m5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization.

The significance of 5-methylcytosine (m5C) methylation in human malignancies has become an increasing focus of investigation. Here, we show that m5C regulators including writers, readers and erasers, are predominantly upregulated in urothelial carcinoma of the bladder (UCB) derived from Sun Yat-sen University Cancer Center and The Cancer Genome Atlas cohort. In addition, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a methyltransferase and Aly/REF export factor (ALYREF) as a nuclear m5C reader, are frequently coexpressed in UCB. By applying patient-derived organoids model and orthotopic xenograft mice model, we demonstrate that ALYREF enhances proliferation and invasion of UCB cells in an m5C-dependent manner. Integration of tanscriptome-wide RNA bisulphite sequencing (BisSeq), RNA-sequencing (RNA-seq) and RNA Immunoprecipitation (RIP)-seq analysis revealed that ALYREF specifically binds to hypermethylated m5C site in RAB, member RAS oncogene family like 6 (RABL6) and thymidine kinase 1 (TK1) mRNA via its K171 domain. ALYREF controls UCB malignancies through promoting hypermethylated RABL6 and TK1 mRNA for splicing and stabilization. Moreover, ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. Clinically, the patients with high coexpression of ALYREF/RABL6/TK1 axis had the poorest overall survival. Our study unveils an m5C dependent cross-regulation between nuclear reader ALYREF and m5C writer NSUN2 in activation of hypermethylated m5C oncogenic RNA through promoting splicing and maintaining stabilization, consequently leading to tumor progression, which provides profound insights into therapeutic strategy for UCB.

Single-cell transcriptomics reveals a low CD8+ T cell infiltrating state mediated by fibroblasts in recurrent renal cell carcinoma.

PURPOSE: Recurrent renal cell carcinoma(reRCC) is associated with poor prognosis and the underlying mechanism is not yet clear. A comprehensive understanding of tumor microenvironment (TME) of reRCC may aid in designing effective anticancer therapies, including immunotherapies. Single-cell transcriptomics holds great promise for investigating the TME, however, this technique has not been used in reRCC. Here, we aimed to explore the difference in the TME and gene expression pattern between primary RCC (pRCC) and reRCC at single-cell level. EXPERIMENTAL DESIGN: We performed single-cell RNA sequencing analyses of 32,073 cells from 2 pRCC, 2 reRCC, and 3 adjacent normal kidney samples. 41 pairs of pRCC and reRCC samples were collected as a validation cohort to assess differences observed in single-cell sequencing. The prognostic significance of related cells and markers were studied in 47 RCC patients underwent immunotherapy. The function of related cells and markers were validated via in vitro and in vivo experiments. RESULTS: reRCC had reduced CD8+ T cells but increased cancer-associated fibroblasts (CAFs) infiltration compared with pRCC. Reduced CD8+ T cells and increased CAFs infiltration were significantly associated with a worse response from immunotherapy. Remarkably, CAFs showed substantial expression of LGALS1 (Gal1). In vitro, CAFs could induce CD8+ T cells apoptosis via Gal1. In vivo, knockdown of Gal1 in CAFs suppressed tumor growth, increased CD8+ T cells infiltration, reduced the proportion of apoptotic CD8+ T cells and enhanced the efficacy of immunotherapy. CONCLUSIONS: We delineated the heterogeneity of reRCC and highlighted an innovative mechanism that CAFs acted as a suppressor of CD8+ T cells via Gal1. Targeting Gal1 combined with anti-PD1 showed promising efficacy in treating RCC.

LRPPRC regulates redox homeostasis via the circANKHD1/FOXM1 axis to enhance bladder urothelial carcinoma tumorigenesis.

Reactive oxygen species (ROS) which are continuously generated mainly by mitochondria, have been proved to play an important role in the stress signaling of cancer cells. Moreover, pentatricopeptide repeat (PPR) proteins have been suggested to take part in mitochondrial metabolism. However, the mechanisms integrating the actions of these distinct networks in urothelial carcinoma of the bladder (UCB) pathogenesis are elusive. In this study, we found that leucine rich pentatricopeptide repeat containing (LRPPRC) was frequently upregulated in UCB and that it was an independent prognostic factor in UCB. We further revealed that LRPPRC promoted UCB tumorigenesis by regulating the intracellular ROS homeostasis. Mechanistically, LRPPRC modulates ROS balance and protects UCB cells from oxidative stress via mt-mRNA metabolism and the circANKHD1/FOXM1 axis. In addition, the SRA stem-loop interacting RNA binding protein (SLIRP) directly interacted with LRPPRC to protect it from ubiquitination and proteasomal degradation. Notably, we showed that LRPPRC modulated the tumorigenesis of UCB cells in a circANKHD1-FOXM1-dependent manner. In conclusion, LRPPRC exerts critical roles in regulating UCB redox homeostasis and tumorigenesis, and is a prognostic factor for UCB; suggesting that LRPPRC may serve as an exploitable therapeutic target in UCB.

Sofosbuvir/velpatasvir plus ribavirin for Child-Pugh B and Child-Pugh C hepatitis C virus-related cirrhosis

Background/Aims@#Real-world studies assessing the effectiveness and safety of sofosbuvir/velpatasvir (SOF/VEL) plus ribavirin (RBV) for Child-Pugh B/C hepatitis C virus (HCV)-related cirrhosis are limited. @*Methods@#We included 107 patients with Child-Pugh B/C HCV-related cirrhosis receiving SOF/VEL plus RBV for 12 weeks in Taiwan. The sustained virologic response rates at off-treatment week 12 (SVR12) for the evaluable population (EP), modified EP, and per-protocol population (PP) were assessed. Thesafety profiles were reported. @*Results@#The SVR12 rates in the EP, modified EP and PP were 89.7% (95% confidence interval [CI], 82.5–94.2%), 94.1% (95% CI, 87.8–97.3%), and 100% (95% CI, 96.2–100%). Number of patients who failed to achieve SVR12 were attributed to virologic failures. The SVR12 rates were comparable regardless of patient characteristics. One patient discontinued treatment because of adverse events (AEs). Twenty-four patients had serious AEs and six died, but none were related to SOF/VEL or RBV. Among the 96 patients achieving SVR12, 84.4% and 64.6% had improved Child-Pugh and model for endstage liver disease (MELD) scores. Multivariate analysis revealed that a baseline MELD score ≥15 was associated with an improved MELD score of ≥3 (odds ratio, 4.13; 95% CI, 1.16–14.71; P=0.02). Patients with chronic kidney disease (CKD) stage 1 had more significant estimated glomerular filtration rate declines than patients with CKD stage 2 (-0.42 mL/min/1.73 m2/month; P=0.01) or stage 3 (-0.56 mL/min/1.73 m2/month; P<0.001). @*Conclusions@#SOF/VEL plus RBV for 12 weeks is efficacious and well-tolerated for Child-Pugh B/C HCV-related cirrhosis.

Sofosbuvir/velpatasvir plus ribavirin for Child-Pugh B and Child-Pugh C hepatitis C virus-related cirrhosis

Background/Aims@#Real-world studies assessing the effectiveness and safety of sofosbuvir/velpatasvir (SOF/VEL) plus ribavirin (RBV) for Child-Pugh B/C hepatitis C virus (HCV)-related cirrhosis are limited. @*Methods@#We included 107 patients with Child-Pugh B/C HCV-related cirrhosis receiving SOF/VEL plus RBV for 12 weeks in Taiwan. The sustained virologic response rates at off-treatment week 12 (SVR12) for the evaluable population (EP), modified EP, and per-protocol population (PP) were assessed. Thesafety profiles were reported. @*Results@#The SVR12 rates in the EP, modified EP and PP were 89.7% (95% confidence interval [CI], 82.5–94.2%), 94.1% (95% CI, 87.8–97.3%), and 100% (95% CI, 96.2–100%). Number of patients who failed to achieve SVR12 were attributed to virologic failures. The SVR12 rates were comparable regardless of patient characteristics. One patient discontinued treatment because of adverse events (AEs). Twenty-four patients had serious AEs and six died, but none were related to SOF/VEL or RBV. Among the 96 patients achieving SVR12, 84.4% and 64.6% had improved Child-Pugh and model for endstage liver disease (MELD) scores. Multivariate analysis revealed that a baseline MELD score ≥15 was associated with an improved MELD score of ≥3 (odds ratio, 4.13; 95% CI, 1.16–14.71; P=0.02). Patients with chronic kidney disease (CKD) stage 1 had more significant estimated glomerular filtration rate declines than patients with CKD stage 2 (-0.42 mL/min/1.73 m2/month; P=0.01) or stage 3 (-0.56 mL/min/1.73 m2/month; P<0.001). @*Conclusions@#SOF/VEL plus RBV for 12 weeks is efficacious and well-tolerated for Child-Pugh B/C HCV-related cirrhosis.

Prognostic significance of tumor-infiltrating immune cells in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) is a lethal disease with poor treatment response and a high death rate. Immune cells infiltrating the tumor tissues have been shown to play a vital role in tumorigenesis and tumor progression, but their prognostic significance in MIBC remains unclear. OBJECTIVES: To explore the landscape and prognostic significance of tumor-infiltrating immune cells (TIICs) in MIBC, and to develop a model to improve the prognostic predictions of MIBC. METHODS AND MATERIALS: The gene expression profile and clinical data of MIBC patients were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas portal. The fractions of 22 TIIC subtypes were calculated using the Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) algorithm. A TIICs-based model was constructed using least absolute shrinkage and selection operator (LASSO) Cox regression in a training cohort and validated in the validation cohort. RESULTS: Ten types of TIICs demonstrated different infiltration abundance between MIBC and normal tissue. We also found 11 types of TIICs that were significantly associated with overall survival (OS). A TIICs-based model was established, consisting of 15 types of immune cells, and an immunoscore was calculated. Significant differences in OS were found between the high and low immunoscore groups, in both training (n = 343) and validation (n = 146) cohorts. The model could identify patients who would have worse OS despite having similar clinical characteristics. Furthermore, multivariate analysis identified the immunoscore as an independent risk factor (hazard ratio, 3.23; 95% confidence interval; 2.22-4.70) for OS in MIBC patients. CONCLUSION: The landscape of immune infiltration is different between MIBC and normal tissue. The TIICs-based model could provide promising predictive value to complement the existing staging system for predicting the OS of MIBC patients.

Kinesin family member C1 accelerates bladder cancer cell proliferation and induces epithelial-mesenchymal transition via Akt/GSK3ß signaling.

Kinesin family member C1 (KIFC1) is implicated in the clustering of multiple centrosomes to maintain tumor survival and is thought to be an oncogene in several kinds of cancers. In our experiments, we first performed bioinformatics analysis to investigate the expression levels of KIFC1 in bladder cancer (BC) specimens and normal bladder epitheliums and then, using our samples, verified findings by quantitative real-time PCR and western blotting assays. All data showed that KIFC1 was significantly upregulated in BC specimens at both the mRNA and protein levels. Immunohistochemical studies in a cohort of 152 paraffin-embedded BC tissues displayed that upregulated expression of KIFC1 clearly correlated with pT status (P = .014) and recurrent status (P = .002). Kaplan-Meier survival analysis and log-rank test indicated that patients with BC with high KIFC1 expression had both shorter cancer-specific survival (P < .001) and recurrence-free survival time (P < .001) than those with low KIFC1 expression. Furthermore, ectopic downregulation of KIFC1 weakened BC cell proliferation and migration both in vitro and in vivo, whereas upregulation of KIFC1 enhanced this in vitro. Overexpression of KIFC1 phosphorylated GSK3ß and promoted Snail through activating AKT (protein kinase B0) to induce proliferation and epithelial-mesenchymal transition (EMT) and, therefore, substantially promoted BC migration and metastasis. Our study revealed an oncogenic role for KIFC1 to promote BC cell proliferation and EMT via Akt/GSK3ß signaling; KIFC1 might be a promising prognostic biomarker as well as a therapeutic target for BC.

5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.

Although 5-methylcytosine (m5C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m5C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m5C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m5C 'reader' recognizing m5C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3' untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m5C-regulated oncogene activation, providing a potential therapeutic strategy for UCB.

PRMT5 Circular RNA Promotes Metastasis of Urothelial Carcinoma of the Bladder through Sponging miR-30c to Induce Epithelial-Mesenchymal Transition.

PURPOSE: Circular RNAs (circRNAs), a novel class of noncoding RNAs, have recently drawn lots of attention in the pathogenesis of human cancers. However, the role of circRNAs in cancer cells epithelial-mesenchymal transition (EMT) remains unclear. In this study, we aimed to identify novel circRNAs that regulate urothelial carcinoma of the bladder (UCB) cells' EMT and explored their regulatory mechanisms and clinical significance in UCBs. EXPERIMENTAL DESIGN: We first screened circRNA expression profiles using a circRNA microarray in paired UCB and normal tissues, and then studied the clinical significance of an upregulated circRNA, circPRMT5, in a large cohort of patients with UCB. We further investigated the functions and underlying mechanisms of circPRMT5 in UCB cells' EMT. Moreover, we evaluated the regulation effect of circPRMT5 on miR-30c, and its target genes, SNAIL1 and E-cadherin, in two independent cohorts from our institute and The Cancer Genome Atlas (TCGA). RESULTS: We demonstrated that upregulated expression of circPRMT5 was positively associated with advanced clinical stage and worse survival in patients with UCB. We further revealed that circPRMT5 promoted UCB cell's EMT via sponging miR-30c. Clinical analysis from two independent UCB cohorts showed that the circPRMT5/miR-30c/SNAIL1/E-cadherin pathway was essential in supporting UCB progression. Importantly, we identified that circPRMT5 was upregulated in serum and urine exosomes from patients with UCB, and significantly correlated with tumor metastasis. CONCLUSIONS: CircPRMT5 exerts critical roles in promoting UCB cells' EMT and/or aggressiveness and is a prognostic biomarker of the disease, suggesting that circPRMT5 may serve as an exploitable therapeutic target for patients with UCB.

TRIM65 supports bladder urothelial carcinoma cell aggressiveness by promoting ANXA2 ubiquitination and degradation.

Clinically, most of human urothelial carcinoma of the bladder (UCB)-related deaths result from tumor metastasis, but the underlying molecular mechanisms are largely unknown. Recently, a growing number of tripartite motif (TRIM) family members have been suggested to be important regulators for tumorigenesis. However, the impact of most TRIM members on UCB pathogenesis is unclear. In this study, TRIM65 was first screened as an important oncogenic factor of UCB from the Cancer Genome Atlas (TCGA) database and was validated by a large cohort of clinical UCB tissues. By in vitro and in vivo experiments, we demonstrated that TRIM65 promotes UCB cell invasive and metastatic capacities. Notably, we showed that TRIM65 modulates cytoskeleton rearrangement and induces UCB cells epithelial-mesenchymal transition by the ubiquitination of ANXA2, ultimately leading to an enhanced invasiveness of UCB cells. Importantly, UCBs with high expression of TRIM65 and low expression of ANXA2 showed the poorest outcome. Collectively, our results suggest that the overexpression of TRIM65 has an essential oncogenic role via ubiquitination of ANXA2 in UCB pathogenesis, and that such could be used as a novel prognostic marker and/or therapeutic target for UCB.

Chromobox homolog 8 is a predictor of muscle invasive bladder cancer and promotes cell proliferation by repressing the p53 pathway.

Chromobox homolog 8 (CBX8), also known as human polycomb 8, is a repressor that maintains the transcriptionally repressive state in various cellular genes, and has been reported to promote tumorigenesis. In the present study, we examined CBX8 expression in eight pairs of muscle invasive bladder cancer tissues and adjacent non-tumor tissues, and found that CBX8 was frequently upregulated in muscle invasive bladder cancer tissues when compared to adjacent non-tumor tissues. Analysis showed that high expression of CBX8 in 152 muscle invasive bladder cancer specimens was associated with progression of the T, N, and M stages (P = 0.004, 0.005, <0.001, respectively). Furthermore, Kaplan-Meier survival analysis and log-rank test showed that muscle invasive bladder cancer patients with high CBX8 expression had a poor rate of overall survival (P < 0.001) and 5-year recurrence-free survival (P < 0.001) compared to patients with low CBX8 expression. High CBX8 expression predicted poor overall survival and 5-year recurrence-free survival in T and N stages of muscle invasive bladder cancer patients. Moreover, knockdown of CBX8 inhibited cell proliferation of urothelial carcinoma of the bladder both in vitro and in vivo. In addition, CBX8 depletion resulted in cell cycle delay of urothelial carcinoma cells of the bladder at the G2/M phase by the p53 pathway. The data suggest that high expression of CBX8 plays a critical oncogenic role in aggressiveness of urothelial carcinoma cells of the bladder through promoting cancer cell proliferation by repressing the p53 pathway, and CBX8 could be used as a novel predictor for muscle invasive bladder cancer patients.