Role In ccRCC Research Articles

Elevated expression of LIF predicts a poor prognosis and promotes cell migration and invasion of clear cell renal cell carcinoma.

BackgroundRenal cell carcinoma (RCC) is the seventh most common cancer in humans, of which clear cell renal cell carcinoma (ccRCC) accounts for the majority. Recently, although there have been significant breakthroughs in the treatment of ccRCC, the prognosis of targeted therapy is still poor. Leukemia inhibitory factor (LIF) is a pleiotropic protein, which is overexpressed in many cancers and plays a carcinogenic role. In this study, we explored the expression and potential role of LIF in ccRCC.MethodsThe expression levels and prognostic effects of the LIF gene in ccRCC were detected using TCGA, GEO, ICGC, and ArrayExpress databases. The function of LIF in ccRCC was investigated using a series of cell function approaches. LIF-related genes were identified by weighted gene correlation network analysis (WGCNA). GO and KEGG analyses were performed subsequently. Cox univariate and LASSO analyses were used to develop risk signatures based on LIF-related genes, and the prognostic model was validated in the ICGC and E-MTAB-1980 databases. Then, a nomogram model was constructed for survival prediction and validation of ccRCC patients. To further explore the drug sensitivity between LIF-related genes, we also conducted a drug sensitivity analysis based on the GDSC database.ResultsThe mRNA and protein expression levels of LIF were significantly increased in ccRCC patients. In addition, a high expression of LIF has a poor prognostic effect in ccRCC patients. LIF knockdown can inhibit the migration and invasion of ccRCC cells. By using WGCNA, 97 LIF-related genes in ccRCC were identified. Next, a prognostic risk prediction model including eight LIF-related genes (TOB2, MEPCE, LIF, RGS2, RND3, KLF6, RRP12, and SOCS3) was developed and validated. Survival analysis and ROC curve analysis indicated that the eight LIF-related-gene predictive model had good performance in evaluating patients’ prognosis in different subgroups of ccRCC.ConclusionOur study revealed that LIF plays a carcinogenic role in ccRCC. In addition, we firstly integrated multiple LIF-related genes to set up a risk-predictive model. The model could accurately predict the prognosis of ccRCC, which offers clinical implications for risk stratification, drug screening, and therapeutic decision.

NCKAP1 is a Prognostic Biomarker for Inhibition of Cell Growth in Clear Cell Renal Cell Carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is the most frequent type of kidney cancer. Nck-associated protein 1 (NCKAP1) is associated with poor prognosis and tumor progression in several cancer types, but the function and prognostic value of NCKAP1 in ccRCC remain poorly understood. Methods: Using the Ualcan database, we evaluated the correlation between NCKAP1 expression and clinical features of ccRCC. These data were validated by immunohistochemical staining for NCKAP1 in a cohort of ccRCC patients. We assessed the prognostic value of NCKAP1 using GEPIA2 survival analysis. NCKAP1 function was characterized in vitro and in vivo using NCKAP1-overexpression ACHN cell lines. The LinkedOmics and GSCALite databases were used to investigate identify potential NCKAP1-targeted medicines that may play a role in the treatment of ccRCC. The impact of NCKAP1 expression on immune infiltration was also evaluated. Results: NCKAP1 was significantly downregulated in ccRCC and correlated with advanced clinicopathological features and poor prognosis. Overexpression of NCKAP1 in ACHN cells reduced proliferation, invasion and migration capacity in vitro and inhibited tumor growth in vivo. According to the LinkedOmics, GSCALite and TIMER databases, NCKAP1 and related genes function primarily in ribosomal signaling, oxidative phosphorylation, TGF-β, and EMT-related signaling pathways. NCKAP1 was also shown to positively correlate with immune cell types, biomarkers, and immune checkpoints in ccRCCs. Conclusions: NCKAP1 may play a vital tumor-suppressive role in ccRCC and is potentially a useful prognostic biomarker.

High expression of eIF4A1 predicts unfavorable prognosis in clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is a worldwide malignancy with high morbidity and mortality. Translation initiation factor 4A1 (eIF4A1), which is an ATP-dependent RNA helicase as a part of eIF4F complex, has been linked to malignant transformation and progression, and a variety of cancers display dysregulation of this enzyme. However, its role in ccRCC remains unclear. In our study, we examined its potential effects in ccRCC. MethodsBased on Proteomic data, TCGA and ONCOMINE database, RCC cell lines and tissues, the expression of eIF4A1 between ccRCC and normal tissues were investigated. A correlation was evaluated between the prognostic model for OS and ccRCC progression. Analysis of functional enrichment and PPI network were performed. After examining differentially expressed genes between the eIF4A1 high and low-expression groups, we performed GSEA analysis. Furthermore, we investigated immune cell infiltration of eIF4A1. Then we determined eIF4A1 functions in the establishment and maintenance of cell viability, migration and invasion of cell lines. Flow cytometry was utilized to detect cell cycle. ResultsThe eIF4A1 was up-regulated in ccRCC tissues and cell lines. An increased level of eIF4A1 was linked to lower survival rates and impaired immunity. Depletion of eIF4A1 could arrest tumor cells in G1 phase, so as to seriously limit cell proliferation and weaken the capacity of cell migration. ConclusionccRCC patients with high eIF4A1 expression are at increased risk of poor prognosis, furthermore eIF4A1 plays a prominent role in facilitating tumor cell proliferation and migration which may further be a potential prognostic biomarker and therapeutic target.

Epigenetically-regulated miR-30a/c-5p directly target TWF1 and hamper ccRCC cell aggressiveness

Clear cell renal cell carcinoma (ccRCC) is highly prone to metastasize and displays an extremely low 5-year survival rate. Not only miRNAs (miRs) are key gene expression regulators but can also be epigenetically modified. Abnormal miR expression has been linked with epithelial-mesenchymal transition (EMT)-driven ccRCC progression. MiR-30a/c-5p were found downregulated in ccRCC and associated with aggressiveness. Herein, we sought to unravel miR-30a/c-5p mechanistic role in ccRCC. RNA sequencing and genome-wide methylome data of ccRCC and normal tissue samples from The Cancer Genome Atlas database were integrated to identify candidate miRs cytosine-phosphate-guanine (CpG) loci deregulated in ccRCC. TargetScan was searched to identify miR putative targets. MiR-30a/c-5p expression and promoter methylation was evaluated in vitro, by PCR. Western blot, functional and luciferase assays were performed after cell transfection with either pre-miR, antimiR, or siRNA against twinfilin-1 (TWF1). Immunohistochemistry (IHC) was performed in ccRCC tissues. We found miR-30c-5p downregulation and aberrant promoter methylation in ccRCC tissues. In vitro studies revealed concomitant miR-30a/c-5p downregulation and increased promoter methylation, as well as a significant re-expression following decitabine treatment. Functional assays demonstrated that both miRs significantly decreased cell aggressiveness and the protein levels of EMT-promoting players, while upregulating epithelial markers, namely Claudin-1 and ZO-1. Importantly, we confirmed TWF1 as a direct target of both miRs, and its potential involvement in epithelial-mesenchymal transition/mesenchymal-epithelial transition regulation. IHC analysis revealed higher TWF1 expression in primary tissues from patients that developed metastases, after surgical treatment. Our results implicate miR-30a/c-5p in ccRCC cells' aggressiveness attenuation by directly targeting TWF1 and hampering EMT.

Molecular Subtypes Based on Genomic and Transcriptomic Features Correlate with the Responsiveness to Immune Checkpoint Inhibitors in Metastatic Clear Cell Renal Cell Carcinoma.

Simple SummaryImmune checkpoint inhibitors (ICIs), such as programmed cell death protein 1 (PD-1) blockade, have proven to be the most effective agents for the management of many cancer types. Although ICIs are the current standard of care for treating metastatic clear cell renal cell carcinoma (ccRCC), 40–60% of patients still have intrinsic resistance to ICIs across multiple clinical trials. Therefore, identifying optimal biomarkers that can predict either responders or non-responders to ICIs has been of tremendous importance. Here, we generated targeted sequencing and whole transcriptomic sequencing of 60 patients with metastatic ccRCC treated with ICIs. Moreover, transcriptomic analysis was integrated to identify molecular subtypes using a total of 177 tumor samples by merging our data and published data derived from the CheckMate 025 trial. Our results show that these molecular subtypes are associated with specific genomic alterations, distinct molecular pathways, and differential clinical outcomes in patients with metastatic ccRCC treated with ICIs.Clear cell renal cell carcinoma (ccRCC) has been reported to be highly immune to and infiltrated by T cells and has angiogenesis features, but the effect of given features on clinical outcomes followed by immune checkpoint inhibitors (ICIs) in ccRCC has not been fully characterized. Currently, loss of function mutation in PBRM1, a PBAF-complex gene frequently mutated in ccRCC, is associated with clinical benefit from ICIs, and is considered as a predictive biomarker for response to anti-PD-1 therapy. However, functional mechanisms of PBRM1 mutation regarding immunotherapy responsiveness are still poorly understood. Here, we performed targeted sequencing (n = 60) and whole transcriptomic sequencing (WTS) (n = 61) of patients with metastatic ccRCC treated by ICIs. By integrating WTS data from the CheckMate 025 trial, we obtained WTS data of 177 tumors and finally identified three molecular subtypes that are characterized by distinct molecular phenotypes and frequency of PBRM1 mutations. Patient clustered subtypes 1 and 3 demonstrated worse responses and survival after ICIs treatment, with a low proportion of PBRM1 mutation and angiogenesis-poor, but were immune-rich and cell-cycle enriched. Notably, patients clustered in the subtype 2 showed a better response and survival after ICIs treatment, with enrichment of PBRM1 mutation and metabolic programs and a low exhausted immune phenotype. Further analysis of the subtype 2 population demonstrated that GATM (glycine amidinotransferase), as a novel gene associated with PBRM1 mutation, plays a pivotal role in ccRCC by using a cell culture model, revealing tumor, suppressive-like features in reducing proliferation and migration. In summary, we identified that metastatic ccRCC treated by ICIs have distinct genomic and transcriptomic features that may account for their responsiveness to ICIs. We also revealed that the novel gene GATM can be a potential tumor suppressor and/or can be associated with therapeutic efficacy in metastatic ccRCC treated by ICIs.

PD01-06 GCN5L1 REGULATES MITOCHONDRIAL ACETYLATION, LIPID ACCUMULATION AND CELL GROWTH IN VHL-DEFICIENT CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD01-06 GCN5L1 REGULATES MITOCHONDRIAL ACETYLATION, LIPID ACCUMULATION AND CELL GROWTH IN VHL-DEFICIENT CLEAR CELL RENAL CELL CARCINOMA Bradley Webster, Allen Seylani, Michael Daneshvar, Alexis Rompre-Brodeur, and Ramaprasad Srinivasan Bradley WebsterBradley Webster More articles by this author , Allen SeylaniAllen Seylani More articles by this author , Michael DaneshvarMichael Daneshvar More articles by this author , Alexis Rompre-BrodeurAlexis Rompre-Brodeur More articles by this author , and Ramaprasad SrinivasanRamaprasad Srinivasan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002516.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lipid accumulation is pathognomonic for clear cell renal cell carcinoma (ccRCC) and occurs due to VHL loss and accumulation of HIF. General Control of Amino Acid Synthesis 5-like 1 (GCN5L1) augments mitochondrial protein acetylation and inhibits beta oxidation leading to lipid accumulation. Furthermore, VHL reconstitution lowers GCN5L1 transcript levels in VHL -/- ccRCC. We hypothesized that GCN5L1 regulates ccRCC lipid metabolism in a VHL-dependent manner. METHODS: GCN5L1 protein levels in various RCC subtypes were assessed. To determine if GCN5L1 expression is dependent on VHL or HIF, we reintroduced wtVHL in VHL -/- ccRCC, knocked down (KD) VHL, HIF1 or HIF2 in RPTEC cells or grew cells in hypoxia to assess GCN5L1 protein levels. GCN5L1 ubiquitination was determined by immunoprecipitation after VHL reconstitution. Mitochondria acetylation was assessed using purified mitochondria. Oil red O staining and cell proliferation were assessed after GCN5L1 KD. Beta oxidation was assessed via Seahorse. Finally, acetylation and activity levels of key beta oxidation enzymes were assessed after GCN5L1 KD. RESULTS: GCN5L1 protein level and mitochondrial acetylation were augmented in ccRCC compared to other RCC subtypes and was dependent on GCN5L1 expression. Re-expression of wtVHL in ccRCC attenuated, while VHL KD, but not HIF1 & 2 KD, in RPTEC augmented levels of GCN5L1. GCN5L1 was not induced in RPTEC under hypoxia. Further, GCN5L1 ubiquitination in ccRCC was elevated with wtVHL reconstitution. Thus, VHL, and not HIF, appears to directly regulate GCN5L1 levels. Functionally, GCN5L1 KD in ccRCC led to increased levels of CPT1a and lower lipid levels as seen with Oil-red O stain. Furthermore, GCN5L1 KD selectively inhibited growth of ccRCC but not other RCC subtypes/RPTEC. Finally, GCN5L1 KD attenuated acetylation levels of HADHA, LCAD and SCAD and augmented beta oxidation. CONCLUSIONS: VHL loss augments GCN5L1 levels through lost ubiquitin mediated degradation. Thus, GCN5L1 and mitochondrial acetylation levels are selectively elevated in VHL-deficient ccRCC. GCN5L1 KD also reverses ccRCC repression of CPT1a, attenuates acetylation (augmenting activity) of key beta oxidation enzymes leading to decreased lipid accumulation. Finally, cell proliferation is selectively attenuated in ccRCC with GCN5L1 KD. GCN5L1 appears to play a unique role in ccRCC vs. non-ccRCC subtypes. Future work will examine GCN5L1’s role in mouse xenografts and patient tumors further highlighting the selective importance of GCN5L1 in ccRCC tumor biology. Source of Funding: NIH © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e31 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bradley Webster More articles by this author Allen Seylani More articles by this author Michael Daneshvar More articles by this author Alexis Rompre-Brodeur More articles by this author Ramaprasad Srinivasan More articles by this author Expand All Advertisement PDF DownloadLoading ...

Survival Prognosis, Tumor Immune Landscape, and Immune Responses of ADAMTS14 in Clear Cell Renal Cell Carcinoma and Its Potential Mechanisms

BackgroundADAMTS14 played a crucial role in the formation and development of various cancers. Currently, no associations had been revealed between ADAMTS14 and clear cell renal cell carcinoma (ccRCC). Hence, this study was designed to assess the prognostic values and immunological roles of ADAMTS14 in ccRCC and to reveal its potential mechanisms.MethodsADAMTS14-related expression profiles and related clinical data were downloaded from The Cancer Genome Atlas (TCGA) dataset, validated by the ICGC dataset, qRT-PCR, and immunohistochemistry. We utilized gene set enrichment analysis (GSEA) to find potentially ADAMTS14-related pathways and applied univariate/multivariate Cox regression analyses to identify independent factors significantly related to overall survival (OS) for ccRCC. A nomogram consisted of independent prognostic factors was also conducted. We further explored the associations between ADAMTS14 with immunity and revealed its potential mechanisms.ResultsADAMTS14 displayed a higher expression in ccRCC tumor than in adjacent normal tissues, and further validated results of the ICGC dataset; qRT-PCR and immunohistochemistry remained consistent (all p < 0.05). Moreover, elevated ADAMTS14 expression was significantly associated with poor OS (p < 0.001). Through univariate/multivariate Cox regression analyses, ADAMTS14 was found to be an independent prognostic factor for ccRCC (both p < 0.05) and GSEA identified several signaling pathways including INSULIN, MTOR, and PPAR pathways. The nomogram based on independent prognostic factors was successfully established and well evaluated. Moreover, the expression of ADAMTS14 was remarkably associated with immune checkpoint molecules, tumor mutational burden (TMB), immune cells, and tumor immune microenvironment (all p < 0.05). Results from TIDE and TCIA showed that highly expressed ADAMTS14 could predict worse efficacy of immunotherapy (all p < 0.05). As for its potential mechanisms, we also revealed several LncRNA/RNA binding protein (RBP)/ADAMTS14 mRNA networks.ConclusionsADAMTS14 was found to play oncogenic roles in ccRCC and to be significantly associated with immunity. Several LncRNA/RBP/ADAMTS14 mRNA networks were also identified for its potential mechanisms.

LINC01535 Attenuates ccRCC Progression through Regulation of the miR-146b-5p/TRIM2 Axis and Inactivation of the PI3K/Akt Pathway.

lncRNAs, a group of eukaryotic cell genome-encoded transcripts, have been demonstrated to exert a notable impact on tumorigenesis. LINC01535, belonging to the lncRNA family, was reported to have an aberrant expression in certain types of cancers and thus affect cancer progression. Nevertheless, the expression pattern and potential roles of LINC01535 in clear cell renal cell carcinoma (ccRCC) remain to be elucidated. Here, LINC01535 expression was detected in ccRCC by RT-qPCR, cell proliferation by CCK-8 assays, and invasion by transwell assays. Besides, effects of LINC01535 on in vivo tumor growth were investigated by xenograft tumor models. The miR-146b-5p/LINC01535/TRIM2 interaction was evaluated via luciferase reporter assays. This study showed downregulation of LINC01535 in ccRCC. Moreover, LINC01535 upregulation attenuated in vitro ccRCC development and hindered in vivo tumor growth. Furthermore, LINC01535 sponged miR-146b-5p which had a negative correlation with LINC01535, and TRIM2 was a direct target of miR-146b-5p and mediated by LINC01535. Mechanically, LINC01535/miR-146b-5p/TRIM2 axis affected ccRCC progression by mediating the PI3K/Akt signaling. All in all, our observations suggest the LINC01535/miR-146b-5p/TRIM2 axis as a crucial role 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.

Molecular subtypes of m6A RNA methylation modification patterns and their clinical significance in clear cell renal cell carcinoma.

BackgroundIn this study, we sought to investigate the association between N6-methyladenosine (m6A) RNA methylation-modification patterns and patient prognosis in clear cell renal cell carcinoma (ccRCC) and construct a ccRCC molecular signature according to expressions of m6A-related genes.MethodsFirst, the clinical data and the transcriptomes of 530 patients with ccRCC were downloaded from The Cancer Genome Atlas (TCGA). The expression patterns of m6A-related genes were extracted, and the differences in m6A-modification patterns between normal and tumor renal tissues were analyzed. To explore the prognostic role of m6A-modification patterns a in ccRCC, the molecular subtypes of ccRCC were identified based on the expression patterns of the m6A-related genes, and survival rates in patients with the different subtypes were compared. According to expressions of m6A-related genes and clinical prognosis data, a prognostic molecular signature was constructed using least absolute shrinkage and selector operation (LASSO)-Cox regression analysis.ResultsAmong the 13 m6A -related genes identified in this study, 8 (YTHDC1, YTHDF2, HNRNPC, METTL14, ZC3H13, FTO, YTHDC2, and YTHDF1) showed significant expression differences between normal and tumor renal tissues. The molecular subtypes of ccRCC identified according to their expression of the 13 m6A-related genes were associated with differential clinical outcomes.ConclusionsFollowing TCGA data-mining, different molecular subtypes of ccRCC based on m6A RNA methylation patterns were found to have different prognoses. The molecular signature constructed according to the expression patterns of m6A-related genes could predict patient prognosis in ccRCC. We believe m6A RNA methylation modification is a potential therapeutic target and may play a crucial role in ccRCC.

NUF2 Drives Clear Cell Renal Cell Carcinoma by Activating HMGA2 Transcription through KDM2A-mediated H3K36me2 Demethylation.

The poor sensitivity of clear cell renal cell carcinoma (ccRCC) to conventional chemotherapy and radiotherapy makes its treatment challenging. The Ndc80 kinetochore complex component (NUF2) is involved in the development and progression of several cancers. However, its role in ccRCC remains unclear. In this study, we investigated the biological functions and underlying mechanism of NUF2 in ccRCC. We found that NUF2 expression was increased in ccRCC and associated with poor prognosis. Altering NUF2 level affected cell proliferation, migration, and invasion. Moreover, NUF2 acted as a potential oncogene to promote the progression of ccRCC through epigenetic activation of high-mobility group AT-hook 2 (HMGA2) transcription by suppressing lysine demethylase 2A expression and affecting its occupancy on the HMGA2 promoter region to regulate histone H3 lysine 36 di-methylation modification. In addition, Kaplan-Meier and multivariate analysis revealed that patients whose NUF2 and HMGA2 were both elevated showed the shortest survival; and the number of upregulated markers acted as an independent predictor to evaluate survival probability. Thus, our results demonstrate that NUF2 promotes ccRCC progression, at least partly by epigenetically regulating HMGA2 transcription, and that the NUF2-HMGA2 axis could be an ideal therapeutic target and a promising prognostic indicator for ccRCC.

Prostate-specific membrane antigen expression in clear-cell renal cell carcinoma

Background Clear-cell renal cell carcinoma (ccRCC) represents the majority of renal neoplasms with usual late presentation. Metastasis of ccRCC is very common owing to the vascular nature of this tumor. Identifying markers that could predict tumor behavior and metastatic potential is crucial to improve patient prognosis. Prostate-specific membrane antigen (PSMA) is a recently identified vascular marker that is expressed in many cancer types. This study aimed at evaluation of its clinicopathologic role in ccRCC. Materials and methods A total of 40 cases of ccRCC were recruited and subjected to immunohistochemical staining for PSMA. Statistical analysis was performed to assess its expression and its relationship with different clinicopathologic parameters. Results PSMA expression was positive in 72.5% of all cases. High expression was detected in 52.5% of cases, with positive correlation with high tumor grade (P=0.01), stage (0.04), and lymphovascular invasion (P=0.03). PSMA expression was positive in 83.3% of cases of tumor thrombus tissue, and 50% of these cases showed high expression. Two (50%) cases of metastatic lesions showed low positive expression for PSMA with high PSMA expression in 75% of the corresponding primary lesions. Conclusion PSMA is specifically expressed in the vasculature of ccRCC, showing a positive correlation with poor prognostic parameters. This renders PSMA as a promising prognostic marker and therapeutic target.

Integrative Bioinformatics Analysis Demonstrates the Prognostic Value of Chromatin Accessibility Biomarkers in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for 75%–85% of renal cell carcinoma (RCC) and has a poor 5-year survival rate. In recent years, medical advancement has promoted the understanding of the histopathological and molecular characterization of ccRCC; however, the carcinogenesis and molecular mechanisms of ccRCC remain unclear. Chromatin accessibility is an essential determinant of cellular phenotype. This study aimed to explore the potential role of chromatin accessibility in the development and progression of ccRCC. By the combination of open-access genome-wide chromatin accessibility profiles and gene expression profiles in ccRCC, we obtained a total of 13,474 crucial peaks, corresponding to 5,120 crucial genes and 9,185 differentially expressed genes. Moreover, two potential function modules (P2 and G4) that contained 129 upregulated genes were identified via the weighted gene co-expression network analysis (WGCNA). Furthermore, we obtained five independent predictors (FSCN1, SLC17A9, ANKRD13B, ADCY2, and MAPT), and a prognostic model was established based on these genes through the least absolute shrinkage and selection operator-proportional hazards model (LASSO-Cox) analysis. This model can stratify the ccRCC samples into a high-risk and a low-risk group, from which the patients have distinct prognosis. Further analysis demonstrated a completely different immune cell infiltration pattern between these two risk groups. This study also suggested that mast cell resting is associated with the prognosis of ccRCC and could be a target of immunotherapy. Overall, this study indicated that chromatin accessibility plays an essential role in ccRCC. The five prognostic chromatin accessibility biomarkers and the prognostic immune cells can provide a new direction for the treatment of ccRCC.

Upregulated GSDMB in Clear Cell Renal Cell Carcinoma Is Associated with Immune Infiltrates and Poor Prognosis.

Gasdermin B (GSDMB) is part of the gasdermin (GSDM) family, and they use varying means of domain interactions in molecules to adjust their pore-forming and lipid-binding actions. The GSDM family has roles in the regulation of cell differentiation and proliferation, particularly in the process of pyroptosis. Nonetheless, the correlation of GSDMB with immune infiltrates and its prognostic values in clear cell renal cell carcinoma (ccRCC) are still undefined. Therefore, we assessed the correlation of GSDMB with immune infiltrates and its prognostic role in ccRCC. The transcriptional expression profiles of GSDMB in ccRCC tissues in addition to normal tissues were retrieved from The Cancer Genome Atlas (TCGA) and additionally verified in a different independent cohort, which was obtained from the Gene Expression Omnibus (GEO) database. The Human Protein Atlas and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) were used to assess the protein expression of GSDMB. To assess the effectiveness of GSDMB in distinguishing ccRCC from normal samples, the receiver operating characteristic (ROC) curve analysis was performed. Relationships between GSDMB expression, clinicopathological variables, and overall survival (OS) were evaluated with multivariate methods as well as Kaplan-Meier survival curves. Protein-protein interaction (PPI) networks were created with STRING. Functional enrichment analyses were conducted by utilizing the “ClusterProfiler” package. The Tumor Immune Estimation Resource (TIMER) and tumor-immune system interaction database (TISIDB) were utilized to determine the association between the mRNA expression of GSDMB and immune infiltrates. GSDMB expression was significantly more upregulated in ccRCC tissues compared to surrounding normal tissues. An increase in the mRNA expression of GSDMB was related to the high pathologic stage and advanced TNM stage. The analysis of the ROC curve indicated that GSDMB had an AUC value of 0.820 to distinguish between ccRCC tissues and adjacent normal controls. Kaplan-Meier survival analysis indicated that ccRCC patients with high GSDMB had a poorer prognosis compared to those with low GSDMB (P < 0.001). Correlation analysis showed that the mRNA expression of GSDMB was associated with immune infiltrates and the purity of the tumor. Upregulation of GSDMB is significantly related to immune infiltrates and poor survival in ccRCC. The results of this study indicate that GSDMB could be regarded as a biomarker for the detection of poor prognosis and potential target of immune treatment in ccRCC.

Mitochondrial Ndufa4l2 Enhances Deposition of Lipids and Expression of Ca9 in the TRACK Model of Early Clear Cell Renal Cell Carcinoma.

Mitochondrial dysfunction and aberrant glycolysis are hallmarks of human clear cell renal cell carcinoma (ccRCC). Whereas glycolysis is thoroughly studied, little is known about the mitochondrial contribution to the pathology of ccRCC. Mitochondrial Ndufa4l2 is predictive of poor survival of ccRCC patients, and in kidney cancer cell lines the protein supports proliferation and colony formation. Its role in ccRCC, however, remains enigmatic. We utilized our established ccRCC model, termed Transgenic Cancer of the Kidney (TRACK), to generate a novel genetically engineered mouse model in which dox-regulated expression of an shRNA decreases Ndufa4l2 levels specifically in the renal proximal tubules (PT). This targeted knockdown of Ndufa4l2 reduced the accumulation of neutral renal lipid and was associated with decreased levels of the ccRCC markers carbonic anhydrase 9 (CA9) and Enolase 1 (ENO1). These findings suggest a link between mitochondrial dysregulation (i.e. high levels of Ndufa4l2), lipid accumulation, and the expression of ccRCC markers ENO1 and CA9, and demonstrate that lipid accumulation and ccRCC development can potentially be attenuated by inhibiting Ndufa4l2.

Prognostic value and immunological role of AXL gene in clear cell renal cell carcinoma associated with identifying LncRNA/RBP/AXL mRNA networks

BackgroundsThis article aimed to explore the prognostic and immunological roles of AXL gene in clear cell renal cell carcinoma (ccRCC) for overall survival (OS) and to identify the LncRNA/RBP/AXL mRNA networks.MethodsAXL-related gene expression matrix and clinical data were obtained from The Cancer Genome Atlas (TCGA) dataset and AXL-related pathways were identified by gene set enrichment analysis (GSEA). We performed univariate/multivariate Cox regression analysis to evaluate independent prognostic factors and the relationships between AXL and immunity were also investigated.ResultsThe outcomes of us indicated that the AXL mRNA expression was up-regulated in ccRCC samples and high expression of AXL was associated with worse OS in TCGA dataset (P < 0.01). Further external verification results from HPA, UALCAN, ICGC dataset, GSE6344, GSE14994, and qRT-PCR remained consistent (all P < 0.05). AXL was also identified as an independent prognostic factor for ccRCC by univariate/multivariate Cox regression analysis (both P < 0.05). A nomogram including AXL expression and clinicopathological factors was established by us and GSEA results found that elevated AXL expression was associated with the JAK-STAT, P53, WNT, VEGF and MAPK signaling pathways. In terms of immunity, AXL was dramatically linked to tumor microenvironment, immune cells, immune infiltration, immune checkpoint molecules and tumor mutational burden (TMB). As for its potential mechanisms, we also identified several LncRNA/RBP/AXL mRNA axes.ConclusionsAXL was revealed to play prognostic and immunological roles in ccRCC and LncRNA/RBP/AXL mRNA axes were also identified by us for its potential mechanisms.

Integrated analysis on the N6-methyladenosine-related long noncoding RNAs prognostic signature, immune checkpoints, and immune cell infiltration in clear cell renal cell carcinoma.

BackgroundPatients with advanced clear cell renal cell carcinoma (ccRCC) have a poor prognosis and lack effective prognostic biomarkers. N6‐methyladenosine‐related lncRNAs (m6A‐related long noncoding RNAs [lncRNAs]) have been confirmed to be associated with the development of multiple tumors, but its role in ccRCC is not clear.MethodsGene expression data and clinical information of ccRCC patients were extracted from The Cancer Genome Atlas Database. The prognostic m6A‐related lncRNAs were obtained by Pearson's correlation analysis and univariate Cox regression analysis. Afterward, the cluster classification and its correlation with prognosis, clinical characteristics, and immunity were analyzed. LASSO regression was used to establish the prognostic risk model. The predictive performance of the prognostic model was evaluated and validated by survival analysis and receiver operating characteristic curve analysis, et al. The expression of immune checkpoints and immune cell infiltration in patients with different risks were systematically analyzed.ResultsA total of 27 prognostic m6A‐related lncRNAs were identified. These m6A‐related lncRNAs were differentially expressed between tumor and normal tissues. Among them, 24 high‐risk m6A‐related lncRNAs were overexpressed in Cluster 2 and correlated with poor prognosis, low stromal score, high expression of immune checkpoints, and immunosuppressive cells infiltration. Based upon, a prognostic risk model composed of seven m6A‐related lncRNAs was constructed. After a series of analyses, it was proved that this model had good sensitivity and specificity, and could predict the prognosis of patients with different clinical stratification. The expression of PD‐1, PD‐L1, CTLA‐4, LAG‐3, TIM‐3, and TIGIT were significantly increased in the high‐risk patients, and there was a correlation between the risk score and immune cell infiltration.ConclusionsThe seven m6A‐related lncRNAs prognostic risk signature showed reliable prognostic predictive power for ccRCC and was associated with the expression of immune checkpoints and immune cell infiltration. This seven m6A‐related lncRNAs signature will be helpful in managing ccRCC and guiding individualized immunotherapy.

Upregulation of ARNTL2 is associated with poor survival and immune infiltration in clear cell renal cell carcinoma

BackgroundAryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) is a member of the PAS superfamily. Previous studies explored the carcinogenic roles of transcription factor ARNTL2 in human malignancies. However, its roles in ccRCC have not been elucidated. This study sought to explore the roles of ARNTL2 in ccRCC and determine its correlations with tumor immunity.MethodsThe expression of ARNTL2 was analyzed using the GEO, TCGA and GTEx database, and verified in ccRCC tissue samples and cell lines by qRT-PCR and western blot analysis. Kaplan–Meier survival curve analysis, Cox regression analysis (including univariate and multivariate analysis) was utilized to evaluate the prognostic values of ARNTL2. Potential biological mechanisms of ARNTL2 were explored using GSEA method. Colony formation and wound healing assays were conducted to explore the oncogenic role of ARNTL2 in ccRCC. ssGSEA and xCell algorithm were used to explore the correlation between ARNTL2 expression and tumor immune microenvironment (TIME).ResultsARNTL2 was significantly upregulated in ccRCC tissues and cell lines compared to normal kidney tissues and cell line. Enhanced expression of ARNTL2 was strongly linked to advanced clinical stage and unfavorable overall survival in ccRCC. ARNTL2 was determined as an independent prognostic marker through cox regression analysis. A prognostic nomogram was constructed to predict 1-, 3- and 5-year overall survival of ccRCC patients by integrating ARNTL2 expression with other clinicopathologic variables. GSEA analysis showed that focal adhesion, T cell receptor, cell cycle, and JAK-STAT signaling pathway were significantly enriched in high ARNTL2 samples. Silencing of ARNTL2 suppressed the colony formation ability and wound healing efficacy of ccRCC cell lines. xCell analysis showed that high expression level of ARNTL2 exhibited an immune infiltration status similar to CD8 + inflamed ccRCC subtype, which was characterized by high infiltration level of CD8 + T cell and high expression level of the immune escape biomarkers such as PD-L1, PD-L2, PD1 and CTLA4.ConclusionARNTL2 is an independent adverse predictor of ccRCC patient survival. High expression level of ARNTL2 is associated with immune infiltration, and may be a novel therapeutic target in ccRCC.