Bladder Cancer Cells Research Articles

NOTCH3 promotes malignant progression of bladder cancer by directly regulating SPP1 and activating PI3K/AKT pathway

AbstractThe biological role and precise molecular mechanisms of Notch receptor 3 (NOTCH3) in the malignant progression of bladder cancer (BLCA) remain unclear. In this study, we found that NOTCH3 was significantly upregulated and associated with poor prognosis in BLCA patients. Functional experiments demonstrated that NOTCH3 knockdown inhibited BLCA cell proliferation, migration, invasion and significantly suppressed tumor growth and metastasis in vivo as well. Mechanically, chromatin immunoprecipitation and dual-luciferase reporter assays confirmed that NOTCH3 could promote the transcription of secreted phosphoprotein 1 (SPP1), a potential downstream target gene of NOTCH3, by binding to the CSL elements in the SPP1 promoter. Moreover, we also found that targeting NOTCH3 inhibited BLCA growth and metastasis by suppressing the SPP1-PI3K/AKT axis. Our study highlights the critical role of NOTCH3-SPP1-PI3K/AKT axis in the malignant progression of BLCA, suggesting that NOTCH3 may be a potential therapeutic target for BLCA.

Regulation of Apoptosis, Autophagy, and Metastasis by Luteolin in Human Bladder Cancer EJ138 Cells: An Experimental Study

Background: Chemotherapy remains a primary approach to cancer treatment, widely applied in bladder cancer (BC). However, the various side effects and resistance associated with chemotherapeutic drugs pose significant challenges in BC therapy, prompting interest in natural compounds like luteolin. Studies focus on its effects on key biological processes involved in BC, including metastasis, apoptosis, and autophagy. Objectives: This study investigated the regulation of mRNA expression of genes associated with apoptosis (BCL2, P53), autophagy (ULK1, ATG12), and metastasis (MMP2, MMP9) in malignant BC cells treated with luteolin. Methods: This was an in vitro experimental study. EJ138 BC cells were treated with various concentrations of luteolin, and its impact on cell viability, proliferation, and gene expression was assessed. The cytotoxic effect of luteolin on EJ138 BC cells was evaluated using the MTT assay after 24- and 48-hour treatments with different luteolin concentrations. Flow cytometry was performed to examine luteolin’s anti-proliferative effect, and RT-PCR was used to analyze mRNA expression of BCL2, P53, ULK1, ATG12, MMP2, and MMP9 genes. Results: MTT assay results confirmed that luteolin reduced the proliferation rate of BC cells. Flow cytometry indicated increased cell death in EJ138 BC cells following luteolin treatment. RT-PCR findings demonstrated that luteolin upregulated P53, ULK1, and ATG12 expression while downregulating BCL2 mRNA expression. However, luteolin treatment in EJ138 cells did not significantly alter MMP2 and MMP9 expression levels. Conclusions: These findings indicate that luteolin exerts cytotoxic effects on EJ138 BC cells by dysregulating mRNA expression of genes involved in apoptosis and autophagy. Therefore, luteolin shows potential as an effective anti-cancer agent for BC therapy.

Tumor-associated macrophages in bladder cancer: roles and targeted therapeutic strategies

Bladder cancer (BC) is the ninth most common and “expensive” cancer in the world. Despite the availability of various treatment modalities such as chemotherapy, immunotherapy and surgery, the overall survival rate of patients with advanced bladder cancer remains low. As one of the most abundant infiltrating immune cells in bladder cancer, tumor-associated macrophages (TAMs) play an important role in the development of BC and in the standard regimen of intravesical BCG therapy. Targeting TAMs have achieved excellent results in clinical trials for a variety of other cancers, but few studies have been conducted for bladder cancer. Further exploration is still needed to develop TAM-related therapeutic strategies for BC treatment, which are expected to improve the therapeutic efficacy and life quality of patients. This review summarizes the relationship between TAMs in bladder cancer and disease staging, evolution, patient prognosis, and treatment outcome. Several potential TAM targets in BC are also pointed, which may help to inhibit tumor-promoting TAMs and provide new therapeutic approaches for advanced BC.

The role of miR-152 in urological tumors: potential biomarkers and therapeutic targets

Urological malignant tumors pose a significant threat to human health, with a high incidence rate each year. Prostate cancer, bladder cancer, and renal cell carcinoma are among the most prevalent and extensively researched urological malignancies. Despite advancements in research, the prognosis for these tumors remains unfavorable due to late detection, postoperative recurrence, and treatment resistance. A thorough investigation into their pathogenesis is crucial for early diagnosis and treatment. Recent studies have highlighted the close association between microRNAs (miRNAs) and cancer progression. miRNAs are small non-coding RNAs composed of 19-23 nucleotides that regulate gene expression by binding to the 3’ untranslated region (3’UTR) of target mRNAs, impacting key cellular processes such as proliferation, differentiation, apoptosis, and migration. Dysregulation of miRNAs can disrupt the expression of oncogenes and tumor suppressor genes, contributing to cancer development. Among the various miRNAs studied, miR-152 has garnered attention for its role in urological malignancies. Several studies have indicated that dysregulation of miR-152 expression is significant in these cancers, warranting a comprehensive review of the evidence. This review focuses on the expression and function of miR-152 in prostate cancer, bladder cancer, and renal cell carcinoma, elucidating its mechanisms in cancer progression and exploring its potential as a therapeutic target and biomarker in urological malignancies.

The potential of ESCO2 as a prognostic and immunotherapeutic marker of pan-cancer and its role in anti-PD1 treatment of bladder cancer.

Background Establishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2), a member of the EFO2 family, is implicated in the pathogenesis and progression of various cancers. However, there has been limited comprehensive pan-cancer analysis conducted on ESCO2 thus far. Methods Publicly available databases, such as the UCSC Xena database, were utilized to examine differential expression patterns across various cancer types. In addition, variations in expression levels were investigated across distinct clinical stages. Univariate Cox regression and Kaplan-Meier survival analyses were conducted to evaluate the impact on overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) at a pan-cancer level. The correlation between ESCO2 expression and immune cell infiltration was examined to gain insight into the tumor microenvironment in different cancers. The results of the bioinformatic analysis were validated using immunotherapy clinical trials and pathological specimens. CCK-8 and transwell assay experiments were performed to investigate the biological function of ESCO2. Results ESCO2 expression was found to be up-regulated in most cancers, with a correlation to TNM stages. Prognostic analysis indicated that overexpression of ESCO2 was associated with poor prognosis in various cancers. Furthermore, the correlation between ESCO2 expression and immune cell infiltration suggested its potential as a predictor for immunotherapy efficacy. Notably, ESCO2 expression showed positive associations with immunoinhibitor, immnostimulator, MHC molecule, chemokine receptor, tumor mutation burden (TMB) and microsatellite instability (MSI) levels in bladder cancer (BLCA). The validation cohort for immunotherapy corroborated these findings and substantiated that ESCO2 could function as an autonomous prognostic biomarker and a promising target for cancer treatment via immunotherapy. In addition, in vitro experiments confirmed the role of ESCO2 in influcing the proliferation, invasion and migration of BLCA cells. Conclusion ESCO2 participates in regulating the immune infiltration and affecting the prognosis of patients in many cancers, especially in BLCA. ESCO2 may serve as a prognostic and immunotherapy biomarker in future treatment of human cancer.

Mevalonate pathway inhibition reduces bladder cancer metastasis by modulating RhoB protein stability and integrin β1 localization

The progression and outcome of bladder cancer (BLCA) are critically affected by the propensity of tumor metastasis. Our previous study revealed that activation of the mevalonate (MVA) pathway promoted migration of BLCA cells; however, the exact mechanism is unclear. Here we show that elevated expression of MVA pathway enzymes in BLCA cells, correlating with poorer patient prognosis by analyzing single-cell and bulk-transcriptomic datasets. Inhibition of the MVA pathway, either through knockdown of farnesyl diphosphate synthase (FDPS) or using inhibitors such as zoledronic acid or simvastatin, led to a marked reduction in BLCA cell migration. Notably, this effect was reversed by administering geranylgeranyl pyrophosphate (GGPP), not farnesyl pyrophosphate (FPP) or cholesterol, indicating the specificity of geranylgeranylation for cell motility. Moreover, we found that RhoB, a Rho GTPase family member, was identified as a key effector of the impact of the MVA pathway on BLCA metastasis. The post-translational modification of RhoB by GGPP-mediated geranylgeranylation influenced its protein stability through the ubiquitin-proteasome pathway. Additionally, overexpression of RhoB was found to block the membrane translocation of integrin β1 in BLCA cells. In summary, our findings underscore the role of the MVA pathway in BLCA metastasis, providing insights into potential therapeutic targets of this malignancy.

RNF19A inhibits bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway

BackgroundThe role of the RING finger protein superfamily in carcinogenesis has been widely studied, but one member of this family, RNF19A, has not yet been thoroughly explored in bladder cancer (BCa).MethodsThe expression levels of RNF19A in BCa samples and cell lines were analysed through data mining of public resources and further experiments. BCa cells in which RNF19A was stably overexpressed or knocked down were generated through lentivirus infection. The effects of RNF19A on cell proliferation, migration, and invasion were explored by performing a series of in vitro experiments, including CCK-8, colony formation, wound healing, and Transwell invasion assays. Using bioinformatics methods and multiple experiments, including western blot, qRT‒PCR, immunoprecipitation, cycloheximide, ubiquitination, and rescue assays, the mechanism underlying the effect of RNF19A on the progression of BCa was investigated.ResultsHere, we found that RNF19A expression was reduced in BCa samples and cell lines and that lower RNF19A expression predicted shorter overall survival of BCa patients. Functionally, forced expression of RNF19A suppressed BCa cell proliferation, migration, and invasion by inactivating the AKT/mTOR signalling pathway, whereas silencing RNF19A had the opposite effects. Mechanistically, RNF19A could directly interact with ILK and promote its ubiquitination and degradation. Rescue experiments revealed that forced ILK expression partially rescued the decreased phosphorylation of AKT, mTOR, and S6K1 caused by RNF19A overexpression and that the increased levels of the p-AKT, p-mTOR, and p-S6K1 proteins induced by RNF19A knockdown were eliminated after silencing ILK. Similarly, the effects of RNF19A overexpression or knockdown on the phenotypes of cell proliferation, migration, and invasion could also be restored by forced or decreased ILK expression.ConclusionsRNF19A suppressed the proliferation, migration, and invasion abilities of BCa cells by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway. RNF19A might be a potential prognostic biomarker and promising therapeutic target for BCa.

The Evolving Molecular Landscape and Actionable Alterations in Urologic Cancers

The genetic landscape of urologic cancers has evolved with the identification of actionable mutations that impact diagnosis, prognosis, and therapeutic strategies. This narrative review consolidates existing literature on genetic mutations across key urologic cancers, including bladder, renal, prostate, upper tract urothelial, testicular, and penile. The review highlights mutations in DNA damage repair genes, such as BRCA1/2 and PTEN, as well as pathway alterations like FGFR and PD-L1 overexpression. These mutations influence tumor behavior and therapeutic outcomes, emphasizing the need for precision oncology approaches. Molecular profiling, through tools like next-generation sequencing, has revolutionized patient care by enabling targeted treatment strategies, especially in cancers with distinct molecular subtypes such as luminal or basal bladder cancer and clear cell renal carcinoma. Emerging therapies, including FGFR inhibitors and immune checkpoint blockade, offer new treatment avenues, although resistance mechanisms remain a challenge. We also emphasize the importance of biomarker identification for personalized management, especially in metastatic settings where treatment intensification is often required. Future research is needed to further elucidate our understanding of the genetics affecting urologic cancers, which will help develop novel, individualized therapies to enhance oncologic outcomes.

Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer.

Bladder cancer (BC) is a common malignancy and its most prevalent type is urothelial carcinoma, which accounts for ~90% of all cases of BC. The current treatment options for BC are limited, which necessitates the development of alternative treatment strategies. Hyperthermia (HT), as an adjuvant cancer therapy, is known to improve the efficacy of chemotherapy or radiotherapy. The present study aimed to investigate the anti‑tumor effects of HT on cell survival, invasiveness, chemoresistance and immune evasion in human BC cell lines (5637, T24 and UMUC3). Calcein AM staining was performed to analyze the cytotoxicity of natural killer (NK) cells against human BC cells following HT treatment. Cell migration and invasion affected by HT were analyzed using Transwell migration and invasion assays. It was found that HT inhibited the proliferation of BC cells by downregulating the phosphorylation of protein kinase B. Moreover, HT effectively enhanced the sensitivity of BC cells to the chemotherapy drug cisplatin (DDP) and reduced the chemoresistance of DDP‑resistant cells by downregulating the expression of cadherin‑11. It was further demonstrated that HT inhibited the migration and invasion of BC cells and enhanced the cytotoxic effects of NK cells. In summary, the antineoplastic effects of HT were mediated through three main mechanisms: Enhancement of the chemosensitivity of BC cells and mitigation of DDP‑induced chemoresistance, suppression of the invasive potential of BC cells and reinforcement of the anticancer response of NK cells. Thus, HT appears to be a promising adjunctive therapy for human BC.

Exploration on the construction of a bladder cancer prognostic model based on disulfidptosis-related lncRNAs and its clinical significance.

Disulfidptosis is a novel programmed cell death mode that has been reported to play a role in oncogenesis. Increasing evidences suggest that the long non-coding RNAs (lncRNAs) play crucial roles in the initiation and progression of bladder cancer (BLCA). However, the role and prognostic value of disulfidptosis-related lncRNAs in BLCA remain unknown.The aim of this study was to construct and validate a disulfidptosis-related lncRNA risk model for predicting the prognosis of BLCA patients. A risk model consisting of 5 disulfidptosis-related lncRNAs was developed to predict the prognosis of BLCA patients. The overall survival (OS) of BLCA patients in the high-risk group was significantly shorter than that in the low-risk group (P < 0.05). The effectiveness of this model was validated using the receiver operating characteristic (ROC) curve analysis, and this model proved superior in prognostic accuracy compared with other clinical features. Furthermore, the tumor immune dysfunction and exclusion (TIDE) score in the high-risk group was significantly higher than that in the low-risk group, suggesting that the high-risk group had a less favorable response to immunotherapy. Simultaneously, patients in the low-risk group exhibited significantly higher sensitivity to CTLA-4 monoclonal antibody therapy compared to those in the high-risk group, suggesting potential benefits of immunotherapy for patients in the low-risk group. The combination of high risk and low tumor mutational burden (TMB) could further shortened the OS of BLCA patients. Lastly, the drug sensitivity analysis revealed that the BLCA cells in the high-risk group showed an increased sensitivity to cisplatin, sunitinib, cetuximab, axitinib, docetaxel, saracatinib, vinblastine and pazopanib compared with those in the low-risk group. According to the Quantitative real time PCR results, we found that five lncRNAs of the risk model were more highly expressed in BCa cell lines than human immortalized uroepithelial cell line. The disulfidptosis-related lncRNA risk model has a valuable effect in assessing the prognosis of BLCA patients.

ISO-upregulated BECN1 specifically promotes LC3B-dependent autophagy and anticancer activity in invasive bladder cancer

Isorhapontigenin (ISO), an active compound isolated from the Chinese herb Gnetum Cleistostachyum, exhibited strong preventive and therapeutic effects on bladder cancer (BC) both in vitro and in vivo. Our previous studies revealed that ISO-induced autophagy is crucial for its anti-cancer activity. However, the underlying mechanism remains unclear. Here, we showed that BECN1, an important autophagic protein, was induced by ISO treatment and played crucial roles in ISO-induced late phase of LC3B-dependent, and LC3A-independent autophagy, as well as anti-cancer activity. Downregulation of BECN1 was observed in human BCs and BBN-induced mouse invasive BC tissues, whereas co-treatment with ISO completely reversed BECN1 downregulation in BBN-induced mouse invasive BCs. Consistently, ISO treatment significantly increased BECN1 expression in vitro in a dose- and time-dependent manner. Depletion of BECN1 significantly impaired LC3B-dependent autophagy following ISO treatment, as well as abolished the inhibitory effect of ISO on anchorage-independent growth of human BC cells. Mechanistic studies revealed that BECN1 induction was mediated by ISO downregulation of c-Myc, which resulted in miR-613 reduction, in turn leading to increased NCL translation and further promoting NCL binding to BECN1 mRNA, subsequently stabilizing BECN1 mRNA. In conclusion, our results demonstrate that by activating c-Myc/miR-613/NCL axis, ISO treatment results in BECN1 posttranscriptional upregulation, which specifically initiates LC3B-dependent autophagy and anti-cancer activity. Our findings further strengths our application of ISO for therapy of high-grade invasive BC (HGIBC) patients.

CRH upregulates supervillin through ERK and AKT pathways to promote bladder cancer cell migration.

Corticotropin-releasing hormone (CRH) has been well documented playing a role in the regulation of cellular processes, immune responses, and inflammatory processes that can influence the occurrence and development of tumors. Supervillin (SVIL) is a membrane-associated and actin-binding protein, which is actively involved in the proliferation, spread, and migration of cancer cells. This work investigated CRH's influence on bladder cancer cells'migration and relevant mechanisms. By using human bladder cancer cells T24 and RT4 in wound healing experiments and transwell assay, we found that the migration ability of the T24 cells was significantly increased after CRH treatment. In vivo experiments showed that CRH significantly promoted the metastases of T24 cells in cell line-derived xenograft (CDX) mouse model. Interestingly, downregulation of SVIL by SVIL-specifc small hairpin RNAs significantly reduced the promoting effect of CRH on bladder cancer cell migration. Furthermore, CRH significantly increased SVILmessenger RNA and protein expression in T24 cells, accompanied with AKT andERK phosphorylation in T24 cells. Pretreatment with AKT inhibitor (MK2206) blocked the CRH-induced SVIL expression and ERK phosphorylation. Also, inhibition of ERK signaling pathway by U0126 significantly reduced the CRH-induced SVIL expression and AKT phosphorylation. It suggested that cross-talking between AKT and ERK pathways was involved in the effect of CRH on SVIL. Taken together, we demonstrated that CRH induced migration of bladder cancer cells, in which AKT andERK pathways -SVIL played a key role.

Cyclanoline Reverses Cisplatin Resistance in Bladder Cancer Cells by Inhibiting the JAK2/STAT3 Pathway.

Cisplatin is a key therapeutic agent for bladder cancer, yet the emergence of cisplatin resistance presents a significant clinical challenge. This study aims to investigate the potential and mechanisms of cyclanoline (Cyc) in overcoming cisplatin resistance. Cisplatin-resistant T24 and BIU-87 cell models (T24/DR and BIU-87/DR) were established by increasing gradual concentration. Western Blot (WB) assessed the phosphorylation of STAT3, JAK2, and JAK3. T24/DR and BIU-87/DR cell lines were treated with selective STAT3 phosphorylation modulators, and cell viability was evaluated by CCK-8. Cells were subjected to cisplatin, Cyc, or their combination. Immunofluorescence (IHC) examined p-STAT3 expression. Protein and mRNA levels of apoptosis-related and cell cycle-related factors were measured. Changes in proliferation, invasion, migration, apoptosis, and cell cycle were monitored. In vivo, subcutaneous tumor transplantation models in nude mice were established, assessing tumor volume and weight. Changes in bladder cancer tissues were observed through HE staining, and the p-STAT3 was assessed via WB and IHC. Cisplatin-resistant cell lines were successfully established, demonstrating increased phosphorylation of STAT3, JAK2, and JAK3. Cisplatin or Cyc treatment decreased p-STAT3, inhibited invasion and migration, and induced apoptosis and cell cycle arrest in the G0/G1 phase in vitro. In vivo, tumor growth was significantly suppressed, with extensive tumor cell death. IHC and WB consistently showed a substantial downregulation of STAT3 phosphorylation. These changes were more pronounced when cisplatin and Cyc were administered in combination. Cyc reverses cisplatin resistance via JAK/STAT3 inhibition in bladder cancer, offering a potential clinical strategy to enhance cisplatin efficacy in treating bladder cancer.

ROR1-AS1: A Meaningful Long Noncoding RNA in Oncogenesis.

Long noncoding RNA (lncRNA) is a non-coding RNA with a length of more than 200 nucleotides, involved in multiple regulatory processes in vivo, and is related to the physiology and pathology of human diseases. An increasing number of experimental results suggest that when lncRNA is abnormally expressed, it results in the development of tumors. LncRNAs can be divided into five broad categories: sense, antisense, bidirectional, intronic, and intergenic. Studies have found that some antisense lncRNAs are involved in a variety of human tumorigenesis. The newly identified ROR1-AS1, which functions as an antisense RNA of ROR1, is located in the 1p31.3 region of the human genome. Recent studies have reported that abnormal expression of lncRNA ROR1-AS1 can affect cell growth, proliferation, invasion, and metastasis and increase oncogenesis and tumor spread, indicating lncRNA ROR1-AS1 as a promising target for many tumor biological therapies. In this study, the pathophysiology and molecular mechanism of ROR1-AS1 in various malignancies are discussed by retrieving the related literature. ROR1-AS1 is a cancer-associated lncRNA, and studies have found that it is either over- or underexpressed in multiple malignancies, including liver cancer, colon cancer, osteosarcoma, glioma, cervical cancer, bladder cancer, lung adenocarcinoma, and mantle cell lymphoma. Furthermore, it has been demonstrated that lncRNA ROR1-AS1 participates in proliferation, migration, invasion, and suppression of apoptosis of cancer cells. Furthermore, lncRNA ROR1-AS1 promotes the development of tumors by up-regulating or downregulating ROR1-AS1 conjugates and various pathways and miR-504, miR-4686, miR-670-3p, and miR-375 sponges, etc., suggesting that lncRNA ROR1-AS1 may be used as a marker in tumors or a potential therapeutic target for a variety of tumors.

TNFRSF11B promotes the progression of bladder cancer through PI3K/AKT signaling pathway

TNFRSF11B contributes to tumorigenesis in many malignancies. Nevertheless, its function and underlying tumorigenic mechanism in bladder cancer (BC) has been rare.The clinical significance and relevant signaling pathway of TNFRSF11B in BC were assessed using bioinformatic analysis. The determination of TNFRSF11B expression was conducted in bladder tissues and BC cells. BC cells were subjected to functional experiments to evaluate their ability to proliferate, migrate, and invade. Cell apoptosis experiments were conducted. The protein levels of markers associated with epithelial-mesenchymal transition (EMT) and molecules linked to the PI3K/AKT pathway were assessed. To evaluate the effect of the PI3K/AKT pathway on TNFRSF11B, LY294002, a PI3K/AKT pathway inhibitor, was utilized. TNFRSF11B exhibited significant upregulation in both BC tissues and various cell lines. Inhibited TNFRSF11B expression impeded the growth, movement, infiltration of BC cells. Conversely, the ultimate outcome varied when TNFRSF11B was overexpressed. In vivo assay further confirmed the above results. Furthermore, TNFRSF11B promoted malignant traits by controlling the PI3K/AKT pathway. In BC, TNFRSF11B exhibits elevated expression levels and has a substantial tumor-promoting role in BC via the PI3K/AKT pathway. Importantly, TNFRSF11B may represent a valuable prognostic tumor marker for BC treatment.

Synergistic Combination of Quercetin and Mafosfamide in Treatment of Bladder Cancer Cells.

Bladder cancer, which has a rising incidence, is the 10th most common cancer. The transitional cell carcinoma histotype is aggressive and often current therapies are ineffective. We investigated the anti-proliferative effect of quercetin, a natural flavonoid, in combination with the alkylating agent mafosfamide (MFA) on two human bladder cancer cell lines, namely RT112 and J82, representing the progression from low-grade to high-grade tumors, respectively. In both cell types, the combined treatment led to a synergic reduction in cell viability confirmed by a combination index of less than one, though different biological responses were noted. In J82 cells, MFA alone and, to a lesser extent, with quercetin caused cell cycle arrest in the G2/M phase, but only the combined treatment triggered apoptotic cell death. In contrast, in RT112 cells, quercetin induced autophagy, evidenced by the autophagosome formation and the increase in LC-3 lipidation. Interestingly, the synergistic effect was observed only when cells were pre-treated with MFA for 24 h before adding quercetin, not in the reverse order. This suggests that quercetin may help overcome MFA resistance to apoptosis. Although further studies are needed, investigating the combined effects of quercetin and MFA could help elucidate the mechanisms of drug resistance in bladder cancer treatment.

Silencing CCT3 induces ferroptosis through the NOD1-NF-κB signaling pathway in bladder cancer.

Bladder cancer (BCa) is a lethal malignancy of the urinary system and exhibits a poor prognosis. Chaperonin-containing tailless complex polypeptide 1 subunit 3 (CCT3) acts as an oncogene in various tumors, whereas its effect on BCa remains unknown. We identified the ferroptosis-associated differentially expressed genes through bioinformatic analysis and selected CCT3 for further verification. The levels of cell viability, apoptosis, migration, invasion, and proliferation were measured to clarify the effect of silencing CCT3 on BCa cells. Then we evaluated the role of CCT3 knockdown in vivo. Ferroptosis was assessed by the expression detection of the ferroptosis-related proteins. The underlying mechanism was predicted by RNA sequencing and verified by an agonist for nucleotide-binding and oligomerization domain 1 (NOD1). Western blotting was conducted to detect the protein expression of NOD1, nuclear factor kappa B (NF-κB) inhibitor alpha (IκBα), and phospho-IκBα (p-IκBα). In vitro, down-regulation of CCT3 suppressed the cell viability, migration, invasion, and proliferation, as well as induced apoptosis of BCa cells. In vivo, silencing CCT3 elevated the body weight of mice and suppressed the BCa progression. In addition, CCT3 knockdown could induce ferroptosis in vitro and in vivo. CCT3 knockdown suppressed the expression of NOD1 and p-IκBα/IκBα and the NOD1 agonist could reverse the effect of CCT3 suppression on BCa in vitro and in vivo. In summary, our findings demonstrate that silencing CCT3 inhibits BCa via induction of ferroptosis and suppression of the NOD1-NF-κB pathway.

Oncogenic role of lncRNA SBF2-AS1 in bladder cancer

IntroductionBladder cancer is a malignant neoplasm with increasing incidence rates. LncRNAs play an important role in cancer, including development, prognosis, and response to therapies. It is known that lncRNA SBF2-AS1 was associated with cell proliferation and worse prognosis in various tumor types, but its role remains incompletely understood in bladder cancer. In this context, our objective was to evaluate the effect of lncRNA SBF2-AS1 silencing on bladder cancer cells. MethodsJ82 and UM-UC-3 high-grade bladder tumor cells were treated with two siRNAs specific for SBF2-AS1 to evaluate cytotoxicity, clonogenic survival, morphology, cell migration, and cell cycle progression. ResultsExpression inhibition of SBF2-AS1 resulted in cytotoxicity, morphological changes, and decreased clone formation and cell migration. Cell cycle alterations were not observed. ConclusionOur study revealed that SBF2-AS1 plays an oncogenic role and holds promise as a potential target for the treatment of bladder cancer.

Ubiquitin-Specific Protease 1 Promotes Bladder Cancer Progression by Stabilizing c-MYC.

Ubiquitination is an important post-transcriptional modification crucial for maintaining cell homeostasis. As a deubiquitination enzyme, ubiquitin-specific protease 1 (USP1) is associated with tumor progression; however, its role in bladder cancer is unknown. This study aimed to analyze USP1 expression and study its roles in bladder cancer. The web server GEPIA was used to analyze the USP1 expression. To explore USP1's function in bladder cancer, we constructed USP1-knockout cell lines in UMUC3 cells. A FLAG-USP1 (WT USP1) plasmid and a plasmid FLAG-USP1 C90S (catalytic-inactive mutant) were used to overexpress USP1 in T24 cells. CCK8, colony formation, and Transwell assays were used to assess cell viability, proliferation, and migration. RNA-sequencing (RNA-seq) and dual-luciferase reporter assays were performed to screen the pathway. Co-immunoprecipitation and immunofluorescence were used to explore the interaction between USP1 and c-MYC. A xenograft mouse model was used to study the role of USP1 in bladder cancer. USP1 expression was upregulated in human bladder cancer cells and correlated with poor patient prognosis. USP1 overexpression promoted cell proliferation, clone formation, and migration, and this was attenuated by genetic ablation of USP1. Furthermore, we observed that USP1 deficiency inhibited tumor formation in vivo. Mechanistically, the c-MYC pathway was remarkably activated compared with the other pathways. Furthermore, USP1 could interact with c-MYC and increase c-MYC's stability depending on the catalytic activity of USP1. Our results suggested that high expression of USP1 promotes bladder cancer progression by stabilizing c-MYC; hence, USP1 may serve as a novel therapeutic target for treating bladder cancer.

Gefitinib Reverses PD-L1-Mediated Immunosuppression Induced by long-term Glutamine blockade in Bladder Cancer.

Glutamine is a major energy source for tumor cells and blocking glutamine metabolism is being investigated as a promising strategy for cancer therapy. However, the antitumor effect of glutamine blockade in bladder cancer remains unclear, necessitating further investigation. Here, we demonstrated that glutamine metabolism was involved in the malignant progression of bladder cancer. Treatment with the glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON) inhibited the growth of bladder cancer cells in vitro in several ways. In addition, we observed inhibition of tumor growth in bladder cancer-bearing mice using JHU083, a prodrug that was designed to prevent DON-induced toxicity. However, the antitumor immune effect of T cells changed from activation to inhibition as the administrated time extended. We found that both in vitro treatment with DON and in vivo prolonged administration of JHU083 led to the upregulation of PD-L1 in bladder cancer cells. Mechanistically, glutamine blockade up-regulated PD-L1 expression in bladder cancer cells by accumulating ROS, subsequently activating the EGFR/ERK/C-Jun signaling pathway. Combination treatment of JHU083 and gefitinib reversed the up-regulation of PD-L1 in bladder cancer cells induced by prolonged glutamine blockade, resulting in the alleviation of T-cell immunosuppression and a significant improvement in therapeutic outcome. These preclinical findings show promise for glutamine metabolism targeting as a viable therapeutic strategy for bladder cancer, with the potential for further enhancement through combined treatment with gefitinib.