ccRCC Cell Lines Research Articles

AC010973.2 promotes cell proliferation and is one of six stemness-related genes that predict overall survival of renal clear cell carcinoma

Extensive research indicates that tumor stemness promotes tumor progression. Nonetheless, the underlying roles of stemness-related genes in renal clear cell carcinoma (ccRCC) are unclear. Data used in bioinformatics analysis were downloaded from The Cancer Genome Atlas (TCGA) database. Moreover, the R software, SPSS, and GraphPad Prism 8 were used for mapping and statistical analysis. First, the stemness index of each patient was quantified using a machine learning algorithm. Subsequently, the differentially expressed genes between high and low stemness index were identified as stemness-related genes. Based on these genes, a stable and effective prognostic model was identified to predict the overall survival of patients using a random forest algorithm (Training cohort; 1-year AUC: 0.67; 3-year AUC: 0.79; 5-year AUC: 0.73; Validation cohort; 1-year AUC: 0.66; 3-year AUC: 0.71; 5-year AUC: 0.7). The model genes comprised AC010973.2, RNU6-125P, AP001209.2, Z98885.1, KDM5C-IT1, and AL021368.3. Due to its highest importance evaluated by randomforst analysis, the AC010973.2 gene was selected for further research. In vitro experiments demonstrated that AC010973.2 is highly expressed in ccRCC tissue and cell lines. Meanwhile, its knockdown could significantly inhibit the proliferation of ccRCC cells based on colony formation and CCK8 assays. In summary, our findings reveal that the stemness-related gene AC01097.3 is closely associated with the survival of patients. Besides, it remarkably promotes cell proliferation in ccRCC, hence a novel potential therapeutic target.

SPRR3, a novel miR-338-3p target, regulates the malignant progression of clear cell renal cell carcinoma in vitro via the PI3K/Akt signaling pathway.

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma and has a poor prognosis. However, its underlying mechanisms remain unclear. The present study aimed to evaluate the role of small proline-rich repeat protein 3 (SPRR3) in the proliferation, migration and invasion of ccRCC cells and to investigate its upstream and downstream regulatory mechanisms. Survival analysis was performed using the UALCAN website based on the The Cancer Genome Atlas database. Normal renal cell line HK-2 and ccRCC cell lines (786-O, CaKi-1 and UMRC-2) were used. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect mRNA and microRNA (miRNA) levels. Western blotting was used to detect protein levels. Cell Counting Kit-8 and colony formation assays, a wound healing assay and a Transwell invasion assay were used to assess the proliferation, migration and invasion of ccRCC cells, respectively. Transfection of overexpression plasmids and small interfering RNAs were used to upregulate and knockdown SPRR3 expression, respectively. Transfection of miRNA-mimics was used to overexpress miR-338-3p. A luciferase reporter gene assay was used to verify the predicted binding relationship between SPRR3 mRNA and miR-338-3p. The results indicated the following: i) SPRR3 was a risk factor for the survival of patients with ccRCC, and was upregulated in ccRCC cell lines; ii) SPRR3 promoted the proliferation, migration and invasion of ccRCC cells; iii) SPRR3 regulated the tumor phenotypes of ccRCC cells via the PI3K/Akt pathway; iv) miR-338-3p directly targeted SPRR3 mRNA and negatively regulated SPRR3 expression; and v) miR-338-3p inhibited the PI3K/Akt pathway and the tumor phenotypes of ccRCC cells by downregulating SPRR3. In conclusion, SPRR3, as a novel target of miR-338-3p, regulated the proliferation, migration and invasion of ccRCC cells via the PI3K/Akt pathway; this finding not only enriches our understanding of the mechanism underlying ccRCC development, but also demonstrates a potential novel therapeutic target for this disease.

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.

Analysis of Ferroptosis-Related LncRNAs Signatures Associated with Tumor Immune Infiltration and Experimental Validation in Clear Cell Renal Cell Carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is the most aggressive subtype of renal cell carcinoma. Ferroptosis is an iron-dependent programmed cell death. Long non-coding RNAs (lncRNAs) emerge as a critical role in regulating cancer progression.ObjectiveThis study aimed to identify molecular regulation of ferroptosis-related lncRNAs (FRLs) in ccRCC.MethodsThe prognostic value of FRLs was investigated in ccRCC samples downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) dataset. The FRLs were screened out by Pearson correlation test. The 465 FRLs confirmed as potential prognostic factors through univariate Cox regression analysis were entered into Lasso and multivariate Cox regression to build a FRLs prognostic signature. A risk score based on the prognostic model divided ccRCC patients into low- and high-risk groups. A prognostic nomogram, derived from the prognostic signature and integrating clinical characteristics, was constructed. Gene set enrichment analysis (GSEA) revealed the immune- and tumor-associated pathways. Two distinct clusters were identified with different immune signatures through consensus clustering analysis. The prognostic value of some hub FRLs was externally validated via three GEO datasets (GSE46699, GSE53757 and GSE66272) and online databases. Finally, the three FRLs (LINC00460, LINC00941 and LINC02027) were verified through in vitro experiments.ResultsThe FRLs prognostic signature, including 7 independent prognostic lncRNAs, exhibited good accuracy in predicting overall survival (OS) of ccRCC patients. This signature was correlated with immune infiltration and immune checkpoint blockade (ICB). We correlated two distinct clusters with immune infiltration signature of ccRCC. The worse prognosis of cluster 2 was probably mediated by immune evasion. We also found that the expression levels of LINC00460 and LINC00941 in ccRCC cell lines were higher than those in HK-2 cells, but LINC02027 showed the inverse trend.ConclusionCollectively, our study demonstrated a FRLs prognostic signature which had great clinical value in prognosis assessment.

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.

Proteogenomic and clinical implications of unique recurrent splice variants in clear cell renal cell carcinoma.

380 Background: Alternative mRNA splicing is recognized as a key driver of proteomic diversity. In cancer, this splicing process can be altered resulting in generation of aberrant splice variants (SvPs) that can contribute to tumor pathogenesis. However, our understanding of the significance of splice variants in clear cell renal cell carcinoma (ccRCC) is currently limited. Given the lack of actionable genomic mutations in ccRCC, aberrant SpVs may be the avenue to new pathogenic mechanisms and biomarkers. Methods: We implemented a novel pipeline to screen for and select SpVs frequent in and relatively specific to ccRCC. We started with RNA-seq data from the Cancer Cell Line Encyclopedia to identify SpVs specific to ccRCC cell lines. These were then screened across normal tissue in the Genotype-Tissue Expression Project (GTEx) and excluded if expressed. We analyzed bulk RNA-seq data of ccRCC primary tumors obtained from our institutional Total Cancer Care cohort (TCC; n = 111), The Cancer Genome Atlas (TCGA; n = 484) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC; n = 110) to analyze SpV expression in these samples. Using raw proteomics files from the CPTAC portal, proteins were identified and quantified using MaxQuant. Associations of SvP with protein expression were filtered by a Spearman correlation cutoff of +/-0.3. The Enrichr R library was used for pathway enrichment. Finally, we correlated splice variant expression with overall (OS) and cancer-specific survival (CSS). Using LASSO Cox regression analysis, we derived a SpV-based risk score trained on OS from the TCGA cohort and validated on the TCC and CPTAC cohorts. Results: Our pipeline selected 16 previously uncharacterized SpVs, including variants of suspected oncogenes and tumor suppressors. Proteogenomic analysis identified interesting biological associations. Among patients with high levels of EGFR SpV, we found significantly higher expression of the protein regulatory T cell marker CD70 (padj = 0.03). MVK SvP was highly correlated with 25 proteins enriched for the mTOR pathway (padj = 0.002). We derived a survival risk score based on expression of 5 SpVs ( PDZD2, COBLL1, PTPN14, RNASET2, FGD1) in the TCGA cohort. This risk score remained significant on multivariate analysis (HR 1.4, p = 0.002) adjusting for covariates including AJCC stage. This was validated on multivariate analysis in the TCC (HR 3.56, p < 0.001) and CPTAC (HR 3.18, p = 0.019) cohorts. Conclusions: Our novel pipeline selected 16 unique SpVs frequent in and relatively specific for ccRCC. Some are associated with proteins expressed in oncogenic pathways, suggesting a potential role in disease pathogenesis. Additionally, our SpV-based risk score is strongly associated with OS and CSS across multiple cohorts. This study provides a template for identifying and characterizing disease-specific aberrant SpVs to aid discovery of new mechanisms and biomarkers.

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.

PTEN loss confers sensitivity to rapalogs in clear cell renal cell carcinoma.

Rapalogs (everolimus and temsirolimus) are allosteric mTORC1 inhibitors and approved agents for advanced clear cell renal cell carcinoma (ccRCC), although only a subset of patients derive clinical benefit. Progress in genomic characterization has made it possible to generate comprehensive profiles of genetic alterations in ccRCC; however, the correlations between recurrent somatic mutations and rapalog efficacy remain unclear. Here, we demonstrate by using multiple patient-derived ccRCC cell lines that compared to PTEN-proficient cells, PTEN-deficient cells exhibit hypersensitivity to rapalogs. Rapalogs inhibit cell proliferation by inducing G0/G1 arrest without inducing apoptosis in PTEN-deficient ccRCC cell lines. Using isogenic cell lines generated by CRISPR/Cas9, we validate the correlation between PTEN loss and rapalog hypersensitivity. In contrast, deletion of VHL or chromatin-modifying genes (PBRM1, SETD2, BAP1, or KDM5C) fails to influence the cellular response to rapalogs. Our mechanistic study shows that ectopic expression of an activating mTOR mutant (C1483F) antagonizes PTEN-induced cell growth inhibition, while introduction of a resistant mTOR mutant (A2034V) enables PTEN-deficient ccRCC cells to escape the growth inhibitory effect of rapalogs, suggesting that PTEN loss generates vulnerability to mTOR inhibition. PTEN-deficient ccRCC cells are more sensitive to the inhibitory effects of temsirolimus on cell migration and tumor growth in zebrafish and xenograft mice, respectively. Of note, PTEN protein loss as detected by immunohistochemistry is much more frequent than mutations in the PTEN gene in ccRCC patients. Our study suggests that PTEN loss correlates with rapalog sensitivity and could be used as a marker for ccRCC patient selection for rapalog therapy.

Identification of the Expression and Clinical Significance of E2F Family in Clear Cell Renal Cell Carcinoma.

BackgroundMultiple studies have identified that E2F transcriptions act as important regulators for the tumorigenesis and progression of several human cancers. However, little is known about the function of E2Fs in clear cell renal cell carcinoma (ccRCC).MethodsWe firstly investigated the expression levels, genetic alteration, and biological function of E2Fs in patients with ccRCC and the connections between the immune cell infiltration and the overall survivals of ccRCC patients with the E2Fs expression levels based on UALCAN, The Cancer Genome Atlas database, Gene Expression Profiling Interactive Analysis, TIMER, STRING, GSCALite and cBioPortal databases.ResultsResults revealed that the expression levels of E2F1/2/3/4/6/7/8 were markedly upregulated in patients with ccRCC, while the expression of E2F5 displayed an opposite trend. We also experimentally validated the overexpression of E2F3/4/7 in human ccRCC tissues and ccRCC cell lines. Furthermore, the high E2F1/2/3/4/7/8 expression levels were clearly associated with worse pathological characteristics of ccRCC, including high pathological stage, poor molecular subtypes and high tumor grade. Meanwhile, high expression levels of E2F1/2/4/7/8 were evidently associated with worse overall survivals (OSs) and progression-free survivals (PFSs) of patients harboring ccRCC. Univariate and multivariate analyses illustrated that the expressions of E2F4/5/7 were independent factors associated with the OSs and PFSs of patients with ccRCC. Meanwhile, the mutations in E2Fs were also significantly related to poor OSs and PFSs of patients with ccRCC. Mechanically, the E2Fs genes synergistically promoted the progression of ccRCC by accelerating the cell cycle and inhibiting DNA damage response and apoptosis after performing the protein structure, functional enrichment, and PPI network analyses. In addition, E2Fs genes were also significantly associated with tumor immune cells infiltration and the drug sensitivity in ccRCC.ConclusionAs a result, E2F4/7 were highly expressed in ccRCC and significantly associated with worse pathological characteristics of ccRCC, including high pathological stage, poor molecular subtypes and high tumor grade, tumor immune cell infiltration, and drug sensitivity, consequently translating into poor OSs and PFSs of patients with ccRCC. Our results indicated that E2F4/7 could be potential biomarkers and therapeutic targets of ccRCC patients.

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.

Spindle pole body component 24 homolog potentiates tumor progression via regulation of SRY-box transcription factor 2 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of human kidney cancer with a high probability of metastasis. To understand the molecular processing essential for ccRCC tumorigenicity, we conducted an integrative in silico analysis of The Cancer Genome Atlas (TCGA) ccRCC dataset and clustered randomly interspersed short palindromic repeats (CRISPR) screening dataset of ccRCC cell lines from Depmap. We identified spindle pole body component 24homolog (SPC24) as an essential gene for ccRCC cell lines with prognostic significance in the TCGA database. Targeting SPC24 by CRISPR/Cas9-mediated gene knockout attenuated ccRCC proliferation, metastasis, and in vivo tumor growth. Furthermore, we found that SPC24 regulates metastasis genes expression in a SRY-box transcription factor 2 (SOX2)-dependent manner. The anti-proliferative effects of SPC24knockout were strengthened with SOX2knockdown. Collectively, our findings suggest SPC24has a pivotal function in promoting ccRCC progression, providing a new insight for the treatment of ccRCC.

Key genes associated with prognosis and metastasis of clear cell renal cell carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is a tumor that frequently shows the hematogenous pathway and tends to be resistant to radiotherapy and chemotherapy. However, the exact mechanism of ccRCC metastasis remains unknown.MethodsDifferentially expressed genes (DEGs) of three gene expression profiles (GSE85258, GSE105288 and GSE22541) downloaded from the Gene Expression Omnibus (GEO) database were analyzed by GEO2R analysis, and co-expressed DEGs among the datasets were identified using a Venn drawing tool. The co-expressed DEGs were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and hub genes were determined based on the protein-protein interaction network established by STRING. After survival analysis performed on UALCAN website, possible key genes were selected and verified in ccRCC cell lines and ccRCC tissues (n = 44). Statistical analysis was conducted using GraphPad Prism (Version 8.1.1).ResultsA total of 104 co-expressed DEGs were identified in the three datasets. Pathway analysis revealed that these genes were enriched in the extracellular matrix (ECM)–receptor interaction, protein digestion and absorption and focal adhesion. Survival analysis on 17 hub genes revealed that four key genes with a significant impact on survival: procollagen C-endopeptidase enhancer (PCOLCE), prolyl 4-hydroxylase subunit beta (P4HB), collagen type VI alpha 2 (COL6A2) and collagen type VI alpha 3 (COL6A3). Patients with higher expression of these key genes had worse survival than those with lower expression. In vitro experiments revealed that the mRNA expression levels of PCOLCE, P4HB and COL6A2 were three times higher and that of COL6A3 mRNA was 16 times higher in the metastatic ccRCC cell line Caki-1 than the corresponding primary cell line Caki-2. Immunohistochemistry revealed higher expression of the proteins encoded by these four genes in metastatic ccRCC compared with tumors from the corresponding primary sites, with statistical significance.ConclusionPCOLCE, P4HB, COL6A2 and COL6A3 are upregulated in metastatic ccRCC and might be related to poor prognosis and distant metastases.

The prognostic value of miR-487a in clear cell renal cell carcinoma and its influence on cell biological behavior.

Clear cell renal cell carcinoma (ccRCC) is a type of kidney cancer that has a poor prognosis. microRNA-487a (miR-487a) plays a role in the prognosis of gastric cancer, liver cancer, and other cancers. The purpose of this study is to explore the role of miR-487a in the generation and progression of ccRCC. The RT-qPCR technology was used to detect the expression levels of miR-487a in ccRCC tissues and cell lines. The association between miR-487a and clinical-pathological characteristics of patients was analyzed using the chi-square test. Kaplan-Meier survival analysis and Cox regression analysis were used to analyze the prognostic significance of miR-487a in ccRCC. CCK-8 and Transwell assays were used to analyze the influences of miR-487a on cell proliferation, migration, and invasion. miR-487a was significantly up-regulated in ccRCC tissues and cell lines. The high expression of miR-487a is related to lymph node metastasis and TNM staging and may be used as an independent prognostic factor related to lower overall survival and disease-free survival rate. Increased expression of miR-487a accelerated the proliferation, migration, and invasion of ccRCC cells. The enhanced expression of miR-487a was related to the prognosis of ccRCC, and it also facilitated cell proliferation, migration, and invasion.

Overexpression CPT1A reduces lipid accumulation via PPARα/CD36 axis to suppress the cell proliferation in ccRCC.

Clear cell renal carcinoma (ccRCC) is histologically defined by its cytoplasmic lipid deposits. Lipid metabolism disorder largely increases the risk of ccRCC. In this study, we aimed to investigate the biological functions and molecular mechanisms of carnitine palmitoyl transferase 1A (CPT1A) in ccRCC. Our results showed that CPT1A is decreased in ccRCC clinical samples and cell lines compared with that in normal samples. Lentivirus overexpressing CPT1A was used to investigate the neoplastic phenotypes of ccRCC, and the results showed that lipid accumulation and tumor growth are attenuated both and . In addition, CPT1A prevents cholesterol uptake and lipid accumulation by increasing the peroxisome proliferator-activated receptor α (PPARα) level through regulation of Class B scavenger receptor type 1 (SRB1) and cluster of differentiation 36 (CD36). Furthermore, PI3K/Akt signaling pathway promotes tumor cell proliferation in ccRCC, which is related to the enhanced expression of CD36. Functionally, weakened CPT1A expression is critical for lipid accumulation to promote ccRCC development. Collectively, our research unveiled a novel function of CPT1A in lipid metabolism via PPARα/CD36 axis, which provides a new theoretical explanation for the pathogenesis of ccRCC. Targeting CPT1A may be a potential therapeutic strategy to treat ccRCC.

MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis.

Objective:In various cancers, migration and invasion inhibitory protein (MIIP) is expressed at low level and is involved in cancer pathogenesis. Herein, we sought to explore the function of MIIP in clear cell renal cell carcinoma (ccRCC).Methods:CCK-8, colony formation, cell cycle, and endothelial cell tube formation assays were performed to evaluate the roles of MIIP in ccRCC proliferation and angiogenesis. To explore the underlying mechanism, we conducted RNA-sequencing, GSEA, qRT-PCR, Western blot, ELISA, cell transfection, coimmunoprecipitation, and ubiquitination assays in ccRCC cell lines. Furthermore, xenograft tumor growth in nude mice, and Ki-67 and CD31 staining in xenograft tissues were examined. Finally, the association of MIIP expression with clinical pathology and the expression status of HIF-2α and cysteine-rich 61 (CYR61) were further analyzed in human RCC tissues through Western blot and immunohistochemistry.Results:Both in vitro and in vivo functional experiments indicated that forced expression of MIIP inhibited ccRCC proliferation and angiogenesis, whereas silencing MIIP either in normal HK-2 cells or in ccRCC cells had the opposite effect (P < 0.05). Mechanistically, CYR61 was identified as a gene significantly downregulated by MIIP overexpression, and was required for the suppressive role of MIIP in ccRCC. MIIP was found to promote HSP90 acetylation and thus impair its chaperone function toward HIF-2α. Consequently, RACK1 binds HIF-2α and causes its ubiquitination and proteasomal degradation, thus decreasing the transcription of its target, CYR61. Finally, analyses of clinical samples demonstrated that MIIP is significantly downregulated in cancer vs. normal tissues in RCC cases, and its expression is negatively associated with histological grade, metastasis, the prognosis of patients with RCC, and the expression of HIF-2α and CYR61 (P < 0.05).Conclusions:MIIP is a novel tumor suppressor in ccRCC via negative regulation of HIF-2α-CYR61 axis.

FGL1 as a Novel Mediator and Biomarker of Malignant Progression in Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), which is the most prevalent renal cell carcinoma subtype, has a poor prognosis. Emerging strategies for enhancing the immune response in ccRCC therapy are currently being investigated. Fibrinogen-like Protein 1(FGL1) is a novel mechanism that tumors may use to evade the immune system by binding LAG-3 and negatively regulating T cells. In this study, we aimed at investigating the underlying mechanism of FGL1 in ccRCC, and its expression and prognostic value. We found that FGL1 was upregulated in tumor tissues and plasma specimens of ccRCC patients. High FGL1 expression predicted a poor prognosis for ccRCC patients. We also discovered that overexpression of FGL1 enhances RCC cell migration, invasion, and metastasis by activating the epithelial-to-mesenchymal transition (EMT). Consistent with these results, we identified a significant positive correlation between expression of FGL1 and EMT-related genes through tissue microarray analysis. Gene-expression analysis revealed that FGL1-deficient ccRCC cell lines had altered transcriptional output in inflammatory response, cell-cell signaling, negative regulation of T cell activation, and intracellular signal transduction. Depletion of FGL1 significantly inhibited tumor growth and lung metastasis in orthotopic xenograft mouse model. Infiltration of myeloid-derived CD11b+ and Ly6G+ immune cells in tumor microenvironment (TME) was strikingly decreased when FGL1 expression reduced. Therefore, increased FGL1 expression in ccRCC is positively correlated with poor prognosis. Mechanistically, FGL1 facilitates the EMT process and modulates TME, which promotes ccRCC progression and metastasis. Consequently, targeting FGL1 can potentially improve clinical outcome of ccRCC patients.

HZIP1 Inhibits Progression of Clear Cell Renal Cell Carcinoma by Suppressing NF-kB/HIF-1α Pathway.

PurposeAccumulating literature has suggested that hZIP1 and HIF-1α play vital roles in the tumor process of clear cell renal cell carcinoma (ccRCC). However, the functional roles of hZIP1 and HIF-1α in ccRCC remain largely unknown.MethodsHIF-1α protein level was evaluated by a western blot in ccRCC tissues and cell lines. ccRCC cell lines were transfected with HIF-1α-siRNA to downregulate the expression level of HIF-1α. Then the proliferative, migratory and invasive abilities of ccRCC cells in vitro were detected by real-time cell analysis (RTCA) assay, wound healing assay and transwell assay, respectively. The role of HIF-1α in vivo was explored by tumor implantation in nude mice. Then the effect on glycolysis‐related proteins was performed by western blot after hZIP1 knockdown (overexpression) or HIF-1α knockdown. The effect on NF‐kB pathway was detected after hZIP1 overexpression.ResultsHIF-1α was markedly downregulated in ccRCC tissues compared with normal areas. But HIF-1α presented almost no expression in HK-2 and ACHN cells. Immunofluorescence indicated HIF-1α and PDK1 expression in both the cytoplasm and nucleus in ccRCC cells. Downregulation of HIF-1α suppressed ccRCC cell proliferation, migration, and invasion and resulted in smaller implanted tumors in nude mice. Furthermore, hZIP1 knockdown elevated HIF-1α protein levels and PDK1 protein levels in ccRCC cells. Interestingly, a sharp downregulated expression of HIF-1α was observed after hZIP1 overexpression in OSRC-2 and 786-O cells, which resulted from a downtrend of NF-kB1 moving into the cell nucleus.ConclusionOur work has vital implications that hZIP1 suppresses ccRCC progression by inhibiting NF-kB/HIF-1α pathway.

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.

Systematic Pan-Cancer Analysis of KIF23 and a Prediction Model Based on KIF23 in Clear Cell Renal Cell Carcinoma (ccRCC).

PurposeThis study aims to carry out a pan-cancer analysis of kinesin family member 23 (KIF23) and construct a predictive model for the prognosis of clear cell renal cell carcinoma (ccRCC) patients.MethodsWe evaluated the differential expression of KIF23 in pan-cancer by The Cancer Genome Atlas (TCGA) and Oncomine database. Then, the correlation between KIF23 with prognosis, clinical grade, stage, immune subtype, tumor mutation burden (TMB), microsatellite instability (MSI) and immune microenvironment was explored by TCGA, an integrated repository portal for tumor-immune system interactions (TISIDB) and cBioPortal. Subsequently, we screened out ferroptosis-related genes (FRGs) related to KIF23 and constructed a risk score model. Univariate Cox analysis was used to determine independent prognostic factors for ccRCC overall survival (OS), and a nomogram was established. Furthermore, gene set enrichment analysis (GSEA) was applied to study the biological functions and pathways of KIF23. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to evaluate the expression of KIF23.ResultsKIF23 was highly expressed in most tumors. Further, KIF23 was strongly correlated with prognosis, clinical grade, stage, immune subtype, TMB, MSI and immune microenvironment in different tumors. We found that KIF23 was significantly associated with all aspects of ccRCC. Then, 8 FRGs were identified to construct a risk score model together with KIF23. And a prognostic nomogram prediction model of OS was established. After GSEA analysis, cell cycle, condensed chromosome and other physiological processes were screened out. Finally, qRT-PCR verified the high expression of KIF23 in ccRCC cell lines than normal kidney cell line.ConclusionKIF23 may act as a pivotal part in occurrence and progression of different tumors. In ccRCC, KIF23 can be a great prognostic biomarker, and the nomogram based on KIF23 may contribute to better treatment plans for ccRCC patients.

ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism

BackgroundKidney cancer is a common adult malignancy in the USA. Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, is characterized by widespread metabolic changes. Urea metabolism is one such altered pathway in ccRCC. The aim of this study was to elucidate the contributions of urea cycle enzymes, argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL) towards ccRCC progression.MethodsWe employed a combination of computational, genetic, and metabolomic tools along with in vivo animal models to establish a tumor-suppressive role for ASS1 and ASL in ccRCC.ResultsWe show that the mRNA and protein expression of urea cycle enzymes ASS1 and ASL are reduced in ccRCC tumors when compared to the normal kidney. Furthermore, the loss of ASL in HK-2 cells (immortalized renal epithelial cells) promotes growth in 2D and 3D growth assays, while combined re-expression of ASS1 and ASL in ccRCC cell lines suppresses growth in 2D, 3D, and in vivo xenograft models. We establish that this suppression is dependent on their enzymatic activity. Finally, we demonstrate that conservation of cellular aspartate, regulation of nitric oxide synthesis, and pyrimidine production play pivotal roles in ASS1+ASL-mediated growth suppression in ccRCC.ConclusionsccRCC tumors downregulate the components of the urea cycle including the enzymes argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). These cytosolic enzymes lie at a critical metabolic hub in the cell and are involved in aspartate catabolism and arginine and nitric oxide biosynthesis. Loss of ASS1 and ASL helps cells redirect aspartate towards pyrimidine synthesis and support enhanced proliferation. Additionally, reduced levels of ASS1 and ASL might help regulate nitric oxide (NO) generation and mitigate its cytotoxic effects. Overall, our work adds to the understanding of urea cycle enzymes in a context-independent of ureagenesis, their role in ccRCC progression, and uncovers novel potential metabolic vulnerabilities in ccRCC.