FGL2 as a predictive biomarker for prognosis and immunotherapy in bladder cancer.

Background: Metastasis and immunosuppression result in unfavorable prognosis in bladder cancer (BLCA). FGL1 and FGL2 are two members of the fibrinogen-related proteins family, but their potential effects on BLCA remain elusive. Methods: The expression profile of FGL1 and FGL2 in BLCA was analyzed in multiple databases. Furthermore, the expression of FGL2 was validated in BLCA tissues. The predictive capability of FGL2 was evaluated by Kaplan-Meier analysis, univariate analysis, and multivariate Cox regression. A nomogram model was constructed based on FGL2 expression and clinicopathological parameters for clinical practice. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analyses (GSEA) were performed to investigate enrichment in the biological processes. In addition, the correlation between FGL2 and immunological characteristics in the BLCA tumor microenvironment (TME), including tumor-infiltrating immune cells (TICs), cancer-immunity cycles, immune checkpoint molecules (ICPs), immunophenoscores (IPS), and response to anti-PD-L1 immunotherapy was further analyzed. Results: FGL2 was found to be downregulated in BLCA due to hypermethylation of the FGL2 promoter region, which was associated with an unfavorable prognosis. Moreover, BLCA patients with high FGL2 expression exhibited better response to immunotherapy. Conclusions: Our research revealed that FGL2 was downregulated in BLCA and was negatively correlated with DNA methylation. High FGL2 expression was confirmed as an independent risk for prognosis. Moreover, FGL2 is a promising indicator for the response to immunotherapy in patients with BLCA.

Promoting Intratumoral Drug Accumulation by Bio-Membrane Regulated Active Targeting for Tumor Photothermal Therapy.

Introduction: Insufficient tumor permeability and inadequate nanoparticle retention continue to be significant limitations in the efficacy of anti-tumor drug therapy. Numerous studies have focused on enhancing tumor perfusion by improvement of tumor-induced endothelial leakage, often known as the enhanced permeability and retention (EPR) effect. However, these approaches have produced suboptimal therapeutic outcomes and have been associated with significant side effects. Therefore, in this study, we prepared tumor cell membrane-coated gold nanorods (GNR@TM) to enhance drug delivery in tumors through homogeneous targeting of tumor cell membranes and in situ real-time photo-controlled therapy. Methods: Here, we fabricated GNR@TM, and characterized it using various techniques including Ultraviolet-Visible (UV-Vis) spectrophotometer, particle size analysis, potential measurement, and transmission electron microscopy (TEM). The cellular uptake and cytotoxicity of GNR@TM were analyzed by flow cytometry, confocal laser scanning microscopy (CLSM), TEM, CCK8 assay and live/dead staining. Tissue drug distribution was determined by inductively coupled plasma mass spectrometry (ICP-MS) and immunofluorescence staining. Furthermore, to evaluate the therapeutic effect, mice bearing MB49 tumors were intravenously administered with GNR@TM. Subsequently, near-infrared (NIR) laser therapy was performed, and the mice's tumor growth and body weight were monitored. Results: The tumor cell membrane coating endowed GNR@TM with extended circulation time in vivo and homotypic targeting to tumor, thereby enhancing the accumulation of GNR@TM within tumors. Upon 780 nm laser, GNR@TM exhibited excellent photothermal conversion capability, leading to increased tumor vascular leakage. This magnification of the EPR effect induced by NIR laser further increased the accumulation of GNR@TM at the tumor site, demonstrating strong antitumor effects in vivo. Conclusion: In this study, we successfully developed a NIR-triggered nanomedicine that increased drug accumulation in tumor through photo-controlled therapy and homotypic targeting of the tumor cell membrane. GNR@TM has been demonstrated effective suppression of tumor growth, excellent biocompatibility, and significant potential for clinical applications.

Mechanisms of Xiaozheng decoction for anti-bladder cancer effects via affecting the GSK3ß/ß-catenin signaling pathways: a network pharmacology-directed experimental investigation.

PURPOSE: The combination of Xiaozheng decoction with postoperative intravesical instillation has been shown to improve the prognosis of bladder cancer patients and prevent recurrence. However, the mechanisms underlying the efficacy of this herbal formula remain largely unclear. This research aims to identify the important components of Xiaozheng decoction and explore their anti-bladder cancer effect and mechanism using network pharmacology-based experiments. METHODS: The chemical ingredients of each herb in the Xiaozheng decoction were collected from the Traditional Chinese Medicine (TCM) database. Network pharmacology was employed to predict the target proteins and pathways of action. Disease databases were utilized to identify target genes associated with bladder cancer. A Protein-Protein Interaction (PPI) network was constructed to illustrate the interaction with intersected target proteins. Key targets were identified using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis. A compound-target-pathway network was established after molecular docking predictions. In vitro experiments with bladder cancer cell lines were conducted using core chemical components confirmed by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-qTOF-MS) to verify the conclusions of network pharmacology. RESULTS: 45 active compounds were extracted, and their relationships with Traditional Chinese Medicines (TCMs) and protein targets were presented, comprising 7 herbs, 45 active compounds, and 557 protein targets. The intersection between potential TCM target genes and bladder cancer-related genes yielded 322 genes. GO and KEGG analyses indicated that these targets may be involved in numerous cancer-related pathways. Molecular docking results showed that candidate compounds except mandenol could form stable conformations with the receptor. In vitro experiments on three bladder cancer cell lines demonstrated that quercetin and two other impressive new compounds, bisdemethoxycurcumin (BDMC) and kumatakenin, significantly promoted cancer cell apoptosis through the B-cell lymphoma 2/Bcl-2-associated X (Bcl-2/BAX) pathway and inhibited proliferation and migration through the glycogen synthase kinase 3 beta (GSK3ß)/ß-catenin pathway. CONCLUSION: By employing network pharmacology and conducting in vitro experiments, the mechanism of Xiaozheng decoction's effect against bladder cancer was tentatively elucidated, and its main active ingredients and targets were identified, providing a scientific basis for future research.

A prognostic model based on necroptosis-related genes for prognosis and therapy in bladder cancer.

Bladder cancer, one of the most prevalent malignant cancers, has high rate of recurrence and metastasis. Owing to genomic instability and high-level heterogeneity of bladder cancer, chemotherapy and immunotherapy drugs sensitivity and lack of prognostic markers, the prognosis of bladder cancer is unclear. Necroptosis is a programmed modality of necrotic cell death in a caspase-independent form. Despite the fact that necroptosis plays a critical role in tumor growth, cancer metastasis, and cancer patient prognosis, necroptosis-related gene sets have rarely been studied in bladder cancer. As a result, the development of new necroptosis-related prognostic indicators for bladder cancer patients is critical. Herein, we assessed the necroptosis landscape of bladder cancer patients from The Cancer Genome Atlas database and classified them into two unique necroptosis-related patterns, using the consensus clustering. Then, using five prognosis-related genes, we constructed a prognostic model (risk score), which contained 5 genes (ANXA1, DOK7, FKBP10, MAP1B and SPOCD1). And a nomogram model was also developed to offer the clinic with a more useful prognostic indicator. We found that risk score was significantly associated with clinicopathological characteristics, TIME, and tumor mutation burden in patients with bladder cancer. Moreover, risk score was a valid guide for immunotherapy, chemotherapy, and targeted drugs. In our study, DOK7 was chosen to further verify our prognosis model, and functional assays indicated that knockdown the expression of DOK7 could prompt bladder cancer proliferation and migration. Our work demonstrated the potential role of prognostic model based on necroptosis genes in the prognosis, immune landscape and response efficacy of immunotherapy of bladder cancer.

Establishment of an optimized orthotopic bladder cancer model in mice.

BACKGROUND: Bladder cancer (BC) is one of the most common malignancies of the genitourinary system. Animal models offer an important tool to explore tumour initiation, progression, and therapeutic mechanisms. Our aim is to construct an optimized orthotopic BC model which is predictable, reproducible, and convenient. METHODS: The optimized orthotopic BC model was constructed in male C57BL/6 mice utilizing microsyringes to inoculate them with a murine BC cell line (MB49). Anesthetised mice were inoculated with an MB49 cell suspension (10 µL) at approximately 5 × 106/mL. The whole process of modelling was observed and monitored every 3 days for 21 days utilizing HE staining and transabdominal ultrasonography (TUS). RESULTS: In this study, the model showed excellent success rates for tumour formation (96.67%) and metastatic rate (89.66%). Compared to the control group (sham operation), mice in the modelling group had serous cachexia, visible haematuresis and weight loss (all P < 0.05). The lungs, liver, ureter and kidneys were found to have tumour metastasis. Moreover, the average survival time (19.73 ± 1.69 d) of modelling mice was significantly shorter than that of the control mice (P < 0.05), which remained alive. CONCLUSION: Our study established a method using microsyringes to inject murine BC cells into the bladder wall, creating a stable transplantable BC model in mice.

Identification of ENO1 as a prognostic biomarker and molecular target among ENOs in bladder cancer.

BACKGROUND: Enolase is an essential enzyme in the process of glycolysis and has been implicated in cancer progression. Though dysregulation of ENOs has been reported in multiple cancers, their prognostic value and specific role in bladder cancer (BLCA) remain unclear. METHODS: Multiple databases were employed to examine the expression of ENOs in BLCA. The expression of ENO1 was also validated in BLCA cell lines and tissue samples by western blotting and immunohistochemistry. Kaplan-Meier analysis, ROC curve, univariate and multivariate Cox regression were performed to evaluate the predictive capability of the ENO1. Gene ontology (GO) and Gene Set Enrichment Analyses (GSEA) analysis were employed to perform the biological processes enrichment. Function experiments were performed to explore the biological role of ENO1 in BLCA. The correlation of ENO1 with immune cell infiltration was explored by CIBERSORT. RESULTS: By analyzing three ENO isoforms in multiple databases, we identified that ENO1 was the only significantly upregulated gene in BLCA. High expression level of ENO1 was further confirmed in BLCA tissue samples. Aberrant ENO1 overexpression was associated with clinicopathological characteristics and unfavorable prognosis. Functional studies demonstrated that ENO1 depletion inhibited cancer cell aggressiveness. Furthermore, the expression level of ENO1 was correlated with the infiltration levels of immune cells and immune-related functions. CONCLUSIONS: Taken together, our results indicated that ENO1 might serve as a promising prognostic biomarker for prognosticating prognosis associated with the tumor immune microenvironment, suggesting that ENO1 could be a potential immune-related target against BLCA.

Pyroptosis-Related Patterns Predict Tumor Immune Landscape and Immunotherapy Response in Bladder Cancer.

Background: Bladder cancer (BC) is a leading cause of death from malignancy, with significant heterogeneity in the immunotherapeutic responsiveness of advanced status. Pyroptosis, a newly discovered inflammatory programmed cell death, is confirmed to play an indispensable role in tumorigenesis and anti-tumor activity. However, the effect of pyroptosis on the tumor-immune landscape remodeling and immunotherapy in BC remains elusive. Methods: We comprehensively evaluated the mRNA expression and genomic alterations of 33 pyroptosis-related genes (PRGs) in BC and evaluated the patterns of pyroptosis in publicly available BC datasets. An unsupervised clustering method was used to classify patients into distinct patterns. Then, we established a pyroptosis-related signature score (PS-score) model to quantify the pyroptosis-related patterns of individual BC patients using principal component analysis. Furthermore, we correlated the patterns with the immune landscape and response efficacy of immunotherapy. Results: Two pyroptosis-related patterns were identified in BC, and distinct patterns showed various immune characteristics. Patterns with a high expression level of PRGs exhibited a survival advantage and showed higher infiltration of cytotoxic lymphocytes. Tumors with a low PS-score were characterized by high tumor-infiltrating lymphocytes and considered "hot." Further analysis revealed that the PS-score was an independent prognostic factor and could predict the response to immunotherapy for patients with advanced BC. We found a significant positive association between AHNAK2, AHNAK nucleoprotein 2, expression, and PS-score. Functional assays showed that AHNAK2 knockdown was correlated with attenuated invasive ability. Conclusion: This work comprehensively demonstrated the potential function of pyroptosis-related patterns in the bladder tumor-immune landscape and identified their therapeutic liability in immunotherapy. Our study enhanced our understanding of the immune landscape and provided a new approach toward more effective immunotherapy strategies.

NLRP3 promotes immune escape by regulating immune checkpoints: A pan-cancer analysis.

NLRP3 plays a pathogenic role in tumorigenesis by regulating innate and acquired immunity, apoptosis, differentiation, and intestinal microbes in tumors. Our research aimed to investigate the role of NLRP3 in pan-cancers based on multi-omics data in the TCGA database. Most types of tumors showed increased expression of NLRP3. Among them, the overexpressed NLRP3 in liver hepatocellular carcinoma (LIHC) and ovarian cancer (OV) indicated worse overall survival (OS). Further analysis also confirmed overexpressed NLRP3 in colon cancer (COAD) indicated a high probability of microsatellite instability (MSI) and low tumor mutational burden (TMB), which indicated a better response to immune checkpoint inhibitors (ICIs). Interestingly, overexpression of NLRP3 was closely related to high infiltration of immune cells (T cells, B cells, etc.) and overexpressed immune checkpoints (PD-1, PD-L1, LAG3, etc.). These results demonstrated NLRP3 promoted immune escape in cancers. Finally, we investigated the expression of various immune checkpoints by treating NLRP3 inhibitor MCC950 during the co-culture of peripheral blood mononuclear cells (PBMC) and LIHC cell line Hep3B. MCC950 significantly repressed the expression of PD-L1 and LAG3, and promoted the apoptosis rate of Hep3B. In conclusion, our research demonstrated the role of NLRP3 in pan-cancer, especially in LIHC. Inhibition of NLRP3 promoted the killing effect of T cells to cancer cells by repressing the expression of immune checkpoints.

A novel cuproptosis-related lncRNA signature predicts prognosis and therapeutic response in bladder cancer.

Bladder cancer (BC) ranks the tenth in the incidence of global tumor epidemiology. LncRNAs and cuproptosis were discovered to regulate the cell death. Herein, we downloaded transcriptome profiling, mutational data, and clinical data on patients from The Cancer Genome Atlas (TCGA). High- and low-risk BC patients were categorized. Three CRLs (AL590428.1, AL138756.1 and GUSBP11) were taken into prognostic signature through least absolute shrinkage and selection operator (LASSO) Cox regression. Worse OS and PFS were shown in high-risk group (p < 0.05). ROC, independent prognostic analyses, nomogram and C-index were predicted via CRLs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated IncRNAs play a biological role in BC progression. Immune-related functions showed the high-risk group received more benefit from immunotherapy and had stronger immune responses, and the overall survival was better (p < 0.05). Finally, a more effective outcome (p < 0.05) was found from clinical immunotherapy via the TIDE algorithm and many potential anti-tumor drugs were identified. In our study, the cuproptosis-related signature provided a novel tool to predict the prognosis in BC patients accurately and provided a novel strategy for clinical immunotherapy and clinical applications.

A Novel Ferroptosis-Related Prognostic Signature Reveals Macrophage Infiltration and EMT Status in Bladder Cancer.

Bladder cancer (BC) belongs to one of the most common and highly heterogeneous malignancies. Ferroptosis is a newly discovered regulated cell death (RCD), characterized by accumulation of toxic lipid peroxides, and plays a crucial role in tumor progression. Here, we conducted a comprehensive analysis on the transcriptomics data of ferroptosis-related genes in BC based on The Cancer Genome Atlas (TCGA) and three Gene Expression Omnibus (GEO) datasets. In our study, a 6-gene signature was identified based on the potential prognostic ferroptotic regulatory genes. Furthermore, our signature revealed a good independent prognostic ability in BC. Patients with low-risk score exhibited higher FGFR3 mutation rates while high risk score had a positive association with higher RB1 mutation rates. Meanwhile, higher proportions of macrophages were observed in high BC risk group simultaneously with four methods. Unexpectedly, the risk score showed a significant positive correlation with epithelial-mesenchymal transition (EMT) status. Functional assays indicated that CRYAB and SQLE knockdown was associated with attenuated invasion capacity. Our study revealed a ferroptosis-related risk model for predicting prognostic and BC progression. Our results indicate that targeting ferroptosis may be a therapeutic strategy for BC.

Antiproliferative effects of mealworm larvae (Tenebrio molitor) aqueous extract on human colorectal adenocarcinoma (Caco-2) and hepatocellular carcinoma (HepG2) cancer cell lines.

Recently, insects have aroused the interest of researchers as potential therapeutic resources against malignant diseases such as cancer. In this study, the effects of aqueous extracts from mysore thorn borer (MTB) (Anoplophora chinensis) and mealworm larvae (MWL) (Tenebrio molitor) against cancer cells were investigated. MWL aqueous extract showed higher antiproliferative effects against Caco-2 and HepG2 cells compared to MTB. The IC50 (48 hr) of MWL aqueous extract were 11.44 and 20 mg/ml for Caco-2 and HepG2 respectively. Flow cytometry analysis showed that MWL aqueous extract induced apoptosis in Caco-2 and HepG2 increasing from 2.06% to 74.34% and from 0.04% to 42.14% after 24 hr respectively. Caspase activity assay showed that apoptosis was mediated via death receptor pathway mediated by caspase-8 and -9 followed by the activation of caspase-3; caspase-3 may have induced DNA damage and cell death. These effects may be correlated to its free amino acids. The results of this study demonstrate the potentials of MWL in the development of natural anticancer therapeutics in the future. PRACTICAL APPLICATIONS: Natural nutraceuticals from insects might be useful for the treatment and prevention of cancers such as colorectal and liver cancer. In recent years, edible insects have caught the attention of researchers, because of their potential as an alternative source of food and nutraceuticals. The results of our study showed that MWL extract might provide important anticancer compounds against colon and liver cancer.

CBX7 suppresses urinary bladder cancer progression via modulating AKR1B10-ERK signaling.

The chromobox (CBX) proteins mediate epigenetic gene silencing and have been implicated in the cancer development. By analyzing eight CBX family members in TCGA dataset, we found that chromobox 7 (CBX7) was the most strikingly downregulated CBX family member in urinary bladder cancer (UBC), as compared to normal tissues. Though dysregulation of CBX7 has been reported in multiple cancers, its specific role and clinical relevance in UBC remain unclear. Herein, we found that frequent downregulation of CBX7 in UBC specimens, which was due to its promoter hypermethylation, was correlated with poor prognosis. The ectopic expression of CBX7 suppressed UBC cell proliferation, migration, invasion, and cancer stemness, whereas CBX7 depletion promoted cancer cell aggressiveness. Importantly, CBX7 overexpression in UBC cells inhibited tumorigenicity, whereas CBX7 depletion promoted the tumor development, indicating its tumor-suppressive role in UBC. Using RNA-seq and chromosome immunoprecipitation (ChIP) assays, we identified aldo-keto reductase family 1 member 10 (AKR1B10) as a novel downstream target of CBX7, which was negatively modulated by CBX7 in a PRC1-dependent manner and involved in stimulating ERK signaling. Consistently, AKR1B10 overexpression induced cancer cell aggressiveness, whereas suppression of AKR1B10 by siRNA or its small molecular inhibitor, oleanolic acid, reversed the CBX7 deficiency-induced cellular effects. AKR1B10 overexpression was negatively associated with CBX7 downregulation and predicted poor clinical outcomes in UBC patients. Taken together, our results indicate that CBX7 functions as a tumor suppressor to downregulate AKR1B10 and further inactivates ERK signaling. This CBX7/AKR1B10/ERK signaling axis may provide a new therapeutic strategy against UBC.

A fish with no sex: gonadal and adrenal functions partition between zebrafish NR5A1 co-orthologs.

People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal-gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.

TEAD4 as a Prognostic Marker Promotes Cell Migration and Invasion of Urinary Bladder Cancer via EMT.

PURPOSE: As a member of TEA Domain Transcription Factors (TEADs), TEAD4 was found to be upregulated in urinary bladder cancer (UBC). This study focused on investigating the clinical value and potential functions of TEAD4 in UBC. MATERIALS AND METHODS: Patients' samples, TCGA-BLCA and multiple GEO datasets were applied to explore the expression pattern of TEAD4 in UBC. Cox regression and Kaplan-Meier survival analyses were carried out to evaluate the prognostic significance of TEAD4 in UBC. Wound healing and transwell assays were performed to explore the biological functions of TEAD4 in UBC cells. RESULTS: The results of TCGA-BLCA, GEO datasets, Western blotting and immunohistochemistry staining (IHC) indicated that TEAD4 was strikingly elevated in UBC tissues as compared to their normal counterparts, and upregulation of TEAD4 was significantly correlated with clinical stage, pathological grade and poor clinical outcome. Functional studies demonstrated that TEAD4 knockdown suppressed cell migration and invasion by reducing the expression of epithelial-mesenchymal transition (EMT) related markers and transcription regulators. CONCLUSION: Our results suggest that TEAD4 may serve as a novel prognostic biomarker and a promising therapeutic target for UBC, and act as a pro-tumorigenic gene to promote cell migration and invasion by inducing EMT.

PDE4B Induces Epithelial-to-Mesenchymal Transition in Bladder Cancer Cells and Is Transcriptionally Suppressed by CBX7.

Urinary bladder cancer (UBC) is a common malignant tumor with high incidence. Advances in the diagnosis and treatment of this disease demand the identification of novel therapeutic targets. Multiple studies demonstrated that PDE4B level was upregulated in malignancies and high PDE4B expression was correlated with poor outcomes. Herein, we identified that PDE4B was a potential therapeutic target in UBC. We confirmed that PDE4B expression was correlated with aggressive clinicopathological characteristics and unfavorable prognosis. Functional studies demonstrated that ectopic expression of PDE4B promoted UBC cells proliferation, migration and invasion, whereas PDE4B depletion suppressed cancer cell aggressiveness. We also identified CBX7 as a regulator of PDE4B to suppress the expression of PDE4B at the transcription level in a PRC1-dependent manner. Moreover, our results indicated that PDE4B induced epithelial-to-mesenchymal transition (EMT) in UBC cells via ß-catenin pathway, whereas inhibition of PDE4B by its small molecule inhibitor, rolipram, effectively reversed the PDE4B overexpression-induced effects. To sum up, our results indicated that PDE4B acts as an oncogene by promoting UBC cell migration and invasion via ß-catenin/EMT pathway.

Identification of glycogene-type and validation of ST3GAL6 as a biomarker predicts clinical outcome and cancer cell invasion in urinary bladder cancer.

Background: Urinary bladder cancer (UBC) is one of the most common causes of morbidity and mortality worldwide characterized by a high risk of invasion and metastasis; however, the molecular classification biomarkers and underlying molecular mechanisms for UBC patient stratification on clinical outcome need to be investigated. Methods: A systematic transcriptomic analysis of 185 glycogenes in the public UBC datasets with survival information and clinicopathological parameters were performed using unsupervised hierarchical clustering. The gene signature for glycogene-type classification was identified using Limma package in R language, and correlated to 8 known molecular features by Gene Set Variation Analysis (GSVA). The clinical relevance and function of a glycogene was characterized by immunohistochemistry in UBC patient samples, and quantitative RT-PCR, Western blotting, promoter activity, MAL II blotting, immunofluorescence staining, wound healing, and transwell assays in UBC cells. Results: A 14-glycogene signature for glycogene-type classification was identified. Among them, ST3GAL6, a glycotransferase to transfer sialic acid to 3'-hydroxyl group of a galactose residue, showed a significant negative association with the subtype with luminal feature in UBC patients (n=2,130 in total). Increased ST3GAL6 was positively correlated to tumor stage, grade, and survival in UBCs from public datasets or our cohort (n=52). Transcription factor GATA3, a luminal-specific marker for UBC, was further identified as a direct upstream regulator of ST3GAL6 to negatively regulate its transactivation. ST3GAL6 depletion decreased MAL II level, cell invasion and migration in 5637 and J82 UBC cells. ST3GAL6 could reverse the effects of GATA3 on global sialylation and cell invasion in SW780 cells. Conclusions: Herein, we successfully identified a novel 14-gene signature for glycogene-type classification of UBC patients. ST3GAL6 gene, from this signature, was demonstrated as a potential biomarker for poor outcomes and cell invasion in UBCs.

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation.

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.

Downregulation of TET1 Promotes Bladder Cancer Cell Proliferation and Invasion by Reducing DNA Hydroxymethylation of AJAP1.

Ten-eleven translocation 1 (TET1) is a member of methylcytosine dioxygenase, which catalyzes 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC) to promote the demethylation process. The dysregulated TET1 protein and 5 hmC level were reported to either suppress or promote carcinogenesis in a cancer type-dependent manner. Currently, the role of TET1 in the development of urinary bladder cancer (UBC) and its underlying molecular mechanisms remain unclear. Herein, we found that TET1 expression was downregulated in UBC specimens compared with normal urothelium and was inversely related to tumor stage and grade and overall survival, suggesting its negative association with UBC progression. TET1 silencing in UBC cells increased cell proliferation and invasiveness while the ectopic expression of wild-type TET1-CD, but not its enzymatic inactive mutant, reversed these effects and suppressed tumorigenicity in vivo. In addition, as a direct regulator of TET1 activity, vitamin C treatment increased 5 hmC level and inhibited the anchorage-independent growth and tumorigenicity of UBC cells. Furthermore, we found that TET1 maintained the hypomethylation in the promoter of the AJAP1 gene, which codes for adherens junction-associated protein 1. The downregulation of AJAP1 reversed TET1-CD-induced nuclear translocation of ß-catenin, thus inhibiting the expression of its downstream genes. In human UBC specimens, AJAP1 is frequently downregulated and positively associated with TET1. Notably, low expression levels of both TET1 and AJAP1 predict poor prognosis in UBC patients. In conclusion, we found that the frequently downregulated TET1 level reduces the hydroxymethylation of AJAP1 promoter and subsequently activates ß-catenin signaling to promote UBC development. The downregulation of both TET1 and AJAP1 might be a promising prognostic biomarker for UBC patients.

Identification of a novel immune microenvironment signature predicting survival and therapeutic options for bladder cancer.

Few studies have investigated the potential of tumor immune microenvironment genes as indicators of urinary bladder cancer. Here, we sought to establish an immune-related gene signature for determining prognosis and treatment options. We developed a ten-gene tumor immune microenvironment signature and evaluated its prognostic capacity on internal and external cohorts. Multivariate Cox regression and nomogram analyses revealed the prognostic risk model as an independent and effective indicator of prognosis. We observed lower proportions of CD8+ T cells, dendritic cells, regulatory T cells, higher proportions of macrophages and neutrophils in high UBC risk group. UBC tissues with high-risk score tend to exhibit high TP53 and RB1 mutation rates, high PD1/PD-L1 expression and poor-survival basal squamous subtypes, while those with low-risk score tend to have high FGFR3 mutation rates and luminal papillary subtypes. Unexpectedly, we found a highly significant positive correlation between glycolytic genes and risk score, highlighting metabolic competition in tumor ecosystem and potential therapeutic avenues. Our study thus revealed a tumor immune microenvironment signature for predicting prognostic and response to immune checkpoint inhibitors against bladder cancer. Prospective studies are required to further test the predictive capacity of this model.

Hepcidin protects against iron overload-induced inhibition of bone formation in zebrafish.

Iron overload increases the risk of osteoporosis, which leads to an increase in the incidences of bone fracture after menopause. In vitro studies have demonstrated that excess iron can inhibit osteoblast activity. Hepcidin, a central regulator of iron homeostasis, prevents iron overload, and thus, it is considered to have anti-osteoporosis effects. In this study, a zebrafish model was employed to investigate the therapeutic role of hepcidin in iron overload-induced inhibition of bone formation. Our results show that ferric ammonium citrate (FAC) treatment decreased osteoblast-specific gene expression (runx2a, runx2b, and bglap) and bone mineralization in the zebrafish embryo, accompanied with increased whole-body iron levels and oxidative stress. Bone mineralization and osteoblast-specific gene expression increased with the microinjection of hepcidin-flag Capped-mRNA into zebrafish embryos. Moreover, the whole-body iron content and oxidative stress in the iron-overloaded zebrafish embryos decreased when microinjection of hepcidin preceded the FAC treatment. Therefore, our study suggests that hepcidin could prevent and rescue reduced bone formation caused by FAC treatment by preventing iron absorption.