor derived organoid has a potential of tumor development in immune-competent mice. These tumors also maintain squamous phenotype and tumor heterogeneity. These findings suggest that the technique is a promising approach toward the establishment of a useful model of human bladder cancer for the elucidation of bladder carcinogenesis including driver genes. In addition, we think our syngeneic mouse model may be useful tool for further research of tumor immunity and immune checkpoint inhibitor drugs. Citation Format: Akihiro Hamada, Yuki Kita, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takeshi Sano, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice [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 6032.

The origin of bladder cancer from mucosal field effects

SummaryWhole-organ mapping was used to study molecular changes in the evolution of bladder cancer from field effects. We identified more than 100 dysregulated pathways, involving immunity, differentiation, and transformation, as initiators of carcinogenesis. Dysregulation of interleukins signified the involvement of inflammation in the incipient phases of the process. An aberrant methylation/expression of multiple HOX genes signified dysregulation of the differentiation program. We identified three types of mutations based on their geographic distribution. The most common were mutations restricted to individual mucosal samples that targeted uroprogenitor cells. Two types of mutations were associated with clonal expansion and involved large areas of mucosa. The α mutations occurred at low frequencies while the β mutations increased in frequency with disease progression. Modeling revealed that bladder carcinogenesis spans 10–15 years and can be divided into dormant and progressive phases. The progressive phase lasted 1-2 years and was driven by β mutations.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

A long noncoding RNA GTF2IRD2P1 suppresses cell proliferation in bladder cancer by inhibiting the Wnt/β‑catenin signaling pathway.

BackgroundThere is growing evidence that long non-coding RNAs (LncRNAs) are key in the development of a variety of human tumors. However, the role of lncRNA GTF2IRD2P1 has not been well studied in cancer. The impact of GTF2IRD2P1 on the biological function and clinical relevance in bladder cancer is largely unknown. This study aimed to investigate the biological role of GTF2IRD2P1 in bladder evolution and carcinogenesis.MethodsWe used bioinformatics to obtain the lncRNA GTF2IRD2P1 from bladder urothelial carcinoma (BLCA) in The Cancer Genome Atlas (TCGA) database. The expression of lncRNA GTF2IRD2P1 was detected by qRT-PCR. The CCK8 assay and flow cytometry were used to detect the lncRNA GTF2IRD2P1 function on the proliferation of bladder cancer cells. A western blot was used to calculate the protein level of cell cycle proteins and Wnt signaling pathway proteins. The effect of lncRNA GTF2IRD2P1 on tumorigenesis of bladder cancer was confirmed by a xenograft nude mouse model.ResultsGTF2IRD2P1 expression was found to be lower in both human bladder cancer tissues and cell lines (UM-UC-3, RT4, and 5637), and elevated in T24 compared to the corresponding normal controls. GTF2IRD2P1 expression was also enhanced after transfection of UM-UC-3 cells with the overexpression vector. Meanwhile, overexpression of GTF2IRD2P1 inhibited the proliferation of UM-UC-3 and prolonged the cell cycle. The silencing of GTF2IRD2P1 significantly increased the proliferation and shortened the cell cycle of T24 cells and induced Wnt signaling activity to promote the progression of bladder cancer. Similarly, the transplanted tumor nude mouse model demonstrated that silencing GTF2IRD2P1 strengthens the progression of bladder cancer by targeting the Wnt signaling pathway.

Cigarette Smoke Containing Acrolein Contributes to Cisplatin Resistance in Human Bladder Cancers through the Regulation of HER2 Pathway or FGFR3 Pathway.

Cisplatin-based chemotherapy is the first-line therapy for bladder cancer. However, cisplatin resistance has been associated with the recurrence of bladder cancer. Previous studies have shown that activation of FGFR and HER2 signaling are involved in bladder cancer cell proliferation and drug resistance. Smoking is the most common etiologic risk factor for bladder cancer, and there is emerging evidence that smoking is associated with cisplatin resistance. However, the underlying mechanism remains elusive. Acrolein, a highly reactive aldehyde, is abundant in tobacco smoke, cooking fumes, and automobile exhaust fumes. Our previous studies have shown that acrolein contributes to bladder carcinogenesis through the induction of DNA damage and inhibition of DNA repair. In this study, we found that acrolein induced cisplatin resistance and tumor progression in both non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) cell lines RT4 and T24, respectively. Activation of HER2 and FGFR3 signaling contributes to acrolein-induced cisplatin resistance in RT4 and T24 cells, respectively. Furthermore, trastuzumab, an anti-HER2 antibody, and PD173074, an FGFR inhibitor, reversed cisplatin resistance in RT4 and T24 cells, respectively. Using a xenograft mouse model with acrolein-induced cisplatin-resistant T24 clones, we found that cisplatin combined with PD173074 significantly reduced tumor size compared with cisplatin alone. These results indicate that differential molecular alterations behind cisplatin resistance in NMIBC and MIBC significantly alter the effectiveness of targeted therapy combined with chemotherapy. This study provides valuable insights into therapeutic strategies for cisplatin-resistant bladder cancer.

A murine MODEL of e-cigarettes exposure to evaluate the carcinogenic effects of vaping on the bladder urothelium.

566 Background: Data evaluating the carcinogenic potential of e-cigarette (ECS) by-products on the bladder urothelium is scarce, and to date, no group has studied the direct impact of ECS on the urothelium at the genomic and epigenetic level. While it has been published that ECS exposure causes bladder hyperplasia in mice, no bladder tumors formed and the field is therefore lacking appropriate preclinical models to be able to study ECS related bladder cancer. Importantly, whole organ mapping studies of tumor bearing human bladders have identified DNA methylation field changes in “adjacent normal” urothelium as early events in bladder carcinogenesis. Whether ECS exposure results in early field alterations of DNA methylation in the urothelium is unknown. Methods: Mice (4 females, 4 males; 35 weeks old) were exposed to 12% nicotine (120mg/ml) in 1:1 polyethylene glycol: vegetable glycerol (PG/VG) or vehicle (PG/VG) daily: 3 hr sessions, 3 sec vape duration, 10 min interval between vapes. Nicotine and cotinine serum concentrations were measured at 3 hrs, and then at 5 days using LC-MS. Bladders were harvested after 4 weeks. DNA was extracted from microdissected urothelial layer. Reduced representation bisulfite sequencing (RRBS) was performed (Zymo Research) using 100ng of DNA input and paired-end chemistry. Alignment was performed using Bismark. Number of loci covered by each sample was determined and data was filtered to only include loci with &gt; 10 reads. Differentially methylated loci were determined. Adjusted p-value &lt; 0.01 was used as threshold to generate heatmaps. Gene Set Enrichment Analysis was performed to contextualize the DNA methylation changes. Results: Acute and repeated exposure resulted in significantly higher levels of nicotine and cotinine serum concentration in the nicotine group vs. the vehicle group without significant adverse events. No tumors were observed on gross visualization at the time of urothelium harvest. The number of loci in the RRBS libraries with &gt; 10 reads ranged from 130,000 to 250,000. 53 loci were differentially methylated between nicotine and vehicle. These loci did not correspond to the top hyper/hypo methylated genes in premalignant bladder tissue from recently reported whole-cystectomy mapping. Conclusions: We established an effective and safe murine model of ECS exposure closely resembling the human experience of vaping. Significant changes of DNA methylation were observed in mice exposed to ECS vapor vs. a vehicle control after four weeks of daily exposure. Ongoing experiments include longer vaping exposures as well as comparison of RRBS to a recently developed commercial mouse methylarray. Understanding epigenetic and genomic changes as a result of ECS exposure in preclinical models can serve as a foundation for studies developing assays to identify ECS users whose bladders contain early field defects that may place them at higher risk for bladder cancer.

Prevalence of bladder cancer in Costello syndrome: New insights to drive clinical decision-making.

Costello syndrome (CS) is a rare disorder affecting development and growth characterized by cancer predisposition and caused by mutations in HRAS proto-oncogene. Somatic HRAS mutations drive bladder carcinogenesis. The aim of this study was to analyze prevalence and histological characterization of bladder cancer (BC) in a cohort of patients with CS to help clinicians plan effective management strategies. This study included 13 patients above 10 years of age with molecular diagnosis of CS. Screening cystoscopies (31 total procedures) were performed to exclude BC. Any lesion was analyzed through cold-cup biopsy or trans-urethral resection of the bladder. According to histology, patients were followed-up with urinalysis and abdominal ultrasound yearly, and cystoscopies every 12-24months. During study enrollment, bladder lesions (often multifocal) were detected in 11/13 patients. Histological analysis documented premalignant lesions in 90% of cystoscopies performed, epithelial dysplasia in 71%, and papillary urothelial neoplasm of low-malignant potential in 19%. BC G1/low grade (Ta) were removed in 10%. Overall, 76% of patients showed a bladder lesion at first cystoscopy. The present findings document that individuals with CS aged 10 years and older have high prevalence of bladder lesions (premalignant/malignant), highlighting the importance of personalized screening protocols.