VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation.

BACKGROUND: Cholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. METHODS: Enabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FINDINGS: Surprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIFα and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. INTERPRETATION: Collectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FUNDING: This work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No. 2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

Single-Port Robot-Assisted Radical Prostatectomy with the Novel Shurui Single-Port Robotic Surgical System.

Purpose: The aim of this study was to explore the safety and efficacy of radical prostatectomy with a novel Shurui single-port (SR-SP) robotic surgical system. Methods: A total of 11 patients with prostate cancer were enrolled in this study. Extraperitoneal radical prostatectomy was performed using the SR-SP robotic surgical system for all patients. Clinicopathologic data, perioperative data, and short-term surgical outcomes were prospectively collected and analyzed. Results: Of the 11 patients, the median age was 65 years (range 52-73), and the median body mass index was 22.6 kg/m2 (range 20.2-26.7). The median operative time was 229 minutes (range 194-317), and the median console time was 167 minutes (range 141-265). The median blood loss was 40 mL (range 10-120), and none of the patients required intraoperative transfusion. There was no conversion to open surgery during the operation, and no assistant ports were added. The surgeons reported a good task load rating with a National Aeronautics and Space Administration Task Load Index (NASA-TLX) score of 25.1 ± 3.3 points. The median postoperative hospital stay time was 7 days (range 4-15). There were no severe intraoperative or postoperative complications (Clavien grade ≥3). Postoperative positive surgical margin occurred in 4 (36.4%) patients. No biochemical recurrence occurred within 1 month of surgery. The continence rate was 72.7% (8/11) 1 month after surgery. Conclusions: The new SR-SP robotic surgical system is safe, effective, flexible, and stable for application in radical prostatectomy.

Preliminary Experience of Partial Nephrectomy Through a New Single-Port Surgical Robot System.

Purpose: To explore the preliminary safety and efficacy of the Shurui single-port (SP) surgical robot in partial nephrectomy (PN). Methods: This study prospectively enrolled patients with T1a renal tumors who met the inclusion criteria from February to July 2022 in The First Affiliated Hospital School of Medicine Zhejiang University. The operative outcomes and perioperative data, including clinical and histological data, were prospectively collected and analyzed. Results: A total of 13 patients were included in this study, including 7 males and 6 females. The median age was 53 (33-74) years, and the average body mass index was 24.9 ± 4.2 kg/m2. There were 6 cases of left kidney tumors and 7 cases of right kidney tumors in the 13 patients. The average tumor diameter was 1.9 ± 0.9 cm. In all operations, the diseased tissue was removed according to the established surgical plan. The average warm ischemia time was 26.2 ± 9.7 minutes; the average device docking time was 3.6 ± 1.8 minutes; and the average robotic arm operation time was 124.7 ± 40.4 minutes. All operations were successfully completed; there was no conversion to open surgery during the operation; and no operation holes were added. The National Aeronautics and Space Administration Task Load Index (NASA-TLX) score was 26.3 ± 2.6 points, and no device-related adverse events occurred during the operation. The median time to discharge was 6 days (range, 4-11 days). Postoperative pathological examination showed that all tumor margins were negative. There were no Clavien grade ≥3 surgical complications in any of the patients during the perioperative period or at the 1-month postoperative follow-up. Conclusion: The new SP surgical robot system is safe, effective, flexible, and stable for application in PN.

Ascorbic acid induced TET2 enzyme activation enhances cancer immunotherapy efficacy in renal cell carcinoma.

Exploring the regulatory mechanism of PD-L1 in renal cancer is one of the key strategies to improve the response of renal cancer patients to checkpoint blockade therapy. In this study, the synergistic effect of ascorbic acid (vitamin C) supplementation and the impact of TET2 depletion on anti-PD-L1 therapy were determined in xenograft model experiments. Lymphocyte infiltration and chemokine expression were determined using flow cytometry and qRT-PCR. To determine the downstream targets of TET2, we performed hMeDip-seq and RNA-seq analyses. The molecular mechanism was further confirmed by hMeDip-qPCR, MeDip-qPCR, bisulfite sequencing, Western blotting, qRT-PCR and xenograft model experiments in vitro and in vivo. The present study demonstrated that ascorbic acid enhanced the efficacy of immunotherapy and that the loss of TET2 function enabled renal cancer cells to evade antitumor immunity. Ascorbic acid treatment significantly increased the intratumoral infiltration of T cells and the expression of cytokines and chemokines, while the loss of TET2 impaired the infiltration of T cells and the expression of cytokines and chemokines. TET2 was recruited to IRF1 by IFN-γ-STAT1 signaling, thereby maintaining IRF1 demethylation and ultimately inducing PD-L1 expression. These results suggest a new strategy of stimulating TET activity to improve immunotherapy for renal cell carcinoma.

P4HB: A novel diagnostic and prognostic biomarker for bladder carcinoma.

Prolyl 4-hydroxylase, beta polypeptide (P4HB) protein is an endoplasmic reticulum (ER) molecular chaperone protein and has been reported to be overexpressed in multiple tumor types. However, the role of P4HB in bladder cancer (BLCA) has not yet been elucidated. The aim of the present study was to investigate the prognostic value of P4HB and the association between clinicopathological characteristics and P4HB in BLCA. P4HB expression levels were assessed through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, and validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in BLCA tissues and cells. A total of 69 pairs of tumor and normal samples were used to analyze the expression of P4HB via immunohistochemical staining. A co-expression network and functional enrichment analyses were conducted to investigate the biological function of P4HB in BLCA. The protein-protein interaction (PPI) network was constructed by Search Tool for the Retrieval of Interacting Genes. The results showed that P4HB was highly expressed in BLCA cells and tissues. The area under the curve value for P4HB expression to discriminate between tumor and normal tissues was up to 0.888 (95% CI: 0.801-0.975; P<0.001) and 0.881 (95% CI: 0.825-0.937; P<0.001) in TCGA database and our database, respectively. Furthermore, the expression level of P4HB was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) by univariate and multivariate analyses. Kaplan-Meier survival analysis demonstrated that high P4HB expression was associated with low OS and RFS. Pathway enrichment analysis suggested that P4HB was involved in protein processing in the endoplasmic reticulum (ER), including N-glycan modification and protein metabolic processes responding to ER stress. PPI analysis revealed that the potential targets of P4HB were mainly involved in posttranslational protein modification and response to ER stress. In conclusion, the expression level of P4HB aid in identifying patients with early-stage BLCA and predicting the prognosis of BLCA. Therefore, P4HB may be a novel diagnostic and prognostic biomarker for BLCA.

Identification of immune-related LncRNA for predicting prognosis and immunotherapeutic response in bladder cancer.

Long noncoding RNAs (lncRNAs) have multiple functions in the cancer immunity response and the tumor microenvironment. To investigate the immune-related lncRNA (IRlncRNA) signature for predicting prognosis and immunotherapeutic response in bladder cancer (BLCA), we extracted BLCA data from The Cancer Genome Atlas (TCGA) database. Finally, a total of 405 cases were enrolled and 8 prognostic IRlncRNAs (MIR181A2HG, AC114730.3, LINC00892, PTPRD-AS1, LINC01013, MRPL23-AS1,LINC01395, AC002454.1) were identified in the training set. Risk scores were calculated to divide patients into high-risk and low-risk groups, and the high-risk patients tended to have a poor overall survival (OS). Multivariate Cox regression analysis confirmed that the IRlncRNA signature could be an independent prognostic factor. The results were subsequently confirmed in the validating set. Additionally, this 8-IRlncRNA classifier was related to recurrence free survival (RFS) of BLCA. Functional characterization revealed this signature mediated immune-related phenotype. This signature was also associated with immune cell infiltration (i.e., macrophages M0, M2, Tregs, CD8 T cells, and neutrophils) and immune checkpoint inhibitors (ICIs) immunotherapy-related biomarkers [mismatch repair (MMR) genes, tumor mutation burden (TMB) and immune checkpoint genes]. The present study highlighted the value of the 8-IRlncRNA signature as a predictor of prognosis and immunotherapeutic response in BLCA.

Biological functions and prognostic value of RNA Binding Proteins in clear cell Renal Cell Carcinoma.

Early detection and accurate evaluation were both critical to improving the prognosis of clear cell Renal Cell Carcinoma (ccRCC) patients. More importantly, RNA Binding Proteins (RBPs) play a vital role in the tumorigenesis and progression of numerous cancers. However, the relationship between RBPs and ccRCC is still unclear. Exploring the potential biological functions of RBPs in ccRCC and establishing a prognostic signature to predict the survival probability remains meaningful. In this study, transcriptome profiling and the corresponding clinical information were obtained from the TCGA database, GEO database, and ICGC database. By using the "edgeR" R package, 200 DERBPs were found, including 128 up-regulated and 72 down-regulated RBPs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DERBPs were mainly involved in regulating transcriptional processes and metabolism. Furthermore, there were 4 hub genes (RPS2, RPS14, RPS20, and RPLP0) were found in the PPI network, which may play vital biological roles among those DERBPs. Then we used LASSO regression to construct a prognostic signature and validated the signature in the GEO and ICGC cohort. The time-dependent receiver operating characteristic (ROC) curve showed that the signature could accurately predict the prognosis of ccRCC patients. Then we established a nomogram, and the calibration curve and ROC curve showed that the nomogram could accurately predict 1-year, 3-year, and 5-year overall survival (OS) of ccRCC patients (The AUC value: 0.871, 0.829, and 0.816). In conclusion, we constructed a 10-RBPs-based prognostic signature integrating clinical parameters to predict the prognosis of ccRCC patients. The prognostic signature based on the differentially expressed RBPs (DERBPs) might serve as promising diagnostic and prognostic biomarkers in ccRCC.

Identification of the Six-RNA-Binding Protein Signature for Prognosis Prediction in Bladder Cancer.

RNA-binding proteins (RBPs) are a kind of gene regulatory factor that presents a significant biological effect in the initiation and development of various tumors, including bladder cancer (BLCA). However, the RBP-based prognosis signature for BLCA has not been investigated. In this study, we attempted to develop an RBP-based classifier to predict overall survival (OS) for BLCA based on transcriptome analysis. We extracted data of BLCA patients from The Cancer Genome Atlas database (TCGA) and UCSC Xena. Finally, a total of 398 cases without missing clinical data were enrolled and six RBPs (FLNA, HSPG2, AHNAK, FASTKD3, POU5F1, and PCSK9) associated with OS of BLCA were identified through univariate and multivariate Cox regression analysis. Online analyses and immunohistochemistry validated the prognostic value and expression of six RBPs. Risk scores were calculated to divide patients into high-risk and low-risk level, and patients in the high-risk group tended to have a poor prognosis. In addition, the receiver operating characteristic (ROC) curve analysis was performed to assess the prognostic value of RBPs, and the area under the curve (AUC) values were 0.711 and 0.706, respectively, in the training set and validating set. The findings were further validated in an external validation set. Subsequently, the 6-RBP-based signature and pathological stage were used to construct the nomogram to predict the 3- and 5-years OS of BLCA patients. Also, this 6-RBP-based signature was highly related to recurrence-free survival of BLCA. Weighted co-expression network analysis (WGCNA) combined with functional enrichment analysis contributed to study the potential pathways of six RBPs, including keratinocyte differentiation, RHO GTPases activate PNKs, epithelial tube morphogenesis, establishment or maintenance of cell polarity, and so on. In summary, the 6-RBP-based signature holds the potentiality to serve as a novel prognostic predictor of OS for BLCA.

Long non-coding RNA SOX2OT promotes the stemness phenotype of bladder cancer cells by modulating SOX2.

BACKGROUND: Accumulating evidence indicates that long non-coding RNAs (lncRNAs) are potential biomarkers and key regulators of tumour development and progression. SOX2 overlapping transcript (SOX2OT) is a novel lncRNA that acts as a potential biomarker and is involved in the development of cancer and cancer stem cells. However, the clinical significance and molecular mechanism of SOX2OT in bladder cancer are still unknown. METHODS: The expression level of SOX2OT was determined by RT-qPCR in a total of 106 patients with urothelial bladder cancer and in different bladder cancer cell (BCC) lines. Bladder cancer stem cells (BCSCs) were isolated from BCCs using flow cytometry based on the stem cell markers CD44 and ALDH1. Loss-of-function experiments were performed to investigate the biological roles of SOX2OT in the stemness phenotype of BCSCs. Comprehensive transcriptional analysis, RNA FISH, dual-luciferase reporter assays and western blots were performed to explore the molecular mechanisms underlying the functions of SOX2OT. RESULTS: SOX2OT was highly expressed in bladder cancer, and increased SOX2OT expression was positively correlated with a high histological grade, advanced TNM stage and poor prognosis. Further experiments demonstrated that knockdown of SOX2OT inhibited the stemness phenotype of BCSCs. Moreover, inhibition of SOX2OT delayed xenograft tumour growth and decreased metastases in vivo. Mechanistically, we found that SOX2OT was mainly distributed in the cytoplasm and positively regulated SOX2 expression by sponging miR-200c. Furthermore, SOX2 overexpression reversed the SOX2OT silencing-induced inhibition of the BCSC stemness phenotype. CONCLUSION: This study is the first to demonstrate that SOX2OT plays an important regulatory role in BCSCs and that SOX2OT may serve as a potential diagnostic biomarker and therapeutic target in bladder cancer.

MTDH promotes metastasis of clear cell renal cell carcinoma by activating SND1-mediated ERK signaling and epithelial-mesenchymal transition.

BACKGROUND: Metastasis is the principal cause of renal cell carcinoma-associated mortality. Metadherin (MTDH) was identified as a vital metastasis driver involved in the metastatic progression of various types of tumors, suggesting that MTDH is a prognostic metastatic biomarker and potential therapeutic target. The role and mechanism of MTDH in the metastatic progression of ccRCC have not yet been adequately explored. RESULTS: MTDH was remarkably elevated in ccRCC tissues, especially in metastatic ccRCC tissues, compared with normal kidney tissues and correlated with advanced clinicopathological features and poor prognosis. MTDH activated ERK signaling and EMT, thus promoting the migration and invasion of ccRCC cells. The interaction between MTDH and SND1 at the protein level was confirmed using immunoprecipitation and immunofluorescence. Based on the analysis of datasets from GEO and TCGA, SND1 was remarkably increased in ccRCC, especially in metastatic ccRCC, and associated with advanced clinicopathological features and poor prognosis. Knockdown of SND1 mainly abolished the migration and invasion of ccRCC cells by blocking MTDH-mediated ERK and EMT signaling activation. CONCLUSION: These results revealed that MTDH may be a prognostic metastatic biomarker of ccRCC that promotes ccRCC metastasis by activating SND1-mediated the ERK and EMT signaling pathways. MTDH may serve as an anti-tumor therapeutic target that can be applied for the clinical treatment of metastatic ccRCC. METHODS: MTDH/SND1 mRNA expression in clear cell renal cell carcinoma (ccRCC) was comprehensively estimated by analysis of GEO-ccRCC and TCGA-KIRC datasets with R software and packages. MTDH protein expression was assessed in a total of 111 ccRCC patients from Peking University First Hospital by immunohistochemistry (IHC). In vitro migration and invasion assays were carried out, and an in vivo metastatic mouse model was developed to investigate the biological functions of MTDH in ccRCC cells. Correlation analysis, immunoprecipitation, western blotting and immunofluorescence were applied to explore the molecular mechanisms of MTDH in ccRCC.

Overexpression of P4HB is correlated with poor prognosis in human clear cell renal cell carcinoma.

Prolyl 4-hydroxylase, beta polypeptide (P4HB) protein has been found to be associated with tumorigenesis in many types of tumor, However, the relationship between P4HB and clear cell renal cell carcinoma (ccRCC) has not been clarified. In this study, we focus on the correlation between P4HB expression and ccRCC. Through the Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) database, our database and immunohistochemical (IHC) staining. Compared with adjacent normal tissues, both the mRNA and protein levels of P4HB in ccRCC tissues were enhanced. The Kaplan-Meier survival analysis showed that high expression of P4HB is correlated with poor prognosis in both TCGA database and our own database. Multivariate survival analysis and Univariate analysis showed that P4HB expression and age are significantly correlative with poor prognose. All the results indicated that P4HB is correlated with poor prognosis in human clear cell renal cell carcinoma.

TGF-ß-induced transgelin promotes bladder cancer metastasis by regulating epithelial-mesenchymal transition and invadopodia formation.

BACKGROUND: Metastatic bladder cancer (BLCA) is a lethal disease with an unmet need for study. Transgelin (TAGLN) is an actin-binding protein that affects the dynamics of the actin cytoskeleton indicating its robust potential as a metastasis initiator. Here, we sought to explore the expression pattern of TAGLN and elucidate its specific functioning and mechanisms in BLCA. METHODS: A comprehensive assessment of TAGLN expression in BLCA was performed in three cohorts with a total of 847 patients. The potential effects of TAGLN on BLCA were further determined using clinical genomic analyses that guided the subsequent functional and mechanistic studies. In vitro migration, invasion assays and in vivo metastatic mouse model were performed to explore the biological functions of TAGLN in BLCA cells. Immunofluorescence, western blot and correlation analysis were used to investigate the molecular mechanisms of TAGLN. FINDINGS: TAGLN was highly expressed in BLCA and correlated with advanced prognostic features. TAGLN promoted cell colony formation and cell migration and invasion both in vitro and in vivo by inducing invadopodia formation and epithelial-mesenchymal transition, during which a significant correlation between TAGLN and Slug was observed. The progression-dependent correlation between TGF-ß and TAGLN was analysed at both the cellular and tissue levels, while TGF-ß-mediated migration was abolished by the suppression of TAGLN. INTERPRETATION: Overall, TAGLN is a promising novel prognosis biomarker of BLCA, and its metastatic mechanisms indicate that TAGLN may represent a novel target agent that can be utilized for the clinical management of invasive and metastatic BLCA. FUND: This work was supported by the National Natural Science Foundation of China [81772703, 81672546, 81602253]; the Natural Science Foundation of Beijing [71772219, 7152146]. and Innovative Fund for Doctoral Students of Peking University Health Science Center (BUM2018BSS002). Funders had no role in the design of the study, data collection, data analysis, interpretation, or the writing of this report.

Prognostic value of long non-coding RNA signatures in bladder cancer.

Bladder cancer (BLCA) is a devastating cancer whose early diagnosis can ensure better prognosis. Aim of this study was to evaluate the potential utility of lncRNAs in constructing lncRNA-based classifiers of BLCA prognosis and recurrence. Based on the data concerning BLCA retrieved from TCGA, lncRNA-based classifiers for OS and RFS were built using the least absolute shrinkage and selection operation (LASSO) Cox regression model in the training cohorts. More specifically, a 14-lncRNA-based classifier for OS and a 12-lncRNA-based classifier for RFS were constructed using the LASSO Cox regression. According to the prediction value, patients were divided into high/low-risk groups based on the cut-off of the median risk-score. The log-rank test showed significant differences in OS and RFS between low- and high-risk groups in the training, validation and whole cohorts. In the time-dependent ROC curve analysis, the AUCs for OS in the first, third, and fifth year were 0.734, 0.78, and 0.78 respectively, whereas the prediction capability of the 14-lncRNA classifier was superior to a previously published lncRNA classifier. As for the RFS, the AUCs in the first, third, and fifth year were 0.755, 0.715, and 0.740 respectively. In summary, the two-lncRNA-based classifiers could serve as novel and independent prognostic factors for OS and RFS individually.