LDHA-mediated M2-type macrophage polarization via tumor-derived exosomal EPHA2 promotes renal cell carcinoma progression.

Lactate dehydrogenase A (LDHA) is known to promote the growth and invasion of various types of tumors, affects tumor resistance, and is associated with tumor immune escape. But how LDHA reshapes the tumor microenvironment and promotes the progression of renal cell carcinoma (RCC) remains unclear. In this study, we found that LDHA was highly expressed in clear cell RCC (ccRCC), and this high expression was associated with macrophage infiltration, while macrophages were highly infiltrated in ccRCC, affecting patient prognosis via M2-type polarization. Our in vivo and in vitro experiments demonstrated that LDHA and M2-type macrophages could enhance the proliferation, invasion, and migration abilities of ccRCC cells. Mechanistically, high expression of LDHA in ccRCC cells upregulated the expression of EPHA2 in exosomes derived from renal cancer. Exosomal EPHA2 promoted M2-type polarization of macrophages by promoting activation of the PI3K/AKT/mTOR pathway in macrophages, thereby promoting the progression of ccRCC. All these findings suggest that EPHA2 may prove to be a potential therapeutic target for advanced RCC.

A Mendelian randomization study on causal effects of leisure sedentary behavior on the risk of erectile dysfunction.

BACKGROUND: Erectile dysfunction has been associated with leisure sedentary behavior in several epidemiological and observational studies. However, the interpretation of these findings is difficult due to residual confounding or reverse causality. OBJECTIVES: To explore the causal association between leisure sedentary behavior and erectile dysfunction, and to explore the underlying mechanism using Mendelian randomization. MATERIALS AND METHODS: In the present study, publicly available large-scale genome-wide association studies of leisure sedentary behaviors (television watching, computer use, and driving), erectile dysfunction, sex hormones (total testosterone, bioactive testosterone, estradiol, follicle-stimulating hormone, luteinizing hormone, prolactin, and sex hormone binding globulin), biomarkers of endothelial function (C reactive protein, E-selectin, and matrix metalloproteinase 7), and psychiatric symptoms (depression and anxiety) were used to perform two-sample Mendelian randomization analyses. The inverse variance weighting method was the main method used to estimate the association, and sensitivity analyses were also performed. RESULTS: A greater risk of erectile dysfunction was significantly associated with a higher genetic susceptibility to leisure computer usage (odds ratio = 3.57; 95% confidence interval = 1.78-7.16; p < 0.001). No evidence was obtained to suggest that watching television or driving for leisure increased the risk of erectile dysfunction. No association was found between computer use and depression, anxiety, C reactive protein, E-selectin, matrix metalloproteinase 7, or other sex hormones, with the exception of follicle-stimulating hormone levels (odds ratio = 0.29; 95% confidence interval = 0.12-0.69; p = 0.01). No indication of heterogeneity or pleiotropy was identified by sensitivity analysis. DISCUSSION: Extended computer usage for leisure raised the likelihood of developing erectile dysfunction, which may be associated to lower follicle-stimulating hormone levels; however, the role of endothelial dysfunction and psychological disorders in the development of erectile dysfunction should not be underestimated. Moderate physical activity may help to correct the dysfunction. CONCLUSION: The present study offered substantial evidence for a positive causal association between computer use and the risk of erectile dysfunction. However, a definitive causal association needs to be established by further research.

Tumor cell-intrinsic SETD2 inactivation sensitizes cancer cells to immune checkpoint blockade through the NR2F1-STAT1 pathway.

BACKGROUND: Cancer immunotherapies can induce durable tumor regression, but most patients do not respond. SETD2 mutation has been linked to the efficacy of immune checkpoint inhibitors (ICIs) immunotherapy. The functional importance of the SETD2 inactivation and how to modulate immunotherapy response remains unclear. METHODS: To explore the function of SETD2 in immunotherapy, knockout and subsequent functional experiments were conducted. Bulk RNA-seq, ATAC-seq, Chip-seq and single-cell RNA-seq were performed to dissect the mechanism and explore the immune microenvironment of mouse tumor. Flow cytometry was used to assess cell surface antigen and intratumoral T cell levels. RESULTS: We comprehensively determine the effect of SETD2 inactivation in ICIs therapy and elucidate the mechanistic impact on tumor immunity. Murine syngeneic tumors harboring Setd2 inactivation are sensitive to ICIs. By bulk and single-cell RNA-seq, we further reveal that SETD2 inactivation reprograms intratumoral immune cells and inflames the tumor microenvironment, which is characterized by high infiltration of T cells and enhanced antigen presentation to activate CD8+ T cell-mediated killing. Mechanistically, via an integrated multiomics analysis using ATAC-seq, ChIP-seq and RNA-seq, we demonstrate that SETD2 inactivation reduces NR2F1 transcription by impairing H3K36me3 deposition and chromatin accessibility, which activates the STAT1 signaling pathway to promote chemokines and programmed cell death protein-1 (PD-1) expression and enhance antigen presentation. All these regulatory mechanisms synergistically promote the effects of anti-programmed cell death ligand 1 immunotherapy in Setd2-knockout syngeneic mouse models. The SETD2-NR2F1-STAT1 regulatory axis is conserved in human and murine cancers. Finally, cancer patients harboring SETD2 mutations who received ICIs show increased durable clinical benefits and survival. CONCLUSIONS: These findings provide novel insights into the biology of SETD2 inactivation regulation and reveal a new potential therapeutic biomarker for ICIs immunotherapy in various refractory cancers.

Integrative transcriptome and proteome analyses of clear cell renal cell carcinoma develop a prognostic classifier associated with thrombus.

Clear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) is associated with poor prognosis. Our integrative analyses of transcriptome and proteome reveal distinctive molecular features of ccRCC with VTT, and yield the development of a prognostic classifier to facilitate ccRCC molecular subtyping and treatment. The RNA sequencing and mass spectrometry were performed in normal-tumor-thrombus tissue triples of five ccRCC patients. Statistical analysis, GO and KEGG enrichment analysis, and protein-protein interaction network construction were used to interpret the transcriptomic and proteomic data. A six-gene-based classifier was developed to predict patients' survival using Cox regression, which was validated in an independent cohort. Transcriptomic analysis identified 1131 tumorigenesis-associated differentially expressed genes (DEGs) and 856 invasion-associated DEGs. Overexpression of transcription factor EGR2 in VTT indicated its important role in tumor invasion. Furthermore, proteomic analysis showed 597 tumorigenesis-associated differentially expressed proteins (DEPs) and 452 invasion-associated DEPs. The invasion-associated DEPs showed unique enrichment in DNA replication, lysine degradation, and PPAR signaling pathway. Integration of transcriptome and proteome reveals 142 tumorigenesis-associated proteins and 84 invasion-associated proteins displaying changes consistent with corresponding genes in transcriptomic profiling. Based on their different expression patterns among normal-tumor-thrombus triples, RAB25 and GGT5 were supposed to play a consistent role in both tumorigenesis and invasion processes, while SHMT2 and CADM4 might play the opposite roles in tumorigenesis and thrombus invasion. A prognostic classifier consisting of six DEGs (DEPTOR, DPEP1, NAT8, PLOD2, SLC7A5, SUSD2) performed satisfactorily in predicting survival of ccRCC patients (HR = 4.41, P < 0.001), which was further validated in an independent cohort of 40 cases (HR = 5.52, P = 0.026). Our study revealed the transcriptomic and proteomic profiles of ccRCC patients with VTT, and identified the distinctive molecular features associated with VTT. The six-gene-based prognostic classifier developed by integrative analyses may facilitate ccRCC molecular subtyping and treatment.

Causal effects of hypertension on risk of erectile dysfunction: A two-sample Mendelian randomization study.

Background: Erection dysfunction has been associated with hypertension in several epidemiological and observational studies. But the causal association between hypertension and erectile dysfunction requires further investigation. Methods: A two-sample Mendelian randomization (MR) was conducted to analyze the causal effect of hypertension on risk of erection dysfunction. Large-scale publicly available genome-wide association study data were used to estimate the putative causality between hypertension and risk of erectile dysfunction. A total of 67 independent single nucleotide polymorphisms were selected as instrumental variables. Inverse-variant weighted, maximum likelihood, weighted median, penalized weighted median, and MR-PRESSO approaches were utilized in MR analyses. Heterogeneity test, horizontal pleiotropy test, and leave-one-out method were used to prove the stability of the results. Results: In total, all P values were less than 0.05, demonstrating a positive causal link between hypertension and risk of erectile dysfunction in multiple MR methods, such as inverse-variant weighted (random and fixed effect) (OR 3.8315, 95% CI 2.3004-6.3817, P = 0.0085), maximum likelihood (OR 3.8877, 95% CI 2.3224-6.5081, P = 0.0085), weighted median (OR 4.9720, 95% CI 2.3645-10.4550, P = 0.0309), penalized weighted median (OR 4.9760, 95% CI 2.3201-10.6721, P = 0.0355), and MR-PRESSO (OR 3.6185, 95% CI 2.2387-5.8488, P = 0.0092). Sensitivity analysis detected no evidence of heterogeneity, pleiotropy, or outlier single nucleotide polymorphisms. Conclusion: The study revealed a positive causal link between the presence of hypertension and the risk of erectile dysfunction. More attention should be paid during the management of hypertension with the purpose of preventing erectile dysfunction or improving erectile function.

Atezolizumab Plus PEGPH20 Versus Chemotherapy in Advanced Pancreatic Ductal Adenocarcinoma and Gastric Cancer: MORPHEUS Phase Ib/II Umbrella Randomized Study Platform.

BACKGROUND: The MORPHEUS platform comprises multiple open-label, randomized, phase Ib/II trials designed to identify early efficacy and safety signals of treatment combinations across cancers. Atezolizumab (anti-programmed cell death 1 ligand 1 [PD-L1]) was evaluated in combination with PEGylated recombinant human hyaluronidase (PEGPH20). METHODS: In 2 randomized MORPHEUS trials, eligible patients with advanced, previously treated pancreatic ductal adenocarcinoma (PDAC) or gastric cancer (GC) received atezolizumab plus PEGPH20, or control treatment (mFOLFOX6 or gemcitabine plus nab-paclitaxel [MORPHEUS-PDAC]; ramucirumab plus paclitaxel [MORPHEUS-GC]). Primary endpoints were objective response rates (ORR) per RECIST 1.1 and safety. RESULTS: In MORPHEUS-PDAC, ORRs with atezolizumab plus PEGPH20 (n = 66) were 6.1% (95% CI, 1.68%-14.80%) vs. 2.4% (95% CI, 0.06%-12.57%) with chemotherapy (n = 42). In the respective arms, 65.2% and 61.9% had grade 3/4 adverse events (AEs); 4.5% and 2.4% had grade 5 AEs. In MORPHEUS-GC, confirmed ORRs with atezolizumab plus PEGPH20 (n = 13) were 0% (95% CI, 0%-24.7%) vs. 16.7% (95% CI, 2.1%-48.4%) with control (n = 12). Grade 3/4 AEs occurred in 30.8% and 75.0% of patients, respectively; no grade 5 AEs occurred. CONCLUSION: Atezolizumab plus PEGPH20 showed limited clinical activity in patients with PDAC and none in patients with GC. The safety of atezolizumab plus PEGPH20 was consistent with each agent's known safety profile. (ClinicalTrials.gov Identifier: NCT03193190 and NCT03281369).

CCNA2 as an Immunological Biomarker Encompassing Tumor Microenvironment and Therapeutic Response in Multiple Cancer Types.

Background: Cancer is a major threat to human health worldwide. Although recent innovations and advances in early detection and effective therapies such as targeted drugs and immune checkpoint inhibitors have saved more lives of cancer patients and improved their quality of life, our knowledge about cancer remains largely unknown. CCNA2 belongs to the cell cyclin family and has been demonstrated to be a tumorigenic gene in multiple solid tumor types. The aim of the present study was to make a comprehensive analysis on the role of CCNA2 at a pancancer level. Methods: Multidatabases were collected to evaluate the different expression, prognostic value, DNA methylation, tumor mutation burden, microsatellite instability, mismatch repair, tumor immune microenvironment, and drug sensitivity of CCNA2 across pancancer. IHC was utilized to validate the expression and prognostic value of CCNA2 in ccRCC patients from SMMU cohort. Results: CCNA2 was differentially expressed in most cancer types vs. normal tissues. CCNA2 may significantly influence the prognosis of multiple cancer types, especially clear cell renal cell carcinoma (ccRCC). CCNA2 was also frequently mutated in most cancer types. Notably, CCNA2 was significantly correlated with immune cell infiltration and immune checkpoint inhibitory genes. In addition, CCNA2 was also strongly related to drug resistance. Conclusion: CCNA2 may prove to be a new biomarker for prognostic prediction, tumor immunity assessment, and drug susceptibility evaluation in pancancer level, especially in ccRCC.

Elevated SNRPA1, as a Promising Predictor Reflecting Severe Clinical Outcome via Effecting Tumor Immunity for ccRCC, Is Related to Cell Invasion, Metastasis, and Sunitinib Sensitivity.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinoma and is associated with poor prognosis and notorious for its immune dysfunction characteristic. SNRPA1 is a spliceosome component responsible for processing pre-mRNA into mRNA, while the biological effect of SNRPA1 in ccRCC remains elusive. The aim of this study was to decipher the effect of SNRPA1 on clinical effect and tumor immunity for ccRCC patients. Multi-databases were collected to evaluate the different expression, prognostic value, DNA methylation, tumor immune microenvironment, and drug sensitivity of SNRPA1 on ccRCC. IHC was utilized to validate the expression and prognostic value of SNRPA1 in ccRCC patients from the SMMU cohort. The knockout expression of SNRPA by sgRNA plasmid inhibited the cell proliferation, migration, and metastasis ability and significantly increased the sensitivity of sunitinib treatment. In addition, we explored the role of SNRPA1 in pan-cancer level. The results indicated that SNRPA1 was differentially expressed in most cancer types. SNRPA1 may significantly influence the prognosis of multiple cancer types, especially in ccRCC patients. Notably, SNRPA1 was significantly correlated with immune cell infiltration and immune checkpoint inhibitory genes. In addition, the aggressive and immune inhibitory effects shown in SNRPA1 overexpression and the effect of SNRPA1 on ccRCC cell line invasion, metastasis, and drug sensitivity in vitro were observed. Moreover, SNRPA1 was related to Myc, MTORC, G2M, E2F, and DNA repair pathways in various cancer types. In all, SNRPA1 may prove to be a new biomarker for prognostic prediction, effect tumor immunity, and drug susceptibility in ccRCC.

Establishment of a Prognostic Prediction and Drug Selection Model for Patients with Clear Cell Renal Cell Carcinoma by Multiomics Data Analysis.

METHODS: This study was based on the multiomics data (including mRNA, lncRNA, miRNA, methylation, and WES) of 258 ccRCC patients from TCGA database. Firstly, we screened the feature values that had impact on the prognosis and obtained two subtypes. Then, we used 10 algorithms to achieve multiomics clustering and conducted pseudotiming analysis to further validate the robustness of our clustering method, based on which the two subtypes of ccRCC patients were further subtyped. Meanwhile, the immune infiltration was compared between the two subtypes, and drug sensitivity and potential drugs were analyzed. Furthermore, to analyze the heterogeneity of patients at the multiomics level, biological functions between two subtypes were compared. Finally, Boruta and PCA methods were used for dimensionality reduction and cluster analysis to construct a renal cancer risk model based on mRNA expression. RESULTS: A prognosis predicting model of ccRCC was established by dividing patients into the high- and low-risk groups. It was found that overall survival (OS) and progression-free interval (PFI) were significantly different between the two groups (p < 0.01). The area under the OS time-dependent ROC curve for 1, 3, 5, and 10 years in the training set was 0.75, 0.72, 0.71, and 0.68, respectively. CONCLUSION: The model could precisely predict the prognosis of ccRCC patients and may have implications for drug selection for ccRCC patients.

Definition and verification of novel metastasis and recurrence related signatures of ccRCC: A multicohort study.

Background: Cancer metastasis and recurrence remain major challenges in renal carcinoma patient management. There are limited biomarkers to predict the metastatic probability of renal cancer, especially in the early-stage subgroup. Here, our study applied robust machine-learning algorithms to identify metastatic and recurrence-related signatures across multiple renal cancer cohorts, which reached high accuracy in both training and testing cohorts. Methods: Clear cell renal cell carcinoma (ccRCC) patients with primary or metastatic site sequencing information from eight cohorts, including one out-house cohort, were enrolled in this study. Three robust machine-learning algorithms were applied to identify metastatic signatures. Then, two distinct metastatic-related subtypes were identified and verified; matrix remodeling associated 5 (MXRA5), as a promising diagnostic and therapeutic target, was investigated in vivo and in vitro. Results: We identified five stable metastasis-related signatures (renin, integrin subunit beta-like 1, MXRA5, mesenchyme homeobox 2, and anoctamin 3) from multicenter cohorts. Additionally, we verified the specificity and sensibility of these signatures in external and out-house cohorts, which displayed a satisfactory consistency. According to these metastatic signatures, patients were grouped into two distinct and heterogeneous ccRCC subtypes named metastatic cancer subtype 1 (MTCS1) and type 2 (MTCS2). MTCS2 exhibited poorer clinical outcomes and metastatic tendencies than MTCS1. In addition, MTCS2 showed higher immune cell infiltration and immune signature expression but a lower response rate to immune blockade therapy than MTCS1. The MTCS2 subgroup was more sensitive to saracatinib, sunitinib, and several molecular targeted drugs. In addition, MTCS2 displayed a higher genome mutation burden and instability. Furthermore, we constructed a prognosis model based on subtype biomarkers, which performed well in training and validation cohorts. Finally, MXRA5, as a promising biomarker, significantly suppressed malignant ability, including the cell migration and proliferation of ccRCC cell lines in vitro and in vivo. Conclusions: This study identified five robust metastatic signatures and proposed two metastatic probability clusters with stratified prognoses, multiomics landscapes, and treatment options. The current work not only provided new insight into the heterogeneity of renal cancer but also shed light on optimizing decision-making in immunotherapy and chemotherapy.

Methyltransferase-like 14 suppresses growth and metastasis of renal cell carcinoma by decreasing long noncoding RNA NEAT1.

Growing evidence supports that N6-methyladenosine (m6A) modification acts as a critical regulator involved in tumorigenesis at the mRNA level. However, the role of m6A modification at the noncoding RNA level remains largely unknown. We found that methyltransferase-like 14 (METTL14) was significantly downregulated in renal cell carcinoma (RCC) tissues (n = 580). Gain-of-function and loss-of-function experiments revealed that METTL14 attenuated the proliferation and migration ability of RCC cells in vivo and in vitro. The methylated RNA immunoprecipitation experiments identified that METTL14 decreased the expression of long noncoding RNA nuclear enriched abundant transcript 1_1 (NEAT1_1) in an m6A-dependent manner. Mechanistically, RNA pull-down assay and RNA immunoprecipitation identified NEAT1_1 directly bound to m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Notably, YTHDF2 accelerated the degradation of NEAT1_1 by selectively recognizing METTL14-mediated m6A marks on NEAT1_1. Multivariate analysis suggested that METTL14 downregulation was associated with malignant characteristics and predicted poor prognosis in RCC patients. In conclusion, our results uncover a newly identified METTL14-YTHDF2-NEAT1_1 signaling axis, which facilitates RCC growth and metastasis and provides fresh insight into RCC therapy.

Establishment of a prognosis Prediction Model Based on Pyroptosis-Related Signatures Associated With the Immune Microenvironment and Molecular Heterogeneity in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Pyroptosis is essential for tumorigenesis and progression of neoplasm. However, the heterogeneity of pyroptosis and its relationship with the tumor microenvironment (TME) in clear cell renal cell carcinoma (ccRCC) remain unclear. The purpose of the present study was to identify pyroptosis-related subtypes and construct a prognosis prediction model based on pyroptosis signatures. METHODS: First, heterogenous pyroptosis subgroups were explored based on 33 pyroptosis-related genes and ccRCC samples from TCGA, and the model established by LASSO regression was verified by the ICGC database. Then, the clinical significance, functional status, immune infiltration, cell-cell communication, genomic alteration, and drug sensitivity of different subgroups were further analyzed. Finally, the LASSO-Cox algorithm was applied to narrow down the candidate genes to develop a robust and concise prognostic model. RESULTS: Two heterogenous pyroptosis subgroups were identified: pyroptosis-low immunity-low C1 subtype and pyroptosis-high immunity-high C2 subtype. Compared with C1, C2 was associated with a higher clinical stage or grade and a worse prognosis. More immune cell infiltration was observed in C2 than that in C1, while the response rate in the C2 subgroup was lower than that in the C1 subgroup. Pyroptosis-related genes were mainly expressed in myeloid cells, and T cells and epithelial cells might influence other cell clusters via the pyroptosis-related pathway. In addition, C1 was characterized by MTOR and ATM mutation, while the characteristics of C2 were alterations in SPEN and ROS1 mutation. Finally, a robust and promising pyroptosis-related prediction model for ccRCC was constructed and validated. CONCLUSION: Two heterogeneous pyroptosis subtypes were identified and compared in multiple omics levels, and five pyroptosis-related signatures were applied to establish a prognosis prediction model. Our findings may help better understand the role of pyroptosis in ccRCC progression and provide a new perspective in the management of ccRCC patients.

The thermogenic activity of adjacent adipocytes fuels the progression of ccRCC and compromises anti-tumor therapeutic efficacy.

Clear cell renal cell carcinoma (ccRCC) preferentially invades into perinephric adipose tissue (PAT), a process associated with poor prognosis. However, the detailed mechanisms underlying this interaction remain elusive. Here, we describe a bi-directional communication between ccRCC cells and the PAT. We found that ccRCC cells secrete parathyroid-hormone-related protein (PTHrP) to promote the browning of PAT by PKA activation, while PAT-mediated thermogenesis results in the release of excess lactate to enhance ccRCC growth, invasion, and metastasis. Further, tyrosine kinase inhibitors (TKIs) extensively used in the treatment of ccRCC enhanced this vicious cycle of ccRCC-PAT communication by promoting the browning of PAT. However, if this cross-communication was short circuited by the pharmacological suppression of adipocyte browning via H89 or KT5720, the anti-tumor efficacy of the TKI, sunitinib, was enhanced. These results suggest that ccRCC-PAT cross-communication has important clinical relevance, and use of combined therapy holds great promise in enhancing the efficacy of TKIs.

DDX39 as a predictor of clinical prognosis and immune checkpoint therapy efficacy in patients with clear cell renal cell carcinoma.

DEAD-box protein 39 (DDX39) has been demonstrated to be a tumorigenic gene in multiple tumor types, but its role in the progression and immune microenvironment of clear cell renal cell cancer (ccRCC) remains unclear. The aim of the present study was to investigate the role of DDX39 in the ccRCC tumor progression, immune microenvironment and efficacy of immune checkpoint therapy. The DDX39 expression level was first detected in tumors in the public data and then verified in ccRCC samples from Changzheng Hospital. The prognostic value of DDX39 expression was assessed in the Cancer Genome Atlas (TCGA) and ccRCC patients from Changhai Hospital. The role of DDX39 in promoting ccRCC was analyzed by bioinformatic analysis and in vitro experiments. The association between DDX39 expression and immune cell infiltration and immune inhibitory markers was analyzed, and its value in predicting the immune checkpoint therapy efficacy in ccRCC were evaluated in the public database. DDX39 expression was elevated in Oncomine, GEO and TCGA ccRCC databases, as well as in Changzheng ccRCC samples. In TCGA ccRCC patients, increased DDX39 expression predicted worse overall survival (OS) (p<0.0001) and progression-free interval (PFI) (p<0.0001), and was shown as an independent predictive factor for OS (p=0.002). These findings were consistent with those from Changhai ccRCC patients. In addition, GO and GSEA analysis identified DDX39 as a pro-ccRCC gene. In vitro experiments confirmed the role of DDX39 in promoting ccRCC cell. Finally, DDX39 was found to be positively correlated with a variety of immune inhibitory markers, and could predict the adverse efficacy of immune checkpoint therapy in TIDE analysis. In conclusion, Increased DDX39 in ccRCC patients predicted worse clinical prognosis, promoted ccRCC cell proliferation, migration and invasion, and also predicted adverse efficacy of immune checkpoint therapy.

EGFR-AS1 Promotes Bladder Cancer Progression by Upregulating EGFR.

Long noncoding RNAs play an essential role in bladder cancer progression. The role of long noncoding RNA EGFR-AS1 in bladder cancer needs further study. We used clinical specimens to analyze the relationship between EGFR-AS1 and bladder cancer patients' characteristics. The functional experiments and mechanism studies were performed using qRT-PCR, transwell assay, survival analysis, and correlation analysis. We found that high expression of EGFR-AS1 was nearly related to aggressive bladder cancer and indicated poor prognosis for patients. The functional experiments in vivo and in vitro suggested that EGFR-AS1 promoted the proliferation and invasion of bladder cancer cells. Mechanically, EGFR-AS1 promoted the expression of EGFR by inhibiting the degradation of EGFR mRNA, thereby promoting the metastasis of bladder cancer. In addition, EGFR-AS1/EGFR may be involved in the immune-related pathways of bladder cancer. These studies indicate that the EGFR-AS1/EGFR pathway may be a potential diagnostic marker and therapeutic target for bladder cancer.

CircRNA cRAPGEF5 inhibits the growth and metastasis of renal cell carcinoma via the miR-27a-3p/TXNIP pathway.

Circular RNAs (circRNAs) are reported to act as important regulators in cancers. CircRNA RAPGEF5 (cRAPGEF5) is derived from exons 2-6 of the RAPGEF5 gene and may promote papillary thyroid cancer progression. However, the role of cRAPGEF5 in renal cell carcinoma (RCC) remains unclear. In this study, we found cRAPGEF5 to be significantly downregulated in RCC tissues. Among 245 RCC cases, cRAPGEF5 downregulation correlated positively with aggressive clinical characteristics and independently predicted poor overall survival and recurrence-free survival. Functional assays demonstrated that cRAPGEF5 suppresses RCC proliferation and migration in vitro and in vivo. Mechanistically, RNA Immunoprecipitation and circRNA in vivo precipitation assays showed that cRAPGEF5 functions as a sponge of oncogenic miR-27a-3p, which targets the suppressor gene TXNIP. Interactions between miR-27a-3p and cRAPGEF5 or TXNIP were confirmed by dual-luciferase reporter assays. In conclusion, cRAPGEF5 plays a role in suppressing RCC via the miR-27a-3p/TXNIP pathway and may serve as a promising prognostic biomarker and novel therapeutic target for RCC patients.

DNA methylation-regulated QPCT promotes sunitinib resistance by increasing HRAS stability in renal cell carcinoma.

Rationale: Although sunitinib has been shown to improve the survival rate of advanced renal cell carcinoma (RCC) patients, poor drug response is a major challenge that reduces patient benefit. It is important to elucidate the underlying mechanism so that the therapeutic response to sunitinib can be restored. Methods: We used an Illumina HumanMethylation 850K microarray to find methylation-differentiated CpG sites between sunitinib-nonresponsive and -responsive RCC tissues and Sequenom MassARRAY methylation analysis to verify the methylation chip results. We verified glutaminyl peptide cyclotransferase (QPCT) expression in sunitinib-nonresponsive and -responsive RCC tissues via qRT-PCR, western blot and immunohistochemical assays. Then, cell counting kit 8 (CCK-8), plate colony formation and flow cytometric assays were used to verify the function of QPCT in RCC sunitinib resistance after QPCT intervention or overexpression. Chromatin immunoprecipitation (ChIP) was performed to clarify the upstream regulatory mechanism of QPCT. A human proteome microarray assay was used to identify downstream proteins that interact with QPCT, and co-immunoprecipitation (co-IP) and confocal laser microscopy were used to verify the protein chip results. Results: We found that the degree of methylation in the QPCT promoter region was significantly different between sunitinib-nonresponsive and -responsive RCC tissues. In the sunitinib-nonresponsive tissues, the degree of methylation in the QPCT promoter region was significantly reduced, and the expression of QPCT was upregulated, which correlated with a clinically poor response to sunitinib. A knockdown of QPCT conferred sunitinib sensitivity traits to RCC cells, whereas an overexpression of QPCT restored sunitinib resistance in RCC cells. Mechanistically, reducing the methylation degree of the QPCT promoter region by 5-aza-2'-deoxycytidine (decitabine) in RCC cells could increase the expression of QPCT and NF-κB (p65) bound to the QPCT promoter region, positively regulating its expression, while the hypermethylation in the QPCT promoter region could inhibit the binding of NF-κB (p65). QPCT could bind to HRAS and attenuate the ubiquitination of HRAS, thus increasing its stability and leading to the activation of the ERK pathway in RCC cells. Conclusion: QPCT may be a novel predictor of the response to sunitinib therapy in RCC patients and a potential therapeutic target.

ANGPTL3 inhibits renal cell carcinoma metastasis by inhibiting VASP phosphorylation.

Angiopoietin-like proteins (ANGPTLs) comprise a group of proteins that are structurally similar to angiopoietins. In our previous studies, we found that ANGPTL3 can inhibit sorafenib resistance in renal cell carcinoma (RCC). According to bioinformatics analysis based on data in the Cancer Genome Atlas (TCGA), we found that expression of ANGPTL3 was significantly lower in RCC tissues than in adjacent tissues and that disease-free survival and overall survival were significantly shorter in patients with lower ANGPTL3 levels than in those with higher ANGPTL3 levels. Consistent with these results, we demonstrated that RCC tissues exhibited lower ANGPTL3 mRNA and protein expression levels than paired adjacent tissues. Moreover, we found that ANGPTL3 upregulation was associated with better clinical outcomes in RCC patients. ANGPTL3 overexpression inhibited the metastatic ability in RCC cells. Mechanistically, ANGPTL3 binds to vasodilator-stimulated phosphoprotein (VASP) and inhibits its phosphorylation at amino acid 157 in RCC cells. Finally, ANGPTL3 expression and VASP-157 phosphorylation may be combined to predict the prognosis of RCC patients. Overall, our findings describe the role of ANGPTL3 in inhibiting RCC metastasis and thus provide new molecular markers for RCC treatment and prognosis.

Cell Adhesion-Related Molecules Play a Key Role in Renal Cancer Progression by Multinetwork Analysis.

Renal cell carcinoma (RCC) is one of the most common malignancies in the urinary system. The study aimed to identify genetic characteristics and reveal the underlying mechanisms in RCC. GSE53757, GSE46699, and TCGA KIRC database (n = 897) were analyzed to screen differentially expressed genes (DEGs) in RCC. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, followed by the analysis of the protein-protein interaction (PPI) network of the DEGs by Cytoscape software. In all, 834 DEGs were identified in RCC, including 416 upregulated genes and 418 downregulated genes. The top 10 hub genes, VEGFA, EGFR, EGF, CD44, CD86, FN1, ITGAM, ITGB2, TLR2, and PTPRC, were identified from the PPI network according to the core degree. The following subnetwork revealed that these significant modules were enriched in positive regulation of response to external stimulus, regulation of leukocyte-mediated immunity, and regulation of exocytosis. The expressions of these hub genes were also validated using qRT-PCR and IHC in Changzheng RCC database (n = 160). We especially found that half of the top ten hub genes were cell adhesion-related molecules, which were associated with RCC progression and poor prognosis. In conclusion, these hub genes, particularly cell adhesion-related molecules, could be used as prognostic biomarkers and potential therapeutic targets for RCC.

Pyk2 promotes tumor progression in renal cell carcinoma.

Proline-rich tyrosine kinase 2 (Pyk2), a member of the focal adhesion kinase family, has recently been associated with tumor development. However, the role of Pyk2 in renal cell carcinoma (RCC) remains unexplored. The present study investigated the expression pattern, clinical significance, and function of Pyk2 in RCC. By using a reverse transcription-quantitative polymerase chain reaction, tissue microarray and immunohistochemistry, it was demonstrated that RCC tissues display a higher Pyk2 expression compared with paired adjacent nontumor tissues. Furthermore, it was revealed that Pyk2 upregulation was associated with poor clinical outcomes in patients with RCC. By using loss-of-function approaches, it was demonstrated that Pyk2 knockdown reduced cell viability, invasive ability and migratory ability, and increased apoptosis in RCC cell lines. In contrast, Pyk2 overexpression promoted tumor cell proliferation, invasion and migration and reduced apoptosis. Collectively, the results of the present study present the tumor-promoting function of Pyk2 in RCC and thus provide molecular evidence for novel tyrosine kinase inhibitors as novel therapeutic options for RCC.