A Multi-Gene Signature of Non-Muscle-Invasive Bladder Cancer Identifies Patients Who Respond to Immunotherapies Including Bacillus Calmette-Guérin and Immune Checkpoint Inhibitors.

Approximately 75% of bladder cancer cases originate as non-muscle-invasive bladder cancer (NMIBC). Despite initial diagnosis, NMIBC commonly recurs, with up to 45% advancing to muscle-invasive bladder cancer (MIBC) and metastatic disease. Treatment for high-risk NMIBC typically includes procedures like transurethral resection and, depending on recurrence risk, intravesical chemotherapy or immunotherapy such as Bacillus Calmette-Guérin (BCG). However, persistent shortages of BCG necessitate alternative first-line treatments. We aim to use a multi-gene signature in high-risk NMIBC patients to determine whether patients may benefit from immune checkpoint inhibitors (ICIs) as an alternative to BCG and to evaluate their clinical utility. The multi-gene signature obtained from the three independent NMIBC cohorts was applied to stratify the UROMOL2016 cohort (n = 476) using consensus clustering. Each subtype was distinguished by biological pathway analysis. Validation analysis using a machine learning algorithm was performed in six independent cohorts including the BRS (n = 283) cohort treated with BCG and the IMvigor210 (n = 298) clinical trials treated with PD-L1 inhibitors. Based on consensus cluster analysis, NMIBC patients in the UROMOL2016 cohort were classified into three classes exhibiting distinguished characteristics, including DNA damage repair (DDR). Survival analysis showed that the NMIBC-DDR class had the highest rates of disease progression (progression-free survival, p = 0.002 by log-rank test) in the UROMOL cohort and benefited from BCG and ICIs (respectively, p = 0.02 and p = 0.03 by log-rank test). This study suggests that the multi-gene signature may have a role in identifying high-risk NMIBC patients and improving the responsiveness of ICIs. Additionally, we propose immunotherapy as a new first-line treatment for patients with high-risk NMIBC because of the shortage of BCG supply. It is important to help more patients prioritize cancer immunotherapy.

Expression of BCL2, TP53, FOXA1, and GATA3 in pTa bladder cancer recurrence.

OBJECTIVES: In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence. METHODS: We analyzed samples of 79 patients with the pTa stage of BC using a real-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue. RESULTS: Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had a significantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant. CONCLUSION: Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had a significantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.

MIR145 Core Promoter Methylation in Pretreatment Cell-Free DNA: A Liquid Biopsy Tool for Muscle-Invasive Bladder Cancer Treatment Outcome.

PURPOSE: The lack of personalized management of bladder cancer (BlCa) results in patients' lifelong post-treatment monitoring with invasive interventions, underlying the urgent need for tailored and minimally invasive health care services. On the basis of our previous findings on miR-143/145 cluster methylation in bladder tumors, we evaluated its clinical significance in pretreatment cell-free DNA (cfDNA) of patients with BlCa. MATERIALS AND METHODS: Methylation analysis was performed in our screening cohort (120 patients with BlCa; 20 age-matched healthy donors) by bisulfite-based pyrosequencing. Tumor recurrence/progression for patients with non-muscle-invasive bladder cancer, and progression and mortality for patients with muscle-invasive bladder cancer (MIBC) were used as clinical end point events in survival analysis. Bootstrap analysis was applied for internal validation of Cox regression models and decision curve analysis for assessment of clinical benefit on disease prognosis. RESULTS: Decreased methylation of MIR145 core promoter in pretreatment cfDNA was associated with short-term disease progression (multivariate Cox: hazard ratio [HR], 2.027 [95% CI, 1.157 to 3.551]; P = .010) and poor overall survival (multivariate Cox: HR, 2.098 [95% CI, 1.154 to 3.817]; P = .009) of patients with MIBC after radical cystectomy (RC). Multivariate models incorporating MIR145 promoter methylation in cfDNA with tumor stage clearly ameliorated patients' risk stratification, highlighting superior clinical benefit in MIBC prognostication. CONCLUSION: Reduced pretreatment cfDNA methylation of MIR145 core promoter was markedly correlated with increased risk for short-term progression and worse survival of patients with MIBC after RC and adjuvant therapy, supporting modern personalized and minimally invasive prognosis. Methylation profiling of MIR145 core promoter in pretreatment cfDNA could serve as a minimally invasive and independent predictor of MIBC treatment outcome and emerge as a promising marker for blood-based test in BlCa.

Investigating the impact of regulatory B cells and regulatory B cell-related genes on bladder cancer progression and immunotherapeutic sensitivity.

BACKGROUND: Regulatory B cells (Bregs), a specialized subset of B cells that modulate immune responses and maintain immune tolerance in malignant tumors, have not been extensively investigated in the context of bladder cancer (BLCA). This study aims to elucidate the roles of Bregs and Breg-related genes in BLCA. METHODS: We assessed Breg infiltration levels in 34 pairs of BLCA and corresponding paracancerous tissues using immunohistochemical staining. We conducted transwell and wound healing assays to evaluate the impact of Bregs on the malignant phenotype of SW780 and T24 cells. Breg-related genes were identified through gene sets and transcriptional analysis. The TCGA-BLCA cohort served as the training set, while the IMvigor210 and 5 GEO cohorts were used as external validation sets. We employed LASSO regression and random forest for feature selection and developed a risk signature using Cox regression. Primary validation of the risk signature was performed through immunohistochemical staining and RT-qPCR experiments using the 34 local BLCA samples. Additionally, we employed transfection assays and flow cytometry to investigate Breg expansion ability and immunosuppressive functions. RESULTS: Breg levels in BLCA tissues were significantly elevated compared to paracancerous tissues (P < 0.05) and positively correlated with tumor malignancy (P < 0.05). Co-incubation of SW780 and T24 cells with Bregs resulted in enhanced invasion and migration abilities (all P < 0.05). We identified 27 Breg-related genes, including CD96, OAS1, and CSH1, which were integrated into the risk signature. This signature demonstrated robust prognostic classification across the 6 cohorts (pooled HR = 2.25, 95% CI = 1.52-3.33). Moreover, the signature exhibited positive associations with advanced tumor stage (P < 0.001) and Breg infiltration ratios (P < 0.05) in the local samples. Furthermore, the signature successfully predicted immunotherapeutic sensitivity in three cohorts (all P < 0.05). Knockdown of CSH1 in B cells increased Breg phenotype and enhanced suppressive ability against CD8 + T cells (all P < 0.05). CONCLUSIONS: Bregs play a pro-tumor role in the development of BLCA. The Breg-related gene signature established in this study holds great potential as a valuable tool for evaluating prognosis and predicting immunotherapeutic response in BLCA patients.

Clinical implications of single cell sequencing for bladder cancer.

Bladder cancer (BC) is the 10th most common cancer worldwide, with about 0.5 million reported new cases and about 0.2 million deaths per year. In this scoping review, we summarize the current evidence regarding the clinical implications of single-cell sequencing for bladder cancer based on PRISMA guidelines. We searched PubMed, CENTRAL, Embase, and supplemented with manual searches through the Scopus, and Web of Science for published studies until February 2023. We included original studies that used at least one single-cell technology to study bladder cancer. Forty-one publications were included in the review. Twenty-nine studies showed that this technology can identify cell subtypes in the tumor microenvironment that may predict prognosis or response to immune checkpoint inhibition therapy. Two studies were able to diagnose BC by identifying neoplastic cells through single-cell sequencing urine samples. The remaining studies were mainly a preclinical exploration of tumor microenvironment at single cell level. Single-cell sequencing technology can discriminate heterogeneity in bladder tumor cells and determine the key molecular properties that can lead to the discovery of novel perspectives on cancer management. This nascent tool can advance the early diagnosis, prognosis judgment, and targeted therapy of bladder cancer.

Plasma metadherin mRNA expression in bladder cancer.

Urinary bladder cancer (BC) is the ninth most common cancer worldwide. At present, the clinical diagnosis of BC depends on self-reported symptoms, tissue biopsy specimens by cystoscopy and from voided urine cytology. However, cystoscopy is an invasive examination and voided urine cytology has low sensitivity, which might provoke misdiagnosis. The search for cancer biomarkers in blood is worthy of intense attention due to patients' comfort and ease of sampling. This work aimed to study expression of mRNA metadherin (MTDH) in plasma, serum BC specific antigen 1 (BLCA-1) and cystatin C as biomarkers of BC and their relation to different disease stages. This study included 59 BC patients, 11 patients with benign bladder lesion and 18 subjects as normal controls. MTDH expression was assessed by real time polymerase chain reaction, BLCA-1, and cystatin C by the enzyme linked immunosorbent assay. The three biomarkers were elevated in BC patients than patients with benign bladder diseases and controls. Patients with BC grade 3 and 4 had higher cystatin C, BLCA-1 and MTDH in comparison to patients with grade 1 and grade 2 (p=0.000). The receiver operating characteristic curve analysis showed that BLCA-1 at a cutoff point of 32.5 ng/ml and area under the curve of 1.00, had 100% accuracy, 100% sensitivity, 100% specificity, 100% positive predictive values and 100% negative predictive value. In conclusion, BLCA-1 was a better biomarker than cystatin C and MTDH. Cystatin C, BLCA-1 and MTDH levels, can differentiate between benign bladder lesion and BC and correlated with tumor grades.especially with OL-HDF compared to HF-HD, with acceptable albumin loss in the dialysate.

Clinical performance and utility of a noninvasive urine-based methylation biomarker: TWIST1/Vimentin to detect urothelial carcinoma of the bladder.

Traditional clinical modalities for diagnosing bladder urothelial carcinoma (BUC) remain limited due to their invasive nature, significant costs, discomfort associated with cystoscopy, and low sensitivity to urine cytology. Therefore, there is an urgent need to identify highly sensitive, specific, and noninvasive biomarkers for the early detection of this neoplasm. Hypermethylated TWIST1/Vimentin promoter may be a noninvasive biomarker using urine sample. We assessed the TWIST1/Vimentin promoter methylation status in urine samples using the Methylated Human TWIST1 and Vimentin Gene Detection Kit (Jiangsu MicroDiag Biomedicine Co., Ltd., China). The samples were collected from five groups: group 1 consisted of patients with BUC, group 2 contained other patients with urologic tumors, group 3 consisted of patients with benign diseases (e.g., urinary tract infections, lithiasis, and benign prostatic hyperplasia), Group 4 included UTUC (upper tract urothelial carcinoma) patients and group5 comprised healthy individuals. The study encompassed 77 BUC patients, and we evaluated the degree of methylation of the TWIST1/Vimentin gene in their urine samples. Notably, TWIST1/Vimentin positivity was significantly elevated in comparison to groups 2, 3 and 5 (all p < 0.001) at a rate of 77.9%, but no significant difference was observed when compared to group 4. In the relationship between TWIST1/Vimentin methylation and clinicopathological features of BC patients from our center, we found there was no significant association between TWIST1/Vimentin status and proteinuria and/or hematuria, and hypermethylation of TWIST1 / VIM genes was found in both high and low tumor grade and in both non-muscle invasive bladder cancer (stages Tis, Ta, or T1) and muscle-invasive bladder cancer (stage T2 or above). In the multivariable analysis for cancer detection, a positive TWIST1/Vimentin methylation were significantly linked to a heightened risk of BC. Moreover, TWIST1/Vimentin promoter methylation demonstrated an ability to detect BUC in urine samples with a sensitivity of 78% and a specificity of 83%. Our findings reveal that hypermethylation of the TWIST1/Vimentin promoter occurs in bladder urothelial carcinoma, and its high sensitivity and specificity suggest its potential as a screening and therapeutic biomarker for urothelial carcinoma of the bladder.

ALYREF m5C RNA methylation reader predicts bladder cancer prognosis by regulating the tumor immune microenvironment.

BACKGROUND: 5-Methylcytidine (m5C) methylation is a recently emerging epigenetic modification that is closely related to tumor proliferation, occurrence, and metastasis. This study aimed to investigate the clinicopathological characteristics and prognostic value of m5C regulators in bladder cancer (BLCA), and their correlation with the tumor immune microenvironment. METHODS: Thirteen m5C RNA methylation regulators were analyzed using RNA-sequencing and corresponding clinical information obtained from the TCGA database. The Cluster Profiler package was used to analyze the gene ontology function of potential targets and enriched the Kyoto Encyclopedia of Genes and Genomes pathway. Kaplan-Meier survival analysis was used to compare survival differences using the log-rank test and univariate Cox proportional hazards regression. The correlation between signature prognostic m5C regulators and various immune cells was analyzed. Univariate and multivariate Cox regression analyses identified independence of the ALYREF gene signature. RESULTS: Nine out of the 13 m5C RNA methylation regulators were differentially expressed in BLCA and normal samples and were co-expressed. These 9 regulators were associated with clinicopathological tumor characteristics, particularly high or low tumor risk, pT or pTNM stage, and migration. Consensus clustering analysis divides the BLCA samples into 4 clusters. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment annotation and gene ontology function analysis identified 273 upregulated and 594 downregulated genes in BLCA. Notably, only ALYREF was significantly correlated with OS (P < .05). ALYREF exhibited significant infiltration levels in macrophage cells. Therefore, we constructed a nomogram for ALYREF as an independent prognostic factor. Additionally, we observed that both the mRNA and protein levels of ALYREF were upregulated, and immunofluorescence showed that ALYREF was mainly distributed in nuclear speckles. ALYREF overexpression was significantly associated with poor OS. CONCLUSION: Our findings demonstrated the potential of ALYREF to predict clinical prognostic risks in BLCA patients and regulate the tumor immune microenvironment. As such, ALYREF may serve as a novel prognostic indicator in BLCA patients.

Development of a prognostic model for muscle-invasive bladder cancer using glutamine metabolism.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is distinguished by its pronounced invasiveness and unfavorable prognosis. Immunotherapy and targeted therapy have emerged as key treatment options for various types of cancer. Altered metabolism is a defining characteristic of cancer cells, and there is mounting evidence suggesting the important role of glutamine metabolism (GM) in tumor metabolism. Nevertheless, the relationship between GM and clinical outcomes, immune microenvironment, and immunotherapy in MIBC remains unknown. METHODS: This study employed Mendelian randomization to explore the causal relationship between blood metabolites and bladder tumors. We systematically evaluated 373 glutamine metabolism-related genes and identified prognostic-related genes, leading to the construction of a glutamine-associated prognostic model. Further analysis confirmed the correlation between high and low-risk groups with the tumor microenvironment, immune cell infiltration, and tumor mutation burden. Subsequently, we assessed the relationship between the risk score and the sensitivity to various immunotherapies and anticancer drugs. RESULTS: We identified 14 blood metabolites at the molecular level that have a causal relationship with bladder tumors. At the gene level, the study discussed differentially expressed GM genes in MIBC. First, we established a risk model predicting overall survival (OS) based on GM genes, confirming its reliable predictive ability in MIBC patients and validated it in a GEO cohort. Additionally, a reliable column line chart was created. Secondly, two distinct molecular subtypes were identified, and the associations between different risk groups and tumor microenvironment and immune infiltration were observed. In addition, the predicted risk values correlated with responses to a broad range of pharmaceutical agents. CONCLUSION: In summary, we confirmed the causal relationship between blood metabolites and bladder tumors. Furthermore, a risk scoring model related to glutamine metabolism consisting of 9 genes was developed. This model could potentially serve as a useful tool for predicting prognosis and guiding the treatment of MIBC patients.

Hyperoside induces cell cycle arrest and suppresses tumorigenesis in bladder cancer through the interaction of EGFR-Ras and Fas signaling pathways.

Hyperoside is a natural flavonol glycoside widely found in plants and has been reported to have a variety of pharmacological effects, including anticancer abilities. In this study, we demonstrated for the first time that hyperoside inhibited the proliferation of bladder cancer cells in vitro and in vivo. Moreover, hyperoside could not only induce cell cycle arrest, but also induce apoptosis of a few bladder cancer cells. Quantitative proteomics, bioinformatics analysis and Western blotting confirmed that hyperoside induced the overexpression of EGFR, Ras and Fas proteins, which affects a variety of synergistic and antagonistic downstream signaling pathways, including MAPKs and Akt, ultimately contributing to its anticancer effects in bladder cancer cells. This study reveals that hyperoside could be a promising therapeutic strategy for the prevention of bladder cancer.

Cytotoxicity and Apoptosis Studies of Brucein D against T24 Bladder Cancer Cells.

OBJECTIVE: Brucein D (BrD), a quassinoid isolated from Brucea javanica fruit, reportedly demonstrates anti-cancer activity. This study's objective is to evaluate the cytotoxicity of Brucein D and its ability to induce apoptosis in T24 bladder cancer cells. METHODS: We investigated the cytotoxic activity of BrD against the T24 cell through the induction of apoptosis in vitro. This cytotoxic activity was evaluated with ΜΤΤ assay and followed by Calcein-AM/PI viability staining. Apoptotic activity was determined with Hoechst 33342 nuclear staining and DNA fragmentation. Doxorubicin and docetaxel were used as a positive control. Evaluation of apoptotic-related gene expression, Bax, Bak, Bcl2, and p53 was also performed using semi-quantitative PCR analysis. Statistical analysis was conducted using One-way ANOVA followed by post hoc test Turkey's HSD (Honestly Significance Difference). RESULTS: Results show that BrD had high toxicity against T24 bladder cancer cells with an IC50 value of 7.65 ± 1.2 µg/mL but relatively less toxic to 1BR3 normal skin fibroblast cells compared to the doxorubicin and docetaxel treated cells. The viability assay shows that BrD significantly increases the percentage of dead cells relative to control in a dose-dependent manner. Furthermore, the percentage of cells with apoptotic appearance was significantly higher in group treated with BrD IC50 (56.04±3.09%) compared to control (9.42±2.88). The result was similar to doxorubicin IC50 (58.97±12.31) but lower than docetaxel IC50 (74.42±9.79). DNA fragmentation in gel electrophoresis was also observed in T24 cells treated with BrD. Apoptosis was also verified by an alteration in the expression of apoptosis-related genes, upregulation of Bax, Bak, and p53, and downregulation of Bcl-2. CONCLUSION: BrD has shown a cytotoxic effect against T24 bladder cancer cells. Hence, it is a promising natural compound for the management of bladder cancer by induction of apoptosis through activation of the intrinsic pathway, with low toxicity to normal cells.

Integrating molecular subtype and CD8+ T cells infiltration to predict treatment response and survival in muscle-invasive bladder cancer.

BACKGROUND: Luminal and Basal are the primary intrinsic subtypes of muscle-invasive bladder cancer (MIBC). The presence of CD8+ T cells infiltration holds significant immunological relevance, potentially influencing the efficacy of antitumor responses. This study aims to synergize the influence of molecular subtypes and CD8+ T cells infiltration in MIBC. METHODS: This study included 889 patients with MIBC from Zhongshan Hospital, The Cancer Genome Atlas, IMvigor210 and NCT03179943 cohorts. We classified the patients into four distinct groups, based on the interplay of molecular subtypes and CD8+ T cells and probed into the clinical implications of these subgroups in MIBC. RESULTS: Among patients with Luminal-CD8+Thigh tumors, the confluence of elevated tumor mutational burden and PD-L1 expression correlated with a heightened potential for positive responses to immunotherapy. In contrast, patients featured by Luminal-CD8+Tlow displayed a proclivity for deriving clinical advantages from innovative targeted interventions. The Basal-CD8+Tlow subgroup exhibited the least favorable three-year overall survival outcome, whereas their Basal-CD8+Thigh counterparts exhibited a heightened responsiveness to chemotherapy. CONCLUSIONS: We emphasized the significant role of immune-molecular subtypes in shaping therapeutic approaches for MIBC. This insight establishes a foundation to refine the process of selecting subtype-specific treatments, thereby advancing personalized interventions for patients.

Clinical effectiveness of a multitarget urine DNA test for urothelial carcinoma detection: a double-blinded, multicenter, prospective trial.

Urine-based testing is promising for noninvasive diagnosis of urothelial carcinoma (UC) but has suboptimal sensitivity for early-stage tumors. Herein, we developed a multitarget urine tumor DNA test, UI-Seek, for UC detection and evaluated its clinical feasibility. The prediction model was developed in a retrospective cohort (n = 382), integrating assays for FGFR3 and TERT mutations and aberrant ONECUT2 and VIM methylation to generate a UC-score. The test performance was validated in a double-blinded, multicenter, prospective trial (n = 947; ChiCTR2300076543) and demonstrated a sensitivity of 91.37% and a specificity of 95.09%. The sensitivity reached 75.81% for low-grade Ta tumors and exceeded 93% in high-grade Ta and higher stages (T1 to T4). Simultaneous identification of both bladder and upper urinary tract tumors was enabled with sensitivities exceeding 90%. No significant confounding effects were observed regarding benign urological diseases or non-UC malignancies. The test showed improved sensitivities over urine cytology, the NMP22 test, and UroVysion FISH alongside comparable specificities. The single-target accuracy was greater than 98% as confirmed by Sanger sequencing. Post-surgery UC-score decreased in 97.7% of subjects. Overall, UI-Seek demonstrated robust performance and considerable potential for the early detection of UC.

Dysregulated FGFR3 signaling alters the immune landscape in bladder cancer and presents therapeutic possibilities in an agent-based model.

Bladder cancer is an increasingly prevalent global disease that continues to cause morbidity and mortality despite recent advances in treatment. Immune checkpoint inhibitors (ICI) and fibroblast growth factor receptor (FGFR)-targeted therapeutics have had modest success in bladder cancer when used as monotherapy. Emerging data suggests that the combination of these two therapies could lead to improved clinical outcomes, but the optimal strategy for combining these agents remains uncertain. Mathematical models, specifically agent-based models (ABMs), have shown recent successes in uncovering the multiscale dynamics that shape the trajectory of cancer. They have enabled the optimization of treatment methods and the identification of novel therapeutic strategies. To assess the combined effects of anti-PD-1 and anti-FGFR3 small molecule inhibitors (SMI) on tumor growth and the immune response, we built an ABM that captures key facets of tumor heterogeneity and CD8+ T cell phenotypes, their spatial interactions, and their response to therapeutic pressures. Our model quantifies how tumor antigenicity and FGFR3 activating mutations impact disease trajectory and response to anti-PD-1 antibodies and anti-FGFR3 SMI. We find that even a small population of weakly antigenic tumor cells bearing an FGFR3 mutation can render the tumor resistant to combination therapy. However, highly antigenic tumors can overcome therapeutic resistance mediated by FGFR3 mutation. The optimal therapy depends on the strength of the FGFR3 signaling pathway. Under certain conditions, ICI alone is optimal; in others, ICI followed by anti-FGFR3 therapy is best. These results indicate the need to quantify FGFR3 signaling and the fitness advantage conferred on bladder cancer cells harboring this mutation. This ABM approach may enable rationally designed treatment plans to improve clinical outcomes.

The mechanism of the contribution of ICAM-1 to epithelial-mesenchymal transition (EMT) in bladder cancer.

Bladder cancer is one of the most prevalent cancers worldwide. Moreover, if not optimally treated, bladder cancer is a significant burden on healthcare systems due to multiple recurrences which often require more aggressive therapies. Therefore, targeted anti-cancer therapies, developed based on an in-depth understanding of specific proteins and molecular mechanisms, are promising in cancer treatment. Here, for the first time, we presented the new approaches indicating that intracellular adhesion molecule-1 (ICAM-1) may play a potential role in enhancing therapeutic effectiveness for bladder cancer. In the present study, we presented that ICAM-1 expression as well as its regulation in bladder cancer is strongly correlated with the high expression of N-cadherin. Importantly, the presence of N-cadherin and its regulator-TWIST-1 was abolished when ICAM-1 was silenced. We identified also that ICAM-1 is capable of regulating cellular migration, proliferation, and EMT progression in bladder cancer cells via the N-cadherin/SRC/AKT/GSK-3ß/ß-catenin signaling axis. Therefore, we propose ICAM-1 as a novel metastatic marker for EMT progression, which may also be used as a therapeutic target in bladder cancer.

Urinary mutagenicity and bladder cancer risk in northern New England.

The etiology of bladder cancer among never smokers without occupational or environmental exposure to established urothelial carcinogens remains unclear. Urinary mutagenicity is an integrative measure that reflects recent exposure to genotoxic agents. Here, we investigated its potential association with bladder cancer in rural northern New England. We analyzed 156 bladder cancer cases and 247 cancer-free controls from a large population-based case-control study conducted in Maine, New Hampshire, and Vermont. Overnight urine samples were deconjugated enzymatically and the extracted organics were assessed for mutagenicity using the plate-incorporation Ames assay with the Salmonella frameshift strain YG1041 + S9. Logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) of bladder cancer in relation to having mutagenic versus nonmutagenic urine, adjusted for age, sex, and state, and stratified by smoking status (never, former, and current). We found evidence for an association between having mutagenic urine and increased bladder cancer risk among never smokers (OR = 3.8, 95% CI: 1.3-11.2) but not among former or current smokers. Risk could not be estimated among current smokers because nearly all cases and controls had mutagenic urine. Urinary mutagenicity among never-smoking controls could not be explained by recent exposure to established occupational and environmental mutagenic bladder carcinogens evaluated in our study. Our findings suggest that among never smokers, urinary mutagenicity potentially reflects genotoxic exposure profiles relevant to bladder carcinogenesis. Future studies are needed to replicate our findings and identify compounds and their sources that influence bladder cancer risk.

TROP-2, NECTIN-4 and predictive biomarkers in sarcomatoid and rhabdoid bladder urothelial carcinoma.

Introduction: The surface protein TROP-2/TACSTD2 and the cell adhesion protein NECTIN-4/NECTIN4 are responsible for the efficacy of anticancer therapies based on antibody-drug conjugates (ADC) targeting intracellular microtubules. In contrast with common histologic subtypes of bladder urothelial carcinoma (BUC), little is known of TROP-2 and NECTIN-4 expression in sarcomatoid and rhabdoid BUC. Aims: In this study, we aimed to analyze TROP-2 and NECTIN-4 expression and additional predictive biomarkers by immunohistochemistry and fluorescence in situ hybridization (FISH) on 35 undifferentiated BUC (28 sarcomatoid and 7 rhabdoid). Wide genomic investigation was also performed on 411 BUC cases of the PanCancer Atlas, focusing on genes related to the microtubule pathways. Results: Seven of 35 (20%) undifferentiated BUC showed expression of TROP-2. NECTIN-4 was expressed in 10 cases (29%). Seven cases (20%) co-expressed TROP-2 and NECTIN-4. HER-2 FISH was amplified in 5 cases (14%) while HER-2 immunoexpression was observed in 14 cases (40%). PD-L1 scored positive for combined proportion score (CPS) in 66% of cases and for tumor proportion score (TPS) in 51% of cases. Pan-NTRK1-2/3 was elevated in 9 cases (26%) and FGFR-2/3 was broken in 7 of 35 cases (20%). Of 28 sarcomatoid BUC, 9 (32%) were negative for all (TROP-2, NECTIN-4, PD-L1, HER-2, FGFR and pan-NTRK) biomarkers and 3 (11%) expressed all five biomarkers. Among cases with rhabdoid dedifferentiation, 1 of 7 (14%) showed activation of all biomarkers, whereas 2 of 7 (28%) showed none. The mRNA analysis identified microtubule-related genes and pathways suitable for combined ADC treatments in BUC. Conclusion: Sarcomatoid and rhabdoid BUC do harbor positive expression of the ADC targets TROP-2 or NECTIN-4 in a relatively modest subset of cases, whereas the majority do not. Different combinations of other positive biomarkers may help the choice of medical therapies. Overall, these findings have important clinical implications for targeted therapy for BUC.

CircMCTP2 enhances the progression of bladder cancer by regulating the miR-99a-5p/FZD8 axis.

BACKGROUND: CircRNAs and miRNAs are involved in the progression of tumor. CircMCTP2 is considered as a novel tumor promoter. However, the exact functions of circMCTP2 in bladder cancer are still unclear. This study was designed to explore the underlying mechanisms of circMCTP2-modulated tumor development in bladder cancer. METHODS: The present study is an original research. The levels of circMCTP2 in a total of 39 bladder cancer specimens and cell lines were determined by RT-qPCR. The expression of FZD8 in T24 and RT-4 cells treated with miR-99a-5p mimics were examined using western blotting. In addition, the proliferative, migrative and invasive abilities of transfected cells were determined by CCK8 and Transwell assays. Furthermore, the apoptosis of transfected cells was evaluated using flow cytometry. Dual luciferase reporter assay was performed to elucidate the relationship between miR-99a-5p and circMCTP2/FZD8. RESULTS: The levels of circMCTP2 were elevated in bladder cancer samples and cells, and this was related to worse survival rate. Downregulation of circMCTP2 suppressed growth and metastasis of cells, whereas the apoptotic rate of cells was enhanced. The levels of miR-99a-5rp was elevated after the downregulation of circMCTP2. Moreover, reverse correlation between the expression of miR-99a-5p and circMCTP2 was revealed in bladder cancer specimens. Additionally, FZD8 was the putative target of miR-99a-5p and the mimics of miR-99a-5p inhibited the proliferation, migration and invasion of bladder cancer cells via the FZD8/Wnt-b-catenin axis. Moreover, circMCTP2 regulated the growth and metastasis of bladder cancer cells potentially through regulating the miR-99a-5p/FZD8/Wnt-b-catenin axis. In summary, circMCTP2 was considered as an oncogenic factor through regulating the miR-99a-5p/FZD8/Wnt-b-catenin axis. CONCLUSIONS: This novel signaling could regulate the biological behaviours of bladder cancer cells, and these findings highlighted circMCTP2 as a critical target for treating bladder cancer.

A first-in-class TIMM44 blocker inhibits bladder cancer cell growth.

Mitochondria play a multifaceted role in supporting bladder cancer progression. Translocase of inner mitochondrial membrane 44 (TIMM44) is essential for maintaining function and integrity of mitochondria. We here tested the potential effect of MB-10 (MitoBloCK-10), a first-in-class TIMM44 blocker, against bladder cancer cells. TIMM44 mRNA and protein expression is significantly elevated in both human bladder cancer tissues and cells. In both patient-derived primary bladder cancer cells and immortalized (T24) cell line, MB-10 exerted potent anti-cancer activity and inhibited cell viability, proliferation and motility. The TIMM44 blocker induced apoptosis and cell cycle arrest in bladder cancer cells, but failed to provoke cytotoxicity in primary bladder epithelial cells. MB-10 disrupted mitochondrial functions in bladder cancer cells, causing mitochondrial depolarization, oxidative stress and ATP reduction. Whereas exogenously-added ATP and the antioxidant N-Acetyl Cysteine mitigated MB-10-induced cytotoxicity of bladder cancer cells. Genetic depletion of TIMM44 through CRISPR-Cas9 method also induced robust anti-bladder cancer cell activity and MB-10 had no effect in TIMM44-depleted cancer cells. Contrarily, ectopic overexpression of TIMM44 using a lentiviral construct augmented proliferation and motility of primary bladder cancer cells. TIMM44 is important for Akt-mammalian target of rapamycin (mTOR) activation. In primary bladder cancer cells, Akt-S6K1 phosphorylation was decreased by MB-10 treatment or TIMM44 depletion, but enhanced after ectopic TIMM44 overexpression. In vivo, intraperitoneal injection of MB-10 impeded bladder cancer xenograft growth in nude mice. Oxidative stress, ATP reduction, Akt-S6K1 inhibition and apoptosis were detected in MB-10-treated xenograft tissues. Moreover, genetic depletion of TIMM44 also arrested bladder cancer xenograft growth in nude mice, leading to oxidative stress, ATP reduction and Akt-S6K1 inhibition in xenograft tissues. Together, targeting overexpressed TIMM44 by MB-10 significantly inhibits bladder cancer cell growth in vitro and in vivo.

Identification of BACH1-IT2-miR-4786-Siglec-15 immune suppressive axis in bladder cancer.

The sialic acid binding Ig like lectin 15 (Siglec-15) was previously identified as tumor immune suppressor gene in some human cancers with elusive molecular mechanism to be elucidated. The continuous focus on both clinical and basic biology of bladder cancer leads us to characterize aberrant abundance of BACH1-IT2 associating with stabilization of Siglec-15, which eventually contributes to local immune suppressive microenvironment and therefore tumor advance. This effect was evidently mediated by miR-4786-5p. BACH1-IT2 functions in this scenario as microRNA sponge, and competitively conceals miR-4786 and up-regulates cancer cell surface Siglec-15. The BACH1-IT2-miR-4786-Siglec-15 axis significantly influences activation of immune cell co-culture. In summary, our data highlights the critical involvements of BACH1-IT2 and miR-4786 in immune evasion in bladder cancer, which hints the potential for both therapeutic and prognostic exploitation.