ZHX3 promotes the progression of urothelial carcinoma of the bladder via repressing of RGS2 and is a novel substrate of TRIM21.

Clinically, patients with urothelial carcinoma of the bladder (UCB) with tumor metastasis are incurable. To find new therapeutic strategies, the mechanisms underlying UCB invasion and metastasis should be further investigated. In this study, zinc finger and homeobox 3 (ZHX3) was first screened as a critical oncogenic factor associated with poor prognosis in a UCB dataset from The Cancer Genome Atlas (TCGA). These results were also confirmed in a large cohort of clinical UCB clinical samples. Next, we found that ZHX3 could promote the migration and invasion capacities of UCB cells both in vitro and in vivo. Mechanistically, coimmunoprecipitation (coIP) and mass spectrometry (MS) analysis indicated that ZHX3 was a target of tripartite motif 21 (TRIM21), which mediates its ubiquitination, and subsequent degradation. Notably, RNA-seq analysis showed that ZHX3 repressed the expression of regulator of G protein signaling 2 (RGS2). Generally, our results suggest that ZHX3 plays an oncogenic role in UCB pathogenesis and might serve as a novel therapeutic target for UCB.

Overexpression of CEP72 Promotes Bladder Urothelial Carcinoma Cell Aggressiveness via Epigenetic CREB-Mediated Induction of SERPINE1.

A vital constituent of the centrosome involved in regulating the activity of the organelle during the cell cycle is centrosomal protein (CEP)-72, whose function in the case of human cancer yet lacks clarity. The expression dynamics of CEP72 and its clinical impact were examined in a large cohort of bladder tissues. Several experiments at both the in vitro and in vivo levels on urothelial carcinoma of the bladder (UCB) cells were conducted to understand the role of this molecule along with the mechanisms. Overexpression of CEP72 in UCB was linked with the acquisition of an aggressive phenotype, which was associated with poor prognosis. In UCB cell lines, knockdown of CEP72 using shRNA was sufficient to inhibit cell invasiveness/metastasis, whereas ectopic overexpression of CEP72 promoted cell invasiveness and/or metastasis both in vitro and in vivo. CEP72 was demonstrated to induce UCB cell aggressiveness via up-regulation of an important target downstream, the serpin family member 1 gene (SERPINE1) (alias plasminogen activator inhibitor, PAI1), ultimately leading to increased cancer cell invasiveness. Particularly, overexpression of CEP72 was associated with a sizable increase in cAMP response element-binding protein binding at the SERPINE1 promoter, leading to increased SERPINE1 transcription. Such observations are suggestive of the potential use of CEP72 as a therapeutic tool for UCB.

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.

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.

[Expression of perforin and granzyme-B in peripheral blood lymphocyte in patients with prostate cancer and the clinical significance].

OBJECTIVE: To explore the expression of perforin and granzyme-B in peripheral blood lymphocyte (PBL) in patients with prostate cancer (PCa) and the clinical significance. METHODS: The expressions of perforin and granzyme-B in PBL were detected by fluorescence quantitative reverse transcription polymerase chain reaction. The results of perforin and granzyme-B expression were compared among patients with PCa (n=60), patients with BPH (benign prostatic hyperplasia, n=40) and healthy controls (n=20). RESULTS: Th e expressions of perforin and granzyme-B in patients with PCa were significantly lower than that in patients with BPH or that in the healthy controls (P<0.05), respectively. Furthermore, in PCa patients with low pathological grade, the expressions of perforin and granzyme-B in PBL was statistically higher than that in patients with high pathological grade (P<0.05). The expressions of perforin and granzyme-B in PCa patients at high clinical stage was statistically lower than that in PCa patients at low clinical stage (P<0.05). CONCLUSION: The results of this study suggest that development and progression of PCa might be associated with poor immune status of patients.

Safety and Efficacy of Second-Line TKI Plus Anti-PD1 in Metastatic Non-Clear Cell Renal Cell Carcinoma: A Real-World Study.

OBJECTIVES: Guidelines recommend clinical trials or tyrosine kinase inhibitor (TKI) as the first-line option for systemic therapy for non-clear cell renal cell carcinoma (nccRCC) with limited efficacy. However, the preferred subsequent options remain unclear when patients progress after first-line treatment. This study aimed to evaluate the efficacy and safety of anti-PD-1 plus TKI therapy as the second-line regimen in nccRCC. PATIENTS AND METHODS: We conducted a retrospective analysis of patients with metastatic nccRCC who failed first-line TKI therapy between October 2011 and September 2020. The baseline characteristics of the patients and adverse events (AEs) were collected. Efficacy measures included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). RESULTS: The current study enrolled 65 patients, with a median age of 48 (interquartile 37-60) years. Among all patients, 21 received TKI monotherapy while 44 patients received combination therapy (TKI plus anti-PD1). The ORR and DCR for the whole cohort were 38.5% and 56.9%, respectively. ORR (50.0% vs. 14.3%, P = .006) and DCR (70.5% vs. 28.6%, P = .001) were improved in the combination group compared with the TKI group. The overall second-line PFS was 7.7 (95% CI: 6.1-9.3) months and OS was 25.2 (19.5-30.8) months. Patients receiving combination therapy had a longer PFS compared with those receiving TKI monotherapy [median PFS (95% CI): 9.2 (5.9-12.4) vs. 5.4 (2.6-8.2) m, Log-rank P = .002]. The incidence of treatment-related AEs of grade 3 or higher was comparable between the 2 groups (56.8% vs. 52.4%). CONCLUSION: Anti-PD-1 plus TKI therapy appeared effective and safe in the treatment of patients with metastatic nccRCC who progressed after first-line TKIs.

Extraperitonealization of the ileal conduit decreases the risk of parastomal hernia: A single-center, randomized clinical trial.

Parastomal hernia (PSH) is a common complication in patients receiving ileal conduit urinary diversion after radical cystectomy. In this randomized controlled clinical trial, we validate our previous finding that extraperitonealization of ileal conduit decreases incidence of PSH. In total, 104 consecutive patients undergoing radical cystectomy at Sun Yat-sen University Cancer Center are randomized 1:1 to receive either modified (extraperitonealized) ileal conduit (n = 52) or conventional ileal conduit (n = 52). Primary endpoint is incidence of radiological PSH during follow-up. Incidence of radiological PSH is lower in the modified group than in the conventional group (11.5% vs. 28.8%; p = 0.028) after a median follow-up of 32 months, corresponding to a hazard ratio of 0.374 (95% confidence interval: 0.145-0.965, p = 0.034) in the modified conduit group. The results support our previous finding that extraperitonealization of the ileal conduit is effective for reducing risk of PSH in patients receiving ileal conduit diversion.

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.

Long-term effect of acute ischemic injury on the kidney underwent clamped partial nephrectomy.

GFR reaches a new baseline, primarily correlating with nephron-mass preservation, 1-12 months after partial nephrectomy (PN). However, does the ipsilateral GFR experience subsequent decline, and does acute ischemic injury has long-term effect on the operated kidney? 319 patients with two kidneys and unilateral clamped PN were analyzed. All had preoperative, new-baseline, and latest follow-up imaging/serum creatinine levels. Annual ipsilateral GFR decline rate (AIGDR) was defined as new-baseline GFR minus latest follow-up GFR normalized by new-baseline GFR, per year. Spectrum score was used to reflect the degree of acute ischemic injury in the operated kidney. 100 subjects searching for health screening served as controls. Predictive factors for AIGDR were assessed. The median AIGDR was 2.25%, significantly higher than controls (0.88%, p = 0.036). With some contralateral hypertrophy, the global annual GFR decline was similar to that of controls (0.81% vs. 0.88%, p = 0.7). Spectrum score correlated significantly with AIGDR (p = 0.037). These results support that acute ischemic injury has long-term effect on the operated kidney.

Long noncoding RNAs to predict postoperative recurrence in bladder cancer and to develop a new molecular classification system.

BACKGROUND: Reliable molecular markers are much needed for early prediction of recurrence in muscle-invasive bladder cancer (MIBC) patients. We aimed to build a long-noncoding RNA (lncRNA) signature to improve recurrence prediction and lncRNA-based molecular classification of MIBC. METHODS: LncRNAs of 320 MIBC patients from the Cancer Genome Atlas (TCGA) database were analyzed, and a nomogram was established. A molecular classification system was created, and immunotherapy and chemotherapy response predictions, immune score analysis, immune infiltration analysis, and mutational data analysis were conducted. Survival analysis validation was also performed. RESULTS: An eight-lncRNA signature classifed the patients into high- and low-risk subgroups, and these groups had significantly different (disease-free survival) DFS. The ability of the eight-lncRNA signature to make an accurate prognosis was tested using a validation dataset from our samples. The nomogram achieved a C-index of 0.719 (95% CI, 0.674-0.764). Time-dependent receiver operating characteristic curve (ROC) analysis indicated the superior prognostic accuracy of nomograms for DFS prediction (0.76, 95% CI, 0.697-0.807). Further, the four clusters (median DFS = 11.8, 15.3, 17.9, and 18.9 months, respectively) showed a high frequency of TTN (cluster 1), fibroblast growth factor receptor-3 (cluster 2), TP53 (cluster 3), and TP53 mutations (cluster 4), respectively. They were enriched with M2 macrophages (cluster 1), CD8+ T cells (cluster 2), M0 macrophages (cluster 3), and M0 macrophages (cluster 4), respectively. Clusters 2 and 3 demonstrated potential sensitivity to immunotherapy and insensitivity to chemotherapy, whereas cluster 4 showed potential insensitivity to immunotherapy and sensitivity to chemotherapy. CONCLUSIONS: The eight-lncRNA signature risk model may be a reliable prognostic signature for MIBC, which provides new insights into prediction of recurrence of MIBC. The model may help clinical decision and eventually benefit patients.

Role of Sam68 in Sunitinib induced renal cell carcinoma apoptosis.

Sunitinib is one of the first-line targeted drugs for metastatic renal cell carcinoma (RCC) with dual effects of antiangiogensis and proapoptosis. Sam68 (Src-associated in mitosis, 68 KDa), is found being involved in cell apoptosis. This article reveals that Sam68 impacts the sensitivity to sunitinib by mediating the apoptosis of RCC cells. Immunohistochemical staining indicated that the Sam68 expression levels in sunitinib sensitive tumor tissues were markedly higher than those in sunitinib resistant tumor tissues. Sunitinib induced RCC cell apoptosis in a concentration-dependent manner and inhibited the expression of total and phosphorylated Sam68 (p-Sam68). Downregulation of Sam68 expression inhibited RCC cell apoptosis induced by sunitinib. While upregulation of Sam68 expression could enhance apoptosis induced by sunitinib. Xenograft models showed that tumors in the Sam68-knockdown group did not shrink as much as those in the control group after treatment with sunitinib for 4 weeks. Together, our results suggest that Sam68 expression is associated with the sensitivity of ccRCC patients to sunitinib. Sam68 may promote cell apoptosis induced by sunitinib, and the Sam68 expression level may be a biomarker for predicting sunitinib sensitivity in ccRCC patients.

FXR1 can bind with the CFIm25/CFIm68 complex and promote the progression of urothelial carcinoma of the bladder by stabilizing TRAF1 mRNA.

RNA-binding proteins (RBPs) are key regulators of gene expression. RBP dysregulation is reported to play essential roles in tumorigenesis. However, the role of RBPs in urothelial carcinoma of the bladder (UCB) is only starting to be unveiled. Here, we comprehensively assessed the mRNA expression landscape of 104 RBPs from two independent UCB cohorts, Sun Yat-sen University Cancer Center (SYSUCC) and The Cancer Genome Atlas (TCGA). Fragile X-related gene 1 (FXR1) was identified as a novel cancer driver gene in UCB. FXR1 overexpression was found to be related to the poor survival rate in the SYSUCC and TCGA cohorts. Functionally, FXR1 promotes UCB proliferation and tumorigenesis. Mechanistically, FXR1 serves as a platform to recruit CFIm25 and CFIm68, forming a novel 3' processing machinery that functions in sequence-specific poly(A) site recognition. FXR1 affects the 3' processing of Tumor necrosis factor receptor-associated factor 1 (TRAF1) mRNA, which leads to nuclear stabilization. The novel regulatory relationship between FXR1 and TRAF1 can enhance cell proliferation and suppress apoptosis. Our data collectively highlight the novel regulatory role of FXR1 in TRAF1 3' processing as an important determinant of UCB oncogenesis. Our study provides new insight into RBP function and provides a potential therapeutic target for UCB.

Genomic Characteristics and Single-Cell Profiles After Immunotherapy in Fumarate Hydratase-Deficient Renal Cell Carcinoma.

PURPOSE: Fumarate hydratase-deficient renal cell carcinoma (FHRCC) is highly malignant, but the urgent need for effective treatment remains unmet. We aimed to analyze the genomic characteristics and microenvironment of FHRCC and the cause of heterogeneous response to immune checkpoint inhibitor (ICI)-based treatment at single-cell level. EXPERIMENTAL DESIGN: Whole-exome sequencing and IHC staining analyses were performed in 30 advanced FHRCC patients. Single-cell RNA sequencing following ICI-based treatment was conducted in 4 patients. The clinical characteristics, therapeutic effect, and follow-up data were analyzed. RESULTS: The median tumor mutation burden was only 0.14 mutations per megabase. IHC staining showed an immune-active tumor microenvironment characterized by extensive CD8+ T-cell infiltration. ATM expression was inversely correlated with percentage of tumor-infiltrating CD8+ T cells. Trajectory analysis indicated gradually upregulated exhausted markers and an increased apoptotic trend of CD8+ T cells despite continuous exposure to ICI-based treatment. ICI-based treatment was associated with improved overall response rate (17.6% vs. 0%, P = 0.046) and disease control rate (DCR; 64.7% vs. 12.5%, P = 0.004) compared with tyrosine kinase inhibitor. Among patients with germline mutation, the ORR (16.7% vs. 0%, P = 0.086) and the DCR (66.7% vs. 14.3%, P = 0.011) were higher after ICI-based treatment. CONCLUSIONS: Immune infiltration is frequent in FHRCC. ICI-based treatment is a promising regimen, and treatment response depends on the functional status of tumor-infiltrating lymphocytes. ICI-based treatment cannot reverse the exhaustion of CD8+ T cells in patients with progressive disease, highlighting the need for additional therapeutic strategies.

Targeting treatment of bladder cancer using PTK7 aptamer-gemcitabine conjugate.

BACKGROUND: Gemcitabine (GEM) is one of the first-line chemotherapies for bladder cancer (BC), but the GEMs cannot recognize cancer cells and have a low long-term response rate and high recurrence rate with side effects during the treatment of BC. Targeted transport of GEMs to mediate cytotoxicity to tumor and avoid the systemic side effects remains a challenge in the treatment of BC. METHODS: Based on a firstly confirmed biomarker in BC-protein tyrosine kinase 7 (PTK7), which is overexpressed on the cell membrane surface in BC cells, a novel targeting system protein tyrosine kinase 7 aptamer-Gemcitabine conjugate (PTK7-GEMs) was designed and synthesized using a specific PTK7 aptamer and GEM through auto-synthesis method to deliver GEM against BC. In addition, the antitumor effects and safety evaluation of PTK7-GEMs was assessed with a series of in vitro and in vivo assays. RESULTS: PTK7-GEMs can specifically bind and enter to BC cells dependent on the expression levels of PTK7 and via the macropinocytosis pathway, which induced cytotoxicity after GEM cleavage from PTK7-GEMs respond to the intracellular phosphatase. Moreover, PTK7-GEMs showed stronger anti-tumor efficacy and excellent biosafety in three types of tumor xenograft mice models. CONCLUSION: These results demonstrated that PTK7-GEMs is a successful targeted aptamer-drug conjugates strategy (APDCs) to treat BC, which will provide new directions for the precision treatment of BC in the field of biomarker-oriented tumor targeted therapy.

Treatment of bladder cancer by geoinspired synthetic chrysotile nanocarrier-delivered circPRMT5 siRNA.

BACKGROUND: Circular RNAs (circRNAs) have important functions in many fields of cancer biology. In particular, we previously reported that the oncogenic circRNA, circPRMT5, has a major role in bladder cancer progression. Therapy based on circRNAs have good prospects as anticancer strategies. While anti-circRNAs are emerging as therapeutics, the specific in vivo delivery of anti-circRNAs into cancer cells has not been reported and remains challenging. METHODS: Synthesized chrysotile nanotubes (SCNTs) with a relatively uniform length (~ 200 nm) have been designed to deliver an siRNA against the oncogenic circPRMT5 (si-circPRMT5) inhibit circPRMT5. In addition, the antitumor effects and safety evaluation of SCNTs/si-circPRMT5 was assessed with a series of in vitro and in vivo assays. RESULTS: The results showed that SCNTs/si-circPRMT5 nanomaterials prolong si-circPRMT5's half-life in circulation, enhance its specific uptake by tumor cells, and maximize the silencing efficiency of circPRMT5. In vitro, SCNTs encapsulating si-circPRMT5 could inhibit bladder cancer cell growth and progression. In vivo, SCNTs/si-circPRMT5 inhibited growth and metastasis in three bladder tumor models (a subcutaneous model, a tail vein injection lung metastatic model, and an in situ model) without obvious toxicities. Mechanistic study showed that SCNTs/si-circPRMT5 regulated the miR-30c/SNAIL1/E-cadherin axis, inhibiting bladder cancer growth and progression. CONCLUSION: The results highlight the potential therapeutic utility of SCNTs/si-circPRMT5 to deliver si-circPRMT5 to treat bladder cancer.

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.

Stereotactic body radiotherapy in combination with non-frontline PD-1 inhibitors and targeted agents in metastatic renal cell carcinoma.

BACKGROUND: Radiotherapy may work synergistically with immunotherapy and targeted agents. We aimed to assess the safety and outcomes of stereotactic body radiotherapy (SBRT) plus non-first-line programmed death-1 (PD-1) inhibitors and targeted agents (TA) in metastatic renal cell carcinoma (mRCC). METHODS: We retrospectively reviewed 74 patients treated with non-first-line PD-1 inhibitors plus TA in non-first-line setting. Survival outcomes were calculated from the anti-PD-1 treatment using the Kaplan-Meier method. Univariate and multivariate analyses were performed by Cox proportional hazards models. RESULTS: Thirty-two (43.2%) patients received anti-PD-1/TA therapy alone (anti-PD-1/TA alone group), and 42 (56.8%) received SBRT in addition (anti-PD-1/TA + SBRT group). The median duration of first-line therapy was 8.6 months. Patients in the anti-PD-1/TA + SBRT group had significantly longer overall survival (OS) (38.5 vs 15.4 months; P = 0.022). On multivariate analysis, oligometastasis, ECOG performance status 0-1, anti-PD-1/TA + SBRT, and duration of first-line therapy ≥ 8.6 months were predictors for OS. The addition of SBRT was associated with improved OS in patients with clear-cell type (HR 0.19; 95% CI 0.07-0.55; P = 0.002) and duration of first-line therapy ≥ 8.6 months (HR 0.22; 95% CI 0.06-0.88; P = 0.032). Grade ≥ 3 toxicities occurred in 23 patients (54.8%) in the anti-PD-1/TA + SBRT group, and in 21 patients (65.6%) in the anti-PD-1/TA alone group. CONCLUSIONS: Incorporating SBRT into anti-PD-1/TA therapy is safe and tolerable. Further investigation is needed, particularly in patients with clear-cell histology and a longer duration of response to first-line antiangiogenic therapy.

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.

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.

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.