Biomarker For ccRCC Research Articles

Integrative analysis of TRPV family to prognosis and immune infiltration in renal clear cell carcinoma

ABSTRACT The transient receptor potential vanilloid (TRPV) family has been preliminarily discovered to play an important role in various cancers, including clear cell renal cell carcinoma (ccRCC), which is closely associated with immune infiltration. However, the expression and prognosis of TRPV family and tumor-infiltrating immune cells in ccRCC are obscure. This study aimed to explore the prognostic and therapeutic value of the TRPV family expression in ccRCC from the perspective of bioinformatics. We analyzed the transcriptome and clinical data of kidney renal clear cell carcinoma (KIRC) from The Cancer Genome Atlas (TCGA) database. A clustering analysis and immune infiltration analysis were conducted to investigate the influence of the TRPV family genes on ccRCC. Our study found that the TRPV family is an excellent prognostic stratification for ccRCC. Among them, TRPV3 is the most significant prognostic marker of ccRCC. In addition, we performed a drug sensitivity analysis to identify the drugs with the strongest association with TRPV3. As a result, the TRPV family, particularly TRPV3, can act as a prognostic biomarker in ccRCC to determine prognosis and levels of immune infiltration.

Assessment of the prognostic value of SPOCK1 in clear cell renal cell carcinoma: a bioinformatics analysis.

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most common urological malignancies, and once metastasis occurs, it often has a poor prognosis and lacks effective treatment. Therefore, there is an urgent need to screen some new biomarkers and explore their molecular mechanisms to improve the early clinical diagnosis and targeted therapy of ccRCC. SPOCK1 (SPARC/osteonectin, CWCV and Kazal-like domains proteoglycan 1) is a conserved multi-domain proteoglycan that plays an important role in the development of multiple cancer types; however, its prognostic value in ccRCC has not been investigated. The study of the prognostic value of SPOCK1 in ccRCC is a good complement to the study of ccRCC biomarkers.MethodsDatabases of this study included Oncomine, Kaplan-Meier Plotter, GEPIA, GeneMANIA, cBioPortal, and TIMER. Student’s t-test was used to analyze the differences in SPOCK1 expression in ccRCC tissues compared with tumor-adjacent normal tissues. Kaplan-Meier curves for survival analysis were used to assess the correlation between the expression of SPOCK1 and the prognostic outcomes. Correlation module drew the expression scatterplots between SPOCK1 and immune cell infiltration in ccRCC, together with the Spearman’s rho value and estimated statistical significance.ResultsThe SPOCK1 mRNA expression was significantly higher in ccRCC tissues (mean expression ± SD: 920.2±195.2) than in normal tissues (mean expression ± SD: 358.4±29.1, P=0.008), and high SPOCK1 expression significantly and positively correlated with the pathological stage of ccRCC patients (F value =10.2, P<0.001). Higher expression of SPOCK1 was also associated with significantly shorter overall survival (OS) and disease-free survival (DFS) in ccRCC patients (GEPIA: P=0.046, P<0.001, respectively; Kaplan-Meier Plotter: P=0.002, P=0.0022, respectively). The function of SPOCK1 is mainly related to tumor development and extracellular matrix remodeling, and it may participate in the epithelial-mesenchymal transition process. SPOCK1 expression significantly and positively correlated with infiltration of several immune cells in ccRCC, including cancer-associated fibroblasts (CAFs) (Rho =0.333, P=2.16×10−13), tumor-associated macrophages (TAMs) (Rho =0.18, P=1.02×10−4), and tumor-associated neutrophils (TANs) (Rho =0.165, P=3.83×10−4). Conversely, there was a significant and negative correlation between SPOCK1 expression and infiltration of CD4+ T cells (Rho =−0.113, P=0.015).ConclusionsSPOCK1 may be a potential prognostic biomarker in ccRCC.

DRAM1 plays a tumor suppressor role in clear cell renal cell carcinoma through modulating Akt signaling

BackgroundClear cell renal carcinoma (ccRCC) is one of the most common malignant tumors worldwide. DNA damage-regulated autophagy modulator1 (DRAM1) plays an important roles in apoptosis and tumor progression. However, the role of DRAM1 in ccRCC is still unknown. In our study, we aimed to investigate the effect of DRAM1 in the progression of ccRCC. MethodsThe expression and prognostic information of DRAM1 in ccRCC were obtained by immunohistochemistry staining and bioinformatics database. Cell proliferation, migration, invasion were detected by CCK-8 assay, wound-healing and transwell assays, and the cell apoptosis was examined by tunel assay and flow cytometry analysis. Western blot was used to detect the expression of DRAM1, Bax, Bcl2, Akt, p53,E-cadherin, N-cadherin of ccRCC cells. ResultsDecreased expression of DRAM1 was found in ccRCC tissues, which predicted a shorter survival rate in ccRCC patient. We confirmed that DRAM1 inhibited the proliferation, migration, invasion and epithelial mesenchymal transformation (EMT), while enhanced the apoptosis of ccRCC cells. In addition, the results of inhibition of Akt signaling were consistent with the above. We further proved that DRAM1 over-expression decreased the phosphorylation of Akt signaling, and overexpression of DRAM1 could reverse oncogenic function induced by the over-activating of Akt in ccRCC cells. Conclusionoverexpression of DRAM1 plays a tumor suppressive role in ccRCC through inactivation of Akt and highlights the potential role of DRAM1 as a prognostic biomarker in ccRCC.

Identification of IL20RB as a Novel Prognostic and Therapeutic Biomarker in Clear Cell Renal Cell Carcinoma.

Background Clear cell renal cell carcinoma (ccRCC) is a type of life-threatening malignant tumor of the urinary system. IL20RB, interleukin 20 receptor subunit beta, is a cytokine receptor subunit coding gene and was initially found to play a vital role in human cancers, while its role in ccRCC still remains unclear. Methods In this work, we explored the prognostic value and therapeutic potential of IL20RB in ccRCC mainly by online tools. Firstly, we used UALCAN and GEPIA to explore the expression profile and prognostic value of IL20RB in various cancers; the expression profile in tumor cell lines was also analysed with CCLE and Expression Atlas. Then, we decided to focus on ccRCC for further analysis; we further demonstrated the significant correlation between expression and clinical features by GEPIA and UALCAN. In order to reveal the potential intrinsic mechanism responsible for the upregulation of IL20RB in ccRCC, we made genetic alternation analysis and methylation analysis. cBioPortal was used for genetic alternation analysis. UALCAN, MethSurv, and Xena were used for methylation analysis. To learn details of how IL20RB might function in ccRCC, we further conducted functional analysis and immune infiltration analysis. STRING and GSEA were used to do functional analysis. TIMER was used for immune infiltration analysis; KM plotter was used for survival analysis. Results Results show that IL20RB is upregulated in ccRCC, and low methylation may be responsible for its upregulation. Both high expression and low methylation of IL20RB predict worse survival, and both have a strong positive correlation with clinical characteristics. In addition, results indicate that there exists a crosstalk between IL20RB and neutrophils. Furthermore, the immune microenvironment could influence the prognosis predicting ability of IL20RB. Conclusions In conclusion, IL20RB plays an important role in ccRCC and is identified as a novel prognostic and potential therapeutic biomarker in ccRCC.

PIWI-Interacting RNA Pathway Genes: Potential Biomarkers for Clear Cell Renal Cell Carcinoma.

Background Clear cell renal cell carcinoma (ccRCC) is one of the most lethal malignancies in the urinary system, yet effective diagnostic and prognostic markers are lacking. Recently, several of piRNA pathway genes have been reported to be associated with cancer diagnosis and prognosis, but their role in ccRCC is still unclear. Methods We analysed the expression of 27 piRNA pathway genes in 539 kidney renal clear cell carcinoma (KIRC) and 72 nontumor tissue samples (data from TCGA), and 12 mRNAs were significantly different. The aim was to sift the piRNA pathway genes that are correlated with ccRCC patient survival and to construct a piRNA pathway gene risk prognostic model using Kaplan-Meier survival curve and ROC curve, respectively. Results 5 piRNA pathway genes (TDRD7, GPAT2, PLD6, SUV39H1, and DOM3Z) were picked out and used to construct the piRNA pathway gene risk model. Kaplan-Meier survival curve analysis showed that compared with that of the low-risk group of ccRCC patients, the OS of the high-risk group of ccRCC patients was significantly reduced. The predictive performance of the prognostic risk model was measured using a ROC curve, which individually showed AUC values for 1 year of 0.707, for 3 years of 0.713, and for 5 years of 0.701. Moreover, the mRNA and protein expression levels of TDRD7 were overexpressed in the ccRCC datasets (data from our cohort, TCGA, GEO, and CPTAC) and ccRCC cell lines, and the expression levels correlated with the clinicopathological characteristics in ccRCC. The Tumor Immune Estimation Resource (TIMER) showed that the mRNA expression level of TDRD7 was positively related to tumor immune infiltrating cells (TICs) in ccRCC. Mechanistically, gene set enrichment analysis (GSEA) was performed to uncover the mechanism of TDRD7 in ccRCC. In summary, the piRNA pathway genes,especially TDRD7, may be potential cancer diagnostic and prognostic biomarkers of ccRCC.

Single cell phenotyping of PDL1 and HLA1 on circulating tumor cells (CTC) in clear cell renal cell carcinoma (ccRCC) on systemic treatment with immunotherapy (IO) and vascular endothelial growth factor receptor tyrosine kinase inhibitors (TKI).

381 Background: Single agent or combination regimens using different IO and TKI agents have dramatically improved survival of patients (pt) with advanced ccRCC. Nevertheless, selecting optimal combination and sequencing of treatments remains challenging given the current absence of predictive biomarkers. Considering substantial disease heterogeneity and tumor clonal evolution, CTCs could provide a more comprehensive and longitudinal assessment of disease and response to treatment when compared to single site biopsy. In this study we explore changes in CTC characteristics in correlation with pt’s response (Resp) to treatment and progression (PD). Methods: 152 blood sample time points from 44 ccRCC pts (mean=3.5 sample/pt, range= 1-9) were included. An automated microfluidic platform using exclusion-based sample technology was employed for high sensitivity enrichment of RCC CTCs using EpCAM and CAIX antibodies. High-specificity CTC identification was achieved with positive staining for pCK or CAXII, and negative staining for CD45, CD34 and CD66b. Single cell phenotyping of PDL1 and HLA1 expression on each CTC was quantified with automated image analysis algorithms followed by manual visual QA of the putative CTCs. Cellular-level expression of PDL1 and HLA1 were compared within treatment-response groupings, using Wilcoxon rank-sum tests (Table). Results: Pts were 70% male, 93% Caucasian, 51% de novo metastatic, 98% received systemic treatment. CTC samples were collected at baseline (12) or while on treatment with IO (71), TKI (55), or IO/TKI (14). Across treatments, the number of CTCs increased from Resp to PD but this trend was not statistically significant. PDL1 expression was higher at PD compared to Resp on IO and IO/TKI but not significantly different on TKI. HLA1 expression was higher at PD compared to Resp on IO and TKI but not significantly different on IO/TKI. Average CTC PD-L1 expression was higher at Resp to IO compared to baseline. Conclusions: Single cell phenotyping of PDL1 and HLA1 were done at Resp compared to PD time points on IO and/or TKI to identify potential mechanisms of resistance. Further evaluation of these preliminary results in prospective trials is ongoing to advance new predictive biomarkers for ccRCC.[Table: see text]

Utilizing the autoantibody immune response to tumor antigens for kidney cancer early detection.

369 Background: Kidney cancer (renal cell carcinoma, RCC), the 8th most common U.S. cancer, is in need for better cure rates through early detection (5-year relative survival for stage I RCC: ̃95%; for stage IV RCC ̃19%). Autoantibodies are common in cancer and result from the altered expression, localization, or post-translational modification of endogenous proteins in tumor cells (autoantigens) and from the expression of mutated genes that give rise to new proteins (neoantigens). In contrast to cellular immune responses in cancer, autoantibodies are less well characterized, yet hold promise to enable cancer early detection by immune amplification of the ‘cancer signal’ while retaining specificity to cancer types including RCC. Autoantibodies may therefore be useful for kidney cancer early detection and diagnosis. Our goal was to profile the autoantibody repertoire in blood from patients with clear cell RCC (ccRCC), the most common form of RCC, in order to: 1) determine if autoantibodies can be detected in patients with early-stage and late-stage ccRCC; 2) identify common epitopes amongst ccRCC patients that could suggest common RCC antigens; and 3) determine specificity and sensitivity of potential autoantibody biomarkers for ccRCC vs. other non-cancer conditions. Methods: We use the SERA platform (https://serimmune.com/publications/) to compare putative autoantibody signal in blood from 177 patients with ccRCC, 23 with benign kidney lesions, and ̃800 healthy controls. SERA utilizes a random bacterial display 12mer peptide library of 1010 diversity in conjunction with next-generation sequencing to ascertain epitope enrichment across the entire human proteome. Results: We find significant differences in epitope repertoires in ccRCC compared to the healthy human cohort. Patients with ccRCC exhibit a rich repertoire of rare, enriched epitopes which may comprise putative autoantibody signal. This epitope signal is present with high abundance in all ccRCC stages, including stage I ccRCC. In contrast, healthy controls and patients with benign kidney lesions demonstrate more restricted repertoires. However, we do not find evidence of common ccRCC antigens: epitopes are not conserved across large subsets of ccRCC patients. Conclusions: Our initial results suggest that each patient may develop an individualized tumor-associated antibody response. Whether assessing a select epitope panel in a patient’s blood could be useful for ccRCC early detection, or even epitope diversity without needing to identify specific epitopes, warrants further study.

OIP5 Is a Novel Prognostic Biomarker in Clear Cell Renal Cell Cancer Correlating With Immune Infiltrates.

Opa interacting protein 5 (OIP5), overexpressed in some types of human cancers, has been reported to be associated with the carcinogenesis of human cancer. However, its contribution to cancer immunity remains unknown. Furthermore, the relationship between OIP5 and cancer immunity remains uncertain. In our research, we explored the different expression of OIP5 between 539 ccRCC and 72 normal renal tissues base on TCGA data set. We analyzed the associations between OIP5 expression with ccRCC progression and survival. Next, we compared immune cell profiles in cancer tissues and normal tissues in the Cancer Genome Atlas (TCGA) ccRCC cohort. We found that the level of immune cell infiltration was correlated with the copy number of OIP5 gene in ccRCC. The effect of OIP5 on immune activity was verified by Gene Set Enrichment Analysis of RNA-seq data from 32 ccRCC cell lines in the public database. Moreover, a pathway enrichment analysis of 49 OIP5-associated immunomodulators demonstrated the involvement of the T cell receptor signaling pathway, the JAK-STAT signaling pathway, the NF-kappa B signaling pathway and the primary immunodeficiency pathway. In addition, using OIP5-associated immunomodulators, we constructed multiple-gene risk prediction signatures using the Cox regression model. Our results provided insights into the role of OIP5 in tumor immunity and revealed that OIP5 may be a potential immunotherapeutic target for ccRCC. Designated immune signature is a promising prognostic biomarker in ccRCC.

CGN Correlates With the Prognosis and Tumor Immune Microenvironment in Clear Cell Renal Cell Carcinoma.

This study aimed to screen and verify the important prognostic genes related to clear cell renal cell carcinoma (ccRCC) and further analyze their relationship with the immune microenvironment. Gene expression profiles from the TCGA-KIRC, GSE46699, GSE36895, and GSE16449 datasets were utilized to explore differentially co-expressed genes in ccRCC. We screened 124 differentially co-expressed genes using a weighted gene co-expression network and differential gene expression analyses. Univariate and multivariate Cox survival analyses revealed that the expressions of genes CGN, FECH, UCHL1, and WT1 were independently related to the overall survival of ccRCC patients. Kaplan–Meier survival analysis was performed, and CGN was found to have the strongest correlation with the prognosis of ccRCC patients and was consequently selected for further analyses and experimental verification. The results showed that NK cell activation, resting dendritic cells, resting monocytes, and resting mast cells were positively correlated with CGN expression; CD4+ memory activated T cells, regulatory T cells, and M0 macrophages were negatively correlated with CGN expression. Finally, using western blotting and reverse transcription polymerase chain reaction, we verified that the CGN protein level was down-regulated in ccRCC samples, which was consistent with the mRNA levels. CGN was thus identified as diagnosis and prognosis biomarker for ccRCC and is related to the immune microenvironment.

Identification of UBE2I as a Novel Biomarker in ccRCC Based on a Large-Scale CRISPR-Cas9 Screening Database and Immunohistochemistry.

Background: The genome-wide CRISPR-cas9 dropout screening has emerged as an outstanding approach for characterization of driver genes of tumor growth. The present study aims to investigate core genes related to clear cell renal cell carcinoma (ccRCC) cell viability by analyzing the CRISPR-cas9 screening database DepMap, which may provide a novel target in ccRCC therapy. Methods: Candidate genes related to ccRCC cell viability by CRISPR-cas9 screening from DepMap and genes differentially expressed between ccRCC tissues and normal tissues from TCGA were overlapped. Weighted gene coexpression network analysis, pathway enrichment analysis, and protein–protein interaction network analysis were applied for the overlapped genes. The least absolute shrinkage and selection operator (LASSO) regression was used to construct a signature to predict the overall survival (OS) of ccRCC patients and validated in the International Cancer Genome Consortium (ICGC) and E-MTAB-1980 database. Core protein expression was determined using immunohistochemistry in 40 cases of ccRCC patients. Results: A total of 485 essential genes in the DepMap database were identified and overlapped with differentially expressed genes in the TCGA database, which were enriched in the cell cycle pathway. A total of four genes, including UBE2I, NCAPG, NUP93, and TOP2A, were included in the gene signature based on LASSO regression. The high-risk score of ccRCC patients showed worse OS compared with these low-risk patients in the ICGC and E-MTAB-1980 validation cohort. UBE2I was screened out as a key gene. The immunohistochemistry indicated UBE2I protein was highly expressed in ccRCC tissues, and a high-level nuclear translocation of UBE2I occurs in ccRCC. Based on the area under the curve (AUC) values, nuclear UBE2I had the best diagnostic power (AUC = 1). Meanwhile, the knockdown of UBE2I can inhibit the proliferation of ccRCC cells. Conclusion: UBE2I, identified by CRISPR-cas9 screening, was a core gene-regulating ccRCC cell viability, which accumulated in the nucleus and acted as a potential novel promising diagnostic biomarker for ccRCC patients. Blocking the nuclear translocation of UBE2I may have potential therapeutic value with ccRCC patients.

Construction of an immune-related prognostic model by exploring the tumor microenvironment of clear cell renal cell carcinoma

ObjectiveIn this study, bioinformatics methods were performed to screen the candidate prognosis-related genes of clear cell renal cell carcinoma (ccRCC) by analyzing the tumor microenvironment (TME). MethodsGene expression and clinical data of ccRCC patients were accessed from TCGA, and R package ESTIMATE was applied to calculate immune, stromal, and ESTIMATE scores of the patients. Survival analysis was conducted per median of these three scores. Based on the scoring results, differentially expressed genes (DEGs) were screened. Regression algorithms were utilized to screen prognostic genes and establish a risk model. Finally, pathway activity differences were analyzed through GSEA. ResultsPatients with the unfavorable prognosis had high immune scores. 619 DEGs (499 up-regulated and 120 down-regulated) were screened based on the differences in gene expression of the patients with high and low immune scores. These genes mainly participated in immune-related signaling pathways. A prognostic risk model for ccRCC patients was constructed and 7 immune-related signature genes (RORB, TNFSF14, UCN2, USP2, TOX3, KLRC2, SLAMF9) were obtained through regression analysis. The constructed prognostic risk model could be used for determining prognoses of patients with ccRCC. ConclusionWe unraveled the association between TME and prognosis of ccRCC patients and established a prognostic risk model based on the differentially expressed genes. These results contributed to understanding of TME that affected patients’ prognosis and progression of ccRCC and conduced to finding potential biomarkers of ccRCC.

ADAM metallopeptidase domain 12 overexpression correlates with prognosis and immune cell infiltration in clear cell renal cell carcinoma

ABSTRACT This study investigated the role of ADAM metallopeptidase domain 12 (ADAM12) in clear cell renal cell carcinoma (ccRCC). The mRNA expression of ADAM12 was analyzed using The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database, and the protein expression level of ADAM12 in renal clear cell carcinoma cell lines was detected by Western blot analysis. The Wilcoxon rank-sum test, logistic regression analysis, Cox regression analysis, and Kaplan-Meier analysis were used to assess the relationship between the clinicopathological characteristics and the prognosis of ccRCC patients and ADAM12 expression. The miRNAs and lncRNAs associated with ADAM12 were predicted, and a ceRNA network was constructed using the Starbase database. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis were used to identify relevant pathways. The relationship between ADAM12 and immune infiltration and checkpoints was analyzed using the TIMER and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The results showed that ADAM12 expression was increased in ccRCC tissues and cells and significantly correlated with patient gender, Tumor stage, Metastasis stage, Node stage, and clinical grade. Survival analysis showed that ccRCC patients with high ADAM12 expression had a low overall survival rate. Univariate and multivariate Cox regression analyses showed that ADAM12 was an independent prognostic factor. Enrichment analysis showed that ADAM12 expression was associated with immune-related pathways. Immune infiltration analysis showed that ADAM12 expression was related to immune cell infiltration, PD-1, PD-L1, and CTLA4. These results suggest that ADAM12 may be a potential diagnostic and prognostic biomarker for ccRCC.

M2-polarization-related CNTNAP1 gene might be a novel immunotherapeutic target and biomarker for clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies, characterized by high mortality rate in urology. Unfortunately, reliable biomarkers for ccRCC diagnosis and prognosis remain lacking. Contactin-associated protein 1 (CNTNAP1) has yet to be thoroughly investigated in cancer, especially its relationship with immune infiltration or clinical outcomes of ccRCC. Here, we explored The Cancer Genome Atlas Kidney Clear Cell Carcinoma database (TCGA-KIRC) for prognostic significance, differential expression, and probable mechanism of CNTNAP1. The aberrant CNTNAP1 expression was also validated by the International Cancer Genome Consortium (ICGC) and ccRCC clinic samples. We used Database for Annotation, Visualization, and Integrated Discovery to perform the GO and KEGG enrichment. TIMER database was further utilized to assess its correlation with immune infiltration in ccRCC. The CellMiner database was used to analyze the relationship between CNTNAP1 expression and drug sensitivity. Results showed CNTNAP1 was upregulated in TCGA-KIRC, ICGC, and clinic samples. And CNTNAP1 expression was positively related to infiltration levels of cancer-associated fibroblast, regulatory T cells, and myeloid-derived suppressor cells, while negatively related to eosinophils. Furthermore, we observed CNTNAP1 was appreciably positively associated with alternatively activated macrophage (M2) in ccRCC. Finally, high CNTNAP1 expression was negatively correlated with nilotinib, crizotinib, eribulin mesylate, and vinorelbine. Collectively, these results strongly suggest that CNTNAP1 might act as an immunotherapeutic target and a promising novel biomarker for ccRCC.

PAQR5 Expression Is Suppressed by TGFβ1 and Associated With a Poor Survival Outcome in Renal Clear Cell Carcinoma.

BackgroundRenal cell carcinoma (RCC) was sex-hormone responsive, and clinical trials using progesterone significantly reduced the incidence of distal metastasis after radical nephrectomy. Recently membrane-bound progesterone receptors (mPRs) were discovered to mediate the non-genomic effect of progesterone. Aberrant expressions of these mPRs were reported in human breast, ovarian, urinary bladder, brain, uterine, and prostate cancers. However, their expression profiles in RCC are yet to be assessed.MethodsMultiple datasets from RNA sequencing (RNA-seq), cDNA microarray, and proteomic analysis were used to compare gene expression between cancerous and normal kidney tissues. Immunohistochemistry was conducted to examine protein expression in kidney tissues. Promoter methylation levels were assessed for correlation analysis with gene expression.ResultsOf the seven membrane-bound progesterone receptor genes, the progestin and adipoQ receptor-5 (PAQR5) gene is predominantly expressed in normal kidney tissue but was significantly downregulated in RCC tissues. PAQR5 downregulation correlated with tumor stage, cancer grade, lymph node invasion, and distal metastasis only in clear cell RCC (ccRCC) tissues. PAQR5 downregulation was associated with an increased promoter DNA methylation and a poor survival outcome in ccRCC patients. In addition, PAQR5 expression inversely correlated with transforming growth factor beta-1 (TGFB1) expression, and TGFβ1 treatment significantly reduced PAQR5 gene expression.ConclusionPAQR5 is a novel prognostic biomarker in ccRCC and is negatively regulated by the TGFβ1 pathway.

Overexpression of Parkin in clear cell renal cell carcinoma decreases tumor aggressiveness by regulating CKS2 levels

Low expression levels of the E3 ubiquitin-protein ligase Parkin (PARK2) are exhibited in several cancer entities, including clear cell renal cell carcinoma (ccRCC), and are associated with poor prognosis; however, PARK2 can also function as a tumor suppressor gene. The aim of the present study was to thoroughly investigate the effects of PARK2 overexpression in ccRCC cell lines and to determine its effects on malignancy by conducting functional assays such as cell cycle analysis, apoptosis analysis, migration and invasion assays. Furthermore, liquid chromatography-mass spectrometry was used to decipher potential targets of PARK2 that may influence the behavior of ccRCC tumor cells. In addition, ccRCC tumor tissues from a patient cohort were examined in tissue microarrays to find correlations between different clinical parameters. In the present study, it was demonstrated that the induction of PARK2 resulted in a less aggressive phenotype, as indicated by lower migration and invasion in ccRCC cell lines. Mass spectrometry revealed decreased levels of 29 proteins in cells with PARK2 overexpression, including CDC28 protein kinase regulatory subunit 2 (CKS2), which is highly expressed in numerous types of cancer. The link between the function of PARK2 as an E3 ubiquitin ligase and the low expression levels of CKS2 was investigated by mutating the catalytic domain of the PARK2 gene, and it was found that the effect of decreased migration was abolished in 786-O and RCC-MH ccRCC cell lines. CKS2 silencing decreased migratory ability of the cells. Furthermore, it was revealed that high CKS2 levels are associated with high tumor grading in patient samples and lower patient survival. In conclusion, the results from the present study indicated that PARK2 may signal via CKS2 to affect tumor behavior. In consequence, CKS2 may be a biomarker in ccRCC and may also serve as potential target for ccRCC therapy.

TLN2 functions as a tumor suppressor in clear cell renal cell carcinoma via inactivation of the Wnt/β-catenin signaling pathway.

BackgroundClear cell renal cell carcinoma (ccRCC) is one of the most prevalent malignancies worldwide, but there is lack of reliable clinical diagnostic biomarkers. We explored the clinical value and functions of Talin 2 (TLN2) in the progression of ccRCC.MethodsA bioinformatic analysis was performed to determine the clinical value of TLN2 in ccRCC. TLN2 expression was evaluated by immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (RT-qPCR) and western blot in ccRCC tissues and cells. The functions of TLN2 in ccRCC were investigated by both in vivo and in vitro studies. The functions of TLN2 in ccRCC cell proliferation was determined by CCK-8 assays and colony formation assays. Transwell assays and wound healing assays were performed to detect the effects of TLN2 on ccRCC cell invasion and migration abilities. Apoptosis assay and cell cycle analysis were used to determine the influence of TLN2 on ccRCC cell apoptosis and cell cycle.ResultsTLN2 was downregulated in ccRCC tissues and cells. Clinically, TLN2 was confirmed to be an independent factor for ccRCC patient prognosis. Results of colony formation and CCK-8 assays showed that TLN2 overexpression inhibited ccRCC cell growth. Moreover, wound healing assays and transwell assays indicated that TLN2 overexpression inhibited ccRCC cell invasion and migration. In vivo assays also indicated that TLN2 played an important role in ccRCC cell growth and metastasis. TLN2 also inhibited cell cycle progression and promoted apoptosis of ccRCC cells. Mechanistically, TLN2 was confirmed to exert anti-ccRCC functions through Wnt/β-catenin signaling.ConclusionsTLN2 served as a tumor regulator of ccRCC via Wnt/β catenin signaling, suggesting that it could be a promising therapeutic and prognostic biomarker for ccRCC.

CD146 as a Prognostic-Related Biomarker in ccRCC Correlating With Immune Infiltrates.

BackgroundsCD146 is highly expressed in various malignant tumors and associated with the poor prognosis. However, the role of CD146 in clear cell renal cell carcinoma (ccRCC) is still unknown. This study aimed to identify the role of CD146 in ccRCC by integrated bioinformatics analysis.MethodsCD146 mRNA expression and methylation data in ccRCC was examined using the TIMER, UALCAN, and MethSurv databases. CD146 expression in paraffin-embedded tissues (140 cancer samples and 140 paracancer tissues) from our cohort were examined by immunohistochemistry assay. The LinkedOmics database was used to study the signaling pathways related to CD146 expression. TIMER and TISIDB were used to analyze the correlations among CD146, CD146-coexpressed genes, tumor-infiltrating immune cells, and immunomodulators. The relationship between CD146 and drug response in renal cancer cell lines was analyzed by the CTRP and CCLE databases.ResultsThe mRNA and protein levels of CD146 were elevated in ccRCC tissues than that in paracancer tissues. The DNA methylation of CD146 in ccRCC tissues were lower than that in normal tissues. Importantly, high CD146 expression was associated with poor prognosis in patients with ccRCC. Furthermore, multivariate Cox regression analysis showed that CD146 was an independent prognostic factor in ccRCC. GO and KEGG pathway analyses indicated the co-expressed genes of CD146 were mainly related to a variety of immune-related pathways, including Th1 and Th2 cell differentiation, Th17 cell differentiation, and leukocyte transendothelial migration. Our data demonstrated that the expression and methylation status of CD146 were strongly correlated with immune infiltration levels, immunomodulators, and chemokines. Further, the sensitivity and resistance of renal cancer cell lines to some drugs were related to CD146 expression.ConclusionsOur study highlights the clinical significance of CD146 in ccRCC and provides novel insights into the immune function of CD146 in the tumor microenvironment.

Analysis and verification of N 6-methyladenosine-modified genes as novel biomarkers for clear cell renal cell carcinoma

ABSTRACT N 6-methyladenosine (m6A) has been involved in diverse biological processes in cancer, but its function and clinical value in clear cell renal cell carcinoma (ccRCC) remain largely unknown. In this study, we found that 1453 m6A-modified differentially expressed genes (DEGs) of ccRCC were mainly enriched in cell cycle, PI3K-AKT, and p53 signaling pathways. Then we constructed a co-expression network of the 1453 m6A-modified DEGs and identified a most clinically relevant module, where NUF2, CDCA3, CKAP2L, KIF14, and ASPM were hub genes. NUF2, CDCA3, and KIF14 could combine with a major RNA m6A methyltransferase METTL14, serving as biomarkers for ccRCC. Real-time quantitative PCR assay confirmed that NUF2, CDCA3, and KIF14 were highly expressed in ccRCC cell lines and ccRCC tissues. Furthermore, these three genes were modified by m6A and negatively regulated by METTL14. This study revealed that NUF2, CDCA3, and KIF14 were m6A-modified biomarkers, representing a potential diagnostic, prognostic, and therapeutic target for ccRCC. Abbreviations: m6A: N 6-methyladenosine; ccRCC: clear cell renal cell carcinoma; DEGs: differentially expressed genes; NUF2: NUF2 component of NDC80 kinetochore complex; CDCA3: cell division cycle associated 3; CKAP2L: cytoskeleton associated protein 2 like; KIF14: kinesin family member 14; ASPM: assembly factor for spindle microtubules; METTL14: methyltransferase 14; OS: overall survival; FPKM: fragments per kilobase million; GEO: gene expression omnibus; TCGA: the Cancer Genome Atlas; RMA: robust multi-array average expression measure; WGCNA: weighted gene co-expression network analysis; GO: gene ontology; KEGG: kyoto encyclopedia of genes and genomes; ROC: receiver operating characteristic curve; AUC: area under the curve; RIP: RNA immunoprecipitation; qPCR: real-time quantitative PCR.

Clinical significance of novel DNA methylation biomarkers for renal clear cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor characterized by the highest mortality rate of the genitourinary cancers, and, therefore, new diagnostic and/or prognostic biomarkers are urgently needed. Based on genome-wide DNA methylation profiling in 11 pairs of ccRCC and non-cancerous renal tissues (NRT), the methylation at regulatory regions of ZNF677, FBN2, PCDH8, TFAP2B, TAC1, and FLRT2 was analyzed in 168 renal tissues and 307 urine samples using qualitative and quantitative methylation-specific PCR (MSP). Significantly higher methylation frequencies for all genes were found in ccRCC tissues compared to NRT (33-60% vs. 0-11%). The best diagnostic performance demonstrated a panel of ZNF677, FBN2, PCDH8, TFAP2B & TAC1 with 82% sensitivity and 96% specificity. Hypermethylation of ZNF677 and PCDH8 in the tissue samples was significantly related to numerous adverse clinicopathologic parameters. For the urine-based ccRCC detection, the highest diagnostic power (AUC = 0.78) was observed for a panel of ZNF677 & PCDH8 (with or without FBN2 or FLRT2) with 69-78% sensitivity and 69-80% specificity, albeit with lower values in the validation cohort. Besides, methylation of PCDH8 was significantly related to higher tumor stage and fat invasion in the study and validation cohorts. Moreover, PCDH8 was strongly predictive for OS (HR, 5.7; 95% CI 1.16-28.12), and its prognostic power considerably increased in combination with ZNF677 (HR, 12.5; 95% CI 1.47-105.58). In summary, our study revealed novel, potentially promising DNA methylation biomarkers of ccRCC with the possibility to be applied for non-invasive urine-based ccRCC detection and follow-up.

PRR11 promotes ccRCC tumorigenesis by regulating E2F1 stability.

Proline rich 11 (PRR11), a novel tumor-related gene, has been identified in different tumors. However, the relevant biological functions of PRR11 in human clear cell renal cell carcinoma (ccRCC) have not been studied. In this study, we first identified PRR11 as a biomarker of ccRCC and predictor of poor prognosis by bioinformatics. Then, we showed that PRR11 silencing substantially reduced ccRCC cell proliferation and migration in vitro and in vivo. Importantly, we found that PRR11 induced the degradation of the E2F1 protein through its interaction with E2F1, and PRR11 reduced the stability of the E2F1 protein in ccRCC cells, thereby affecting cell cycle progression. Further results indicated that the downregulation of E2F1 expression partially reversed the changes in ccRCC cell biology caused by PRR11 deletion. In addition, we showed that PRR11 was a target gene of c-Myc. The transcription factor c-Myc may have promoted the expression of PRR11 in ccRCC cells by binding to the PRR11 promoter region, thereby accelerating the progression of ccRCC. In summary, we found that PRR11 served as an oncogene in ccRCC, and PRR11 reduced the protein stability of E2F1 and could be activated by c-Myc.