UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer.

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

Construction and validation of a prognostic model for bladder cancer based on disulfidptosis-related lncRNAs.

BACKGROUND: Bladder cancer (BLCA) is a prevalent and aggressive cancer associated with high mortality and poor prognosis. Currently, studies on the role of disulfidptosis-related long non-coding RNAs (DRLs) in BLCA are limited. This study aims to construct a prognostic model based on DRLs to improve the accuracy of survival predictions for patients and identify novel targets for therapeutic intervention in BLCA management. METHODS: Transcriptomic and clinical datasets for patients with BLCA were obtained from The Cancer Genome Atlas. Using multivariate Cox regression and least absolute shrinkage and selection operator techniques, a risk prognostic signature defined by DRLs was developed. The model's accuracy and prognostic relevance were assessed through Kaplan-Meier survival plots, receiver operating characteristic curves, concordance index, and principal component analysis. Functional and pathway enrichment analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis, were conducted to elucidate the underlying biological processes. Immune cell infiltration was quantified using the CIBERSORT algorithm. Differences and functions of immune cells in different risk groups were evaluated through single-sample Gene Set Enrichment Analysis. The Tumor Immune Dysfunction and Exclusion predictor and tumor mutational burden (TMB) assessments were utilized to gauge the likelihood of response to immunotherapy. Drug sensitivity predictions were made using the Genomics of Drug Sensitivity in Cancer database. RESULTS: A robust 8-DRL risk prognostic model, comprising LINC00513, SMARCA5-AS1, MIR4435-2HG, MIR4713HG, AL122035.1, AL359762.3, AC006160.1, and AL590428.1, was identified as an independent prognostic indicator. This model demonstrated strong predictive power for overall survival in patients with BLCA, revealing significant disparities between high- and low-risk groups regarding tumor microenvironment, immune infiltration, immune functions, TMB, Tumor Immune Dysfunction and Exclusion scores, and drug susceptibility. CONCLUSION: This study introduces an innovative prognostic signature of 8 DRLs, offering a valuable prognostic tool and potential therapeutic targets for bladder carcinoma. The findings have significant implications for TMB, the immune landscape, and patient responsiveness to immunotherapy and targeted treatments.

Concomitant decrease of E- and A-FABP expression predicts worse survival in urothelial bladder cancer patients.

Non-muscle invasive bladder cancers (NMIBC) pTa-pT1 are depicted by a high risk of recurrence and/or progression with an unpredictable clinical evolution. Our aim was to identify, from the original resection specimen, tumors that will progress to better manage patients. We previously showed that A-FABP (Adipocyte- Fatty Acid Binding Protein) loss predicted NMIBC progression. Here we determined by immunohistochemistry the prognostic value of E-FABP (Epidermal-Fatty Acid Binding Protein) expression in 210 tumors (80 pTa, 75 pT1, 55 pT2-T4). Thus, E-FABP low expression was correlated with a high grade/stage, the presence of metastatic lymph nodes, and visceral metastases (p < 0.001). Unlike A-FABP in NMIBC, E-FABP low expression was not associated with RFS or PFS in Kaplan-Meier analysis. But patients of the overall cohort with a high E-FABP expression had a longer mOS (53.8 months vs. 29.3 months, p = 0.029). The immunohistochemical analysis on the same NMIBC tissue sections revealed that when A-FABP is absent, a high E-FABP expression is detected. E-FABP could compensate A-FABP loss. Interestingly, patients, whose original tumor presents both low E-FABP and negative A-FABP, had the worse survival, those maintaining the expression of both markers had better survival. To conclude, the combined evaluation of A- and E-FABP expression allowed to stratify patients with urothelial carcinoma for optimizing treatment and follow-up.

Nuclear translocation of ISG15 regulated by PPP2R2B inhibits cisplatin resistance of bladder cancer.

Cisplatin resistance is a major challenge for systemic therapy against advanced bladder cancer (BC). Little information is available on the regulation of cisplatin resistance and the underlying mechanisms require elucidation. Here, we detected that downregulation of the tumor suppressor, PPP2R2B (a serine/threonine protein phosphatase 2 A regulatory subunit), in BC promoted cell proliferation and migration. What's more, low PPP2R2B expression was correlated with cisplatin resistance. In vitro and in vivo experiments verified that PPP2R2B could promote BC sensitivity to cisplatin. In terms of mechanism, we identified a novel function of PPP2R2B as a nucleocytoplasmic transport molecule. PPP2R2B promoted ISG15 entry into the nucleus by mediating binding of IPO5 with ISG15. Nuclear translocation of ISG15 inhibited DNA repair, further increasing ISG15 expression through activation of the STING pathway. Besides, PPP2R2B was down-regulated by SUV39H1-mediated histone 3 lysine 9 trimethylation, which could be restored by the SUV39H1-specific inhibitor, chaetocin. Our data suggest that PPP2R2B expression level is a potential biomarker for chemotherapy response and that chemotherapy in combination with chaetocin may be a feasible treatment strategy for patients with BC.

Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

PTBP1-mediated biogenesis of circATIC promotes progression and cisplatin resistance of bladder cancer.

Background: Cisplatin (DDP) based combination chemotherapy is a vital method for the treatment of bladder cancer (BLca). Chemoresistance easily occurs in the course of cisplatin chemotherapy, which is one of the important reasons for the unfavorable prognosis of BLca patients. Circular RNAs (circRNAs) are widely recognized for their role in the development and advancement of BLca. Nevertheless, the precise role of circRNAs in DDP resistance for BLca remains unclear. Methods: To study the properties of circATIC, sanger sequencing, agarose gel electrophoresis and treatment with RNase R/Actinomycin D were utilized. RT-qPCR assay was utilized to assess the expression levels of circRNA, miRNA and mRNA in BLca tissues and cells. Functional experiments were conducted to assess the function of circATIC in BLca progression and chemosensitivity in vitro. Various techniques such as FISH, Dual-luciferase reporter assay, TRAP, RNA digestion assay, RIP and ChIRP assay were used to investigate the relationships between PTBP1, circATIC, miR-1247-5p and RCC2. Orthotopic bladder cancer model, xenograft subcutaneous tumor model and xenograft lung metastasis tumor model were performed to indicate the function and mechanism of circATIC in BLca progression and chemosensitivity in vivo. Results: In our study, we observed that circATIC expression was significantly enhanced in BLca tissues and cells and DDP resistant cells. Patients with higher circATIC expression have larger tumor diameter, higher incidence of postoperative metastasis and lower overall survival rate. Further experiments showed that circATIC accelerated BLca cell growth and metastasis and induced DDP resistance. Mechanistically, alternative splicing enzyme PTBP1 mediated the synthesis of circATIC. circATIC could enhance RCC2 mRNA stability via sponging miR-1247-5p or constructing a circATIC/LIN28A/RCC2 RNA-protein ternary complex. Finally, circATIC promotes RCC2 expression to enhance Epithelial-Mesenchymal Transition (EMT) progression and activate JNK signal pathway, thus strengthening DDP resistance in BLca cells. Conclusion: Our study demonstrated that circATIC promoted BLca progression and DDP resistance, and could serve as a potential target for BLca treatment.

Similar genetic profile in early and late stage urothelial tract cancer.

INTRODUCTION: Urothelial tract cancer (UTC) ranks as the tenth most prevalent cancer and holds the seventh position in terms of mortality worldwide. Despite its prevalence and mortality ranking, there are still gaps in the knowledge of the mutational landscape in patients with advanced disease who have limited therapeutic options after multiple lines of prior treatment. This study compares the genomic and transcriptomic landscape, and targeted treatment options between metastatic UTC (mUTC) patients treated with multiple lines of therapy compared to newly diagnosed, untreated Muscle Invasive Bladder Cancer (MIBC). METHODS: We compared genomic and clinical data from two cohorts: mUTC patients who received multiple lines of therapy and were referred to the Copenhagen Prospective Personalized Oncology (CoPPO) project at Rigshospitalet, University of Copenhagen. Data for MIBC UTC patients were acquired from the Cancer Genome Atlas Bladder Cancer (TCGA BLCA) cohort. Biopsies in CoPPO were performed at the time of enrollment. 523 highly important cancer-related genes (TrueSight Oncology-500 targeted sequencing panel) were used from both cohorts for comparative analysis. Analyses included RNA count data to compare predicted molecular subtypes in each cohort separately. RESULTS: Patients from the CoPPO cohort had a lower median age at first-line treatment than the TCGA BLCA cohort, with no significant gender disparity. The predominant histology was urothelial cell carcinoma in both cohorts. Genomic analysis revealed no significant difference between the top mutated genes in the two cohorts, specifically looking into DNA damage repair genes. Molecular subtyping indicated a higher frequency of neuroendocrine differentiation in the CoPPO cohort. 13% of patients in the CoPPO cohort received targeted therapy based on genomic findings, and 16% received non-targeted treatment, totaling 29% receiving CoPPO treatment (9 patients). The remaining 71% received best supportive care. Kaplan-Meier analysis showed a non-significant survival benefit for the intervention group in the CoPPO cohort. CONCLUSION: When focusing on 523 highly relevant cancer genes, the mutational profile of mUTC patients who have undergone numerous treatment lines resembles that of newly diagnosed MIBC. These alterations can be targeted, indicating the potential advantage of early genomic testing for personalized treatment within clinical trials.

m6A reader IGF2BP2 promotes M2 macrophage polarization and malignant biological behavior of bladder cancer by stabilizing NRP1 mRNA expression.

BACKGROUND: Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) has been confirmed to play oncogenic role in many cancers. However, the role and mechanism of IGF2BP2 in bladder cancer (BCa) still deserves to be further revealed. METHODS: The mRNA and protein levels of IGF2BP2 and neuronilin-1 (NRP1) were detected by real-time quantitative PCR (RT-qPCR) and western blot. Cell proliferation, apoptosis, migration and invasion were determined using colony formation assay, EdU assay, CCK8 assay, flow cytometry and transwell assay. Xenograft tumor model was conducted to evaluate the role of IGF2BP2 in vivo. THP-1-M0 macrophages were co-cultured with the condition medium (CM) of BCa cells to induce polarization. M2 macrophage polarization was assessed by detecting the mRNA levels of M2 macrophage markers using RT-qPCR and measuring the proportion of M2 macrophage markers using flow cytometry. Moreover, MeRIP and RIP assay were performed to assess m6A level and the interaction between IGF2BP2 and NRP1. RESULTS: IGF2BP2 and NRP1 were upregulated in BCa tissues and cells. IGF2BP2 knockdown suppressed BCa cell growth and metastasis, as well as inhibited BCa tumor growth. After THP-1-M0 macrophages were co-cultured with the CM of BCa cells, the levels of M2 macrophage markers were markedly enhanced, while this effect was abolished by IGF2BP2 knockdown. IGF2BP2 level was positively correlated with NRP1 level, and it could increase NRP1 mRNA stability. NRP1 overexpression reversed the suppressive effect of IGF2BP2 knockdown on M2 macrophage polarization and BCa cell progression. CONCLUSION: m6A-reader IGF2BP2 enhanced M2 macrophage polarization and BCa cell progression by promoting NRP1 mRNA stability.

PD-L1 expression and its correlation with tumor biomarkers in Chinese urothelial bladder cancer.

Data on prevalence of programmed death ligand-1 (PD-L1) expression and its correlation with tumor biomarkers in Chinese patients with muscle-invasive urothelial bladder cancer (MIUBC) are scarce. We investigated the prevalence of PD-L1 expression, PD-L1 expression in tumor cells (TC) and immune cells (IC), and its correlation with tumor biomarkers (CD8+ T cells and tumor mutation burden [TMB]) in Chinese patients with newly diagnosed MIUBC (NCT03433924). Of 248 patients enrolled, 229 with PD-L1 data available were analysed. High PD-L1 expression (≥ 25% of TC or IC with PD-L1 expression) was observed in 120 (52.4%) patients. 59 cases showed positive staining in ≥ 25% of TC, and 82 cases had positive staining in ≥ 25% of IC. High expression of CD8+ T cell and TMB (> 10 mutations/megabase) was observed in 44.5% and 54.1% patients, respectively. A positive correlation was observed between percentage of TC with membrane PD-L1 positivity and CD8+ T cells (0.34; P < 0.001) and between IC with membrane PD-L1 positivity and CD8+ T cells (0.44; P < 0.001). There is high prevalence of PD-L1 expression in Chinese patients with MIUBC, suggesting that a sizable subset of patients could benefit from immunotherapy. The correlation of PD-L1 expression with tumor biomarkers provide clues for mechanisms underlying the effects of biomarkers for predicting efficacy.

Prognostic analysis of anoikis-related genes in bladder cancer: An observational study.

Anoikis is proved to play a crucial role in the development of cancers. However, the impact of anoikis on the prognosis of bladder cancer (BLCA) is currently unknown. Thus, this study aimed to find potential effect of anoikis in BLCA. The Cancer Genome Atlas (TCGA)-BLCA and GSE13507 cohorts were downloaded from TCGA and the Gene Expression Omnibus (GEO) databases, respectively. Differentially expressed genes (DEGs) were screened between BLCA and normal groups, which intersected with anoikis-related genes to yield anoikis-related DEGs (AR DEGs). Univariate COX, rbsurv, and multivariate COX analyses were adopted in order to build a prognostic risk model. The differences of risk score in the different clinical subgroups and the relevance between survival rate and clinical characteristics were explored as well. Finally, chemotherapy drug sensitivity in different risk groups was analyzed. In total, 78 AR DEGs were acquired and a prognostic signature was build based on the 6 characteristic genes (CALR, FASN, CSPG4, HGF, INHBB, SATB1), where the patients of low-risk group had longer survival time. The survival rate of BLCA patients was significantly differential in different groups of age, stage, smoking history, pathologic-T, and pathologic-N. The IC50 of 56 drugs showed significant differences between 2 risk groups, such as imatinib, docetaxel, and dasatinib. At last, the results of real time quantitative-polymerase chain reaction (RT-qPCR) demonstrated that the expression trend of CALR, HGF, and INHBB was consistent with the result obtained previously based on public databases. Taken together, this study identified 6 anoikis-related characteristic genes (CALR, FASN, CSPG4, HGF, INHBB, SATB1) for the prognosis of BLCA patients, providing a scientific reference for further research on BLCA.

Identification and validation of basement membrane-related genes predicting prognosis and immune infiltration associated with bladder cancer.

Bladder cancer (BC) is fatal during muscle invasion and treatment progress is limited. In this study, we aimed to construct and validate basement membrane (BM)-associated gene prognosis to predict BC progression and tumor immune infiltration correlation. We choreographed BM-related genes in the Cancer Genome Atlas (TCGA) database using COX regression and least absolute shrinkage and selection operator (LASSO) analysis, and the predictive value of BM-related genes was further validated by the GSE32548, GSE129845, and immunohistochemistry staining. All analyses were performed with R-version 4.2.2, and its appropriate packages. Three genes were identified to construct a gene signature to predictive of BC prognosis. We divided the TCGA database into 2 groups, and patients in the high-risk group had worse overall survival (OS) than those in the low-risk group. In GSE32548, we confirmed that patients in the high-risk group had a poorer prognosis compared to those in the low-risk group in terms of OS. Immunohistochemical staining of EPEMP1, GPC2, and ITGA3 showed significantly higher expression at the protein level in BC tissues than in normal tissues. The Spearman analysis showed risk score was positively correlated with B cell naïve, Macrophages M2, and Mast cells resting. stromal score, immune score, and ESTIMATE scores were significantly higher in the high-risk group. drugs sensitivity analysis showed IC50 of Cisplatin, Gemcitabine, and Methotrexate in the high-risk group was significantly higher than that in the low-risk group. We identified 3 prognostic genes from a novel perspective of BM genes as effective risk stratification tools for BC patients.

The Prognostic Hub Gene POLE2 Promotes BLCA Cell Growth via the PI3K/AKT Signaling Pathway.

BACKGROUND: BLCA is a common urothelial malignancy characterized by a high recurrence rate. Despite its prevalence, the molecular mechanisms underlying its development remain unclear. AIMS: This study aimed to explore new prognostic biomarkers and investigate the underlying mechanism of bladder cancer (BLCA). OBJECTIVE: The objective of this study is to identify key prognostic biomarkers for BLCA and to elucidate their roles in the disease. METHODS: We first collected the overlapping DEGs from GSE42089 and TCGA-BLCA samples for the subsequent weighted gene co-expression network analysis (WGCNA) to find a key module. Then, key module genes were analyzed by the MCODE algorithm, prognostic risk model, expression and immunohistochemical staining to identify the prognostic hub gene. Finally, the hub gene was subjected to clinical feature analysis, as well as cellular function assays. RESULTS: In WGCNA on 1037 overlapping genes, the blue module was the key module. After a series of bioinformatics analyses, POLE2 was identified as a prognostic hub gene in BLCA from potential genes (TROAP, POLE2, ANLN, and E2F8). POLE2 level was increased in BLCA and related to different clinical features of BLCA patients. Cellular assays showed that si-POLE2 inhibited BLCA proliferation, and si-POLE2+ 740Y-P in BLCA cells up-regulated the PI3K and AKT protein levels. CONCLUSION: In conclusion, POLE2 was identified to be a promising prognostic biomarker as an oncogene in BLCA. It was also found that POLE2 exerts a promoting function by the PI3K/AKT signaling pathway in BLCA.

Trends and Disparities in Next-Generation Sequencing in Metastatic Prostate and Urothelial Cancers.

Importance: Targeted therapies based on underlying tumor genomic susceptible alterations have been approved for patients with metastatic prostate cancer (mPC) and advanced urothelial carcinoma (aUC). Objective: To assess trends and disparities in next-generation sequencing (NGS) testing among patients with mPC and aUC. Design, Setting, and Participants: This retrospective cohort study used an electronic health record-derived database to extract deidentified data of patients receiving care from US physician practices, hospital-affiliated clinics, and academic practices. Patients diagnosed with mPC or aUC between March 1, 2015, and December 31, 2022, were included. Exposures: Social determinants of health evaluated by race and ethnicity, socioeconomic status (SES), region, insurance type, and sex (for aUC). Main Outcomes and Measures: The primary outcomes were (1) NGS testing rate by year of mPC and aUC diagnosis using Clopper-Pearson 2-sided 95% CIs and (2) time to NGS testing, which considered death as a competing risk. Cumulative incidence functions were estimated for time to NGS testing. Disparities in subdistributional incidence of NGS testing were assessed by race and ethnicity, SES, region, insurance type, and sex (for aUC) using the Fine-Gray modified Cox proportional hazards model, assuming different subdistribution baseline hazards by year of mPC and aUC diagnosis. Results: A total of 11 927 male patients with mPC (167 Asian [1.6%], 1236 Black [11.6%], 687 Hispanic or Latino [6.4%], 7037 White [66.0%], and 1535 other [14.4%] among 10 662 with known race and ethnicity) and 6490 patients with aUC (4765 male [73.4%]; 80 Asian [1.4%], 283 Black [4.8%], 257 Hispanic or Latino [4.4%], 4376 White [74.9%], and 845 other [14.5%] among 5841 with known race and ethnicity) were eligible and included. Both cohorts had a median age of 73 years (IQR, 66-80 years), and most underwent NGS testing before first-line treatment in the mPC cohort (1502 [43.0%]) and before second-line treatment in the aUC cohort (1067 [51.3%]). In the mPC cohort, the rates of NGS testing increased from 19.0% in 2015 to 27.1% in 2022, but Black patients (hazard ratio [HR], 0.75; 95% CI, 0.67-0.84) and Hispanic or Latino patients (HR, 0.70; 95% CI, 0.60-0.82) were less likely to undergo NGS testing. Patients with mPC who had low SES (quintile 1: HR, 0.74 [95% CI, 0.66-0.83]; quintile 2: HR, 0.89 [95% CI, 0.80-0.99]), had Medicaid (HR, 0.53; 95% CI, 0.38-0.74) or Medicare or other government insurance (HR, 0.89; 95% CI, 0.82-0.98), or lived in the West (HR, 0.81; 95% CI, 0.70-0.94) also were less likely to undergo testing. In the aUC cohort, the NGS rate increased from 14.1% in 2015 to 46.6% in 2022, but Black patients (HR, 0.76; 95% CI, 0.61-0.96) and those with low SES (quintile 1: HR 0.77 [95% CI, 0.66-0.89]; quintile 2: HR, 0.87 [95% CI, 0.76-1.00]) or Medicaid (HR, 0.72; 95% CI, 0.53-0.97) or Medicare or other government insurance (HR, 0.88; 95% CI, 0.78-0.99) were less likely to undergo NGS testing. Patients with aUC living in the South were more likely to undergo testing (HR, 1.29; 95% CI, 1.12-1.49). Conclusions and Relevance: These findings suggest that although NGS tumor testing rates improved over time, the majority of patients still did not undergo testing. These data may help with understanding current disparities associated with NGS testing and improving access to standard-of-care health care services.

Association of interleukin6 rs1800796 gene polymorphism and serum level with bladder cancer in Egyptian population.

BACKGROUND: Urinary bladder cancer (UBC)is a common tumor of the urinary tract. OBJECTIVES: To assess the diagnostic significance of IL6 rs1800796 gene polymorphism and IL6 serum level among Egyptian patients with UBC. DESIGN AND METHODS: One hundred patients with UBC were selected from the Mansoura Urology and Nephrology Center, in addition to 100 healthy control subjects; using PCR and ELISA techniques for IL6 detection. RESULTS: The rs1800796 GC, CC genotypes, and C allele were significantly more prevalent in the cases with bladder cancer compared to the healthy group (p < 0.001, = 0.021, < 0.001 respectively). There was a clear association between elevated levels of IL6 and bladder cancer versus the control group (median = 4.2, 0.89 respectively, p < 0.001). Serum IL6 levels showed significantly higher levels in patients carrying CC, followed by GC then GG genotypes. No significant association was found between IL6 rs1800796 gene polymorphism or serum level with demographic or laboratory data. CONCLUSION: It is suggested that there is a clear link between elevated IL6 levels as well as IL6 rs1800796 gene polymorphism with bladder cancer, suggesting their potential utility as biomarkers for the disease.

MyD88 Signaling Accompanied by Microbiota Changes Supports Urinary Bladder Carcinogenesis.

Urinary bladder cancer (BC) inflicts a significant impairment of life quality and poses a high mortality risk. Schistosoma haematobium infection can cause BC, and the urinary microbiota of BC patients differs from healthy controls. Importantly, intravesical instillation of the bacterium Bacillus Calmette-Guerin stands as the foremost therapy for non-muscle invasive BC. Hence, studying the receptors and signaling molecules orchestrating bacterial recognition and the cellular response in the context of BC is of paramount importance. Thus, we challenged Toll-like receptor 4 (Tlr4) and myeloid differentiation factor 88 (Myd88) knock-out (KO) mice with N-butyl-N-(4-hydroxylbutyl)-nitrosamine (BBN), a well-known urinary bladder carcinogen. Gut microbiota, gene expression, and urinary bladder pathology were followed. Acute exposure to BBN did not reveal a difference in bladder pathology despite differences in the animal's ability to recognize and react to bacteria. However, chronic treatment resulted in reduced cancer invasiveness among Myd88KO mice while the absence of functional Tlr4 did not influence BC development or progression. These differences correlate with a heightened abundance of the Faecalibaculum genus and the lowest microbial diversity observed among Myd88KO mice. The presented data underscore the important role of microbiota composition and MyD88-mediated signaling during bladder carcinogenesis.

Knockdown of AMIGO2 suppresses proliferation and migration through regulating PPAR-γ in bladder cancer.

PURPOSE: This study aims to reveal the relationship between AMIGO2 and proliferation, migration and tumorigenicity of bladder cancer, and explore the potential molecular mechanisms. METHODS: The expression level of AMIGO2 is measured by qRT-PCR and immunohistochemistry (IHC). Stable AMIGO2 knockdown cell lines T24 and 5637 were established by lentivirus transfection. Cell Counting Kit (CCK-8 assay) was produced to determine cell proliferation, flow cytometry analysis was utilized to detect cell cycle, and wound healing assay was proceeded to test migration ability of bladder cancer cells. Xenograft mouse model was established for investigating the effect of AMIGO2 on tumor formation in vivo. The RNA Sequencing technology was applied to explore the underlying mechanisms. The expression level of PPAR-γ was measured by Western Blot. RESULTS: AMIGO2 was upregulated in bladder cancer cells and tissues. Inhibited expression of AMIGO2 suppresses cell proliferation and migration. Low AMIGO2 expression inhibited tumorigenicity of 5637 in nude mice. According to RNA-Seq and bioinformatics analysis, 917 DEGs were identified. The DEGs were mainly enriched in cell-cell adhesion, peroxisome proliferators-activated receptors (PPARs) signaling pathway and some other pathways. PPAR-γ is highly expressed in bladder cancer cell lines T24 and 5637, but when AMIGO2 is knocked down in T24 and 5637, the expression level of PPAR-γ is also decreased, and overexpression of PPAR-γ could reverse the suppression effect of cell proliferation and migration caused by the inhibition of AMIGO2. CONCLUSION: AMIGO2 is overexpressed in bladder cancer cells and tissues. Knockdown of AMIGO2 suppresses bladder cancer cell proliferation and migration. These processes might be regulated by PPAR-γ signaling pathway.

Bladder Cancer Patients with Elevated SPRR1B Expression Experiencing a Poor Prognosis.

BACKGROUND: SPRR1B, a member of the small proline-rich protein family, is implicated in various epithelial cancers as a potential oncogene linked to tumour growth and poor survival outcomes. However, its role in urothelial bladder carcinoma (UBC) remains to be fully elucidated. METHODS: Transcriptional profiling data from The Cancer Genome Atlas grouped UBC samples in accordance with SPRR1B expression. Bioinformatic analysis was conducted to evaluate whether SPRR1B is a prognostic factor and a survival factor in UBC. Gene set enrichment analysis (GSEA) was performed to study immune cells and pathways. Reverse transcription quantitative real-time polymerase chain reaction detected gene expression. Immunohistochemistry assessed protein expression. Spearman correlation test analysed the correlation between SPRR1B and the protein p53. RESULTS: The bioinformatics results indicated that the expression level of SPRR1B in UBC tissues was significantly increased compared with that in normal bladder tissues, correlating with clinical characteristics. A high expression predicted poor prognosis and survival. Univariate Cox statistics showed that a high expression level of SPRR1B was correlated with UBC patients having poor overall survival (OS) (p < 0.05). In addition, on the basis of the multivariate Cox analysis, SPRR1B expression was independently correlated with OS (p = 0.005). GSEA analysis revealed enrichment in the p53, apoptosis, and cell cycle signalling pathways, and an association with B cells, lymphocytes, and natural killer cells. In addition, SPRR1B was found to be associated with immune infiltration based on the analysis of immune cell infiltration. Performing corresponding verification on a small number of tissues collected from bladder cancer patients revealed that the expression of this protein was negatively correlated with the expression of p53. CONCLUSIONS: SPRR1B overexpression predicts poor UBC outcomes, suggesting its role as a prognostic marker and therapeutic target. Further research is necessary to elucidate its role in UBC progression.

Hypermethylated TAGMe as a universal-cancer-only methylation marker and its application in diagnosis and recurrence monitoring of urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is the second most common urological malignancy. Despite numerous molecular markers have been evaluated during the past decades, no urothelial markers for diagnosis and recurrence monitoring have shown consistent clinical utility. METHODS: The methylation level of tissue samples from public database and clinical collected were analyzed. Patients with UC and benign diseases of the urinary system (BUD) were enrolled to establish TAGMe (TAG of Methylation) assessment in a training cohort (n = 567) using restriction enzyme-based bisulfite-free qPCR. The performance of TAGMe assessment was further verified in the validation cohort (n = 198). Urine samples from 57 UC patients undergoing postoperative surveillance were collected monthly for six months after surgery to assess the TAGMe methylation. RESULTS: We identified TAGMe as a potentially novel Universal-Cancer-Only Methylation (UCOM) marker was hypermethylated in multi-type cancers and investigated its application in UC. Restriction enzyme-based bisulfite-free qPCR was used for detection, and the results of which were consistent with gold standard pyrosequencing. Importantly, hypermethylated TAGMe showed excellent sensitivity of 88.9% (95% CI: 81.4-94.1%) and specificity of 90.0% (95% CI: 81.9-95.3%) in efficiently distinguishing UC from BUD patients in urine and also performed well in different clinical scenarios of UC. Moreover, the abnormality of TAGMe as an indicator of recurrence might precede clinical recurrence by three months to one year, which provided an invaluable time window for timely and effective intervention to prevent UC upstaging. CONCLUSION: TAGMe assessment based on a novel single target in urine is effective and easy to perform in UC diagnosis and recurrence monitoring, which may reduce the burden of cystoscopy. Trial registration ChiCTR2100052507. Registered on 30 October 2021.

Durable Objective Response to Lurbinectedin in Small Cell Bladder Cancer with TP53 Mutation: A Molecular-Directed Strategy.

Small cell bladder cancer (SCBC) is a rare and aggressive disease, often treated with platinum/etoposide-based chemotherapy. Key molecular drivers include the inactivation of onco-suppressor genes (TP53, RB1) and amplifications in proto-oncogenes (MYC). We report a patient with SCBC who achieved an objective and prolonged response to lurbinectedin, which has been approved for metastatic small cell lung cancer, after developing disease progression on cisplatin/etoposide and nivolumab/ipilimumab. A genomic analysis of a metastatic biopsy prior to lurbinectedin initiation revealed a TP53 mutation and amplification of the cell cycle regulators E2F3 and MYCL. A repeat biopsy following the development of lurbinectedin resistance showed a new actionable ERBB2 alteration without significant change in the tumor mutation burden (six mutations/Mb). The present report suggests that lurbinectedin may be active and should be further explored in SCBC harboring TP53 mutations and amplifications in E2F3 and MYC family complexes.

Identification of bladder cancer subtypes and predictive signature for prognosis, immune features, and immunotherapy based on immune checkpoint genes.

Immunotherapy based on immune checkpoint genes (ICGs) has recently made significant progress in the treatment of bladder cancer patients, but many patients still cannot benefit from it. In the present study, we aimed to perform a comprehensive analysis of ICGs in bladder cancer tissues with the aim of evaluating patient responsiveness to immunotherapy and prognosis. We scored ICGs in each BLCA patient from TCGA and GEO databases by using ssGSEA and selected genes that were significantly associated with ICGs scores by using the WCGNA algorithm. NMF clustering analysis was performed to identify different bladder cancer molecular subtypes based on the expression of ICGs-related genes. Based on the immune related genes differentially expressed among subgroups, we further constructed a novel stratified model containing nine genes by uni-COX regression, LASSO regression, SVM algorithm and multi-COX regression. The model and the nomogram constructed based on the model can accurately predict the prognosis of bladder cancer patients. Besides, the patients classified based on this model have large differences in sensitivity to immunotherapy and chemotherapy, which can provide a reference for individualized treatment of bladder cancer.