Identification of genes associated with renal cell carcinoma using gene expression profiling analysis

miRNA Profiling for Clear Cell Renal Cell Carcinoma: Biomarker Discovery and Identification of Potential Controls and Consequences of miRNA Dysregulation

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan. Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined. Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%). In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.

Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†.

BackgroundIt is estimated that there are 338,000 new renal-cell carcinoma releases every year in the world. Renal cell carcinoma (RCC) is a heterogeneous tumor, of which more than 70% is clear cell renal cell carcinoma (ccRCC). It is estimated that about 30% of new renal-cell carcinoma patients have metastases at the time of diagnosis. However, the pathogenesis of renal clear cell carcinoma has not been elucidated. Therefore, it is necessary to further study the pathogenesis of ccRCC. MethodsTwo expression profiling datasets (GSE68417, GSE71963) were downloaded from the GEO database. Differentially expressed genes (DEGs) between ccRCC and normal tissue samples were identified by GEO2R. Functional enrichment analysis was made by the DAVID tool. Protein-protein interaction (PPI) network was constructed. The hub genes were excavated. The clustering analysis of expression level of hub genes was performed by UCSC (University of California Santa Cruz) Xena database. The hub gene on overall survival rate (OS) in patients with ccRCC was performed by Kaplan-Meier Plotter. Finally, we used the ccRCC renal tissue samples to verify the hub genes. Results1182 common DEGs between the two datasets were identified. The results of GO and KEGG analysis revealed that variations in were predominantly enriched in intracellular signaling cascade, oxidation reduction, intrinsic to membrane, integral to membrane, nucleoside binding, purine nucleoside binding, pathways in cancer, focal adhesion, cell adhesion molecules. 10 hub genes ITGAX, CD86, LY86, TLR2, TYROBP, FCGR2A, FCGR2B, PTPRC, ITGB2, ITGAM were identified. FCGR2B and TYROBP were negatively correlated with the overall survival rate in patients with ccRCC (P < 0.05). RT-qPCR analysis showed that the relative expression levels of CD86, FCGR2A, FCGR2B, TYROBP, LY86, and TLR2 were significantly higher in ccRCC samples, compared with the adjacent renal tissue groups. ConclusionsIn summary, bioinformatics technology could be a useful tool to predict the progression of ccRCC. In addition, there are DEGs between ccRCC tumor tissue and normal renal tissue, and these DEGs might be considered as biomarkers for ccRCC.

Our study aims to investigate the mechanisms through which Fc receptor-like A (FCRLA) promotes renal cell carcinoma (RCC) and to examine its significance in relation to tumor immune infiltration. The correlation between FCRLA and data clinically related to RCC was explored using The Cancer Genome Atlas (TCGA), then validated using Gene Expression Omnibus (GEO) gene chip data. Enrichment and protein-protein interaction (PPI) network analyses were performed for FCRLA and its co-expressed genes. FCRLA was knocked down in RCC cell lines to evaluate its impact on biological behavior. Then the potential downstream regulators of FCRLA were determined by western blotting, and rescue experiments were performed for verification. The relevance between FCRLA and various immune cells was analyzed through GSEA, TIMER, and GEPIA tools. TIDE and ESTIMATE algorithms were used to predict the effect of FCRLA in immunotherapy. Fc receptor-like A was associated with clinical and T stages and could predict the M stage (AUC = 0.692) and 1-3- and 5-year survival rates (AUC = 0.823, 0.834, and 0.862) of RCC patients. Higher expression of FCLRA predicted an unfavorable overall survival (OS) in TCGA-RCC and GSE167573 datasets (p = 0.03, p = 0.04). FCRLA promoted the malignant biological behavior of RCC cells through the pERK1/2/-MMP2 pathway and was associated with tumor immune microenvironment in RCC. Fc receptor-like A is positively correlated with poor outcomes in RCC patients and plays an oncogenic role in RCC through the pERK1/2-MMP2 pathway. Patients with RCC might benefit from immunotherapy targeting FCRLA.

Introduction: tyrosine kinases receptors (RTKs) play an important role in the pathogenesis of renal cell carcinoma (RCC). RTKs were studied on tumor and endothelial cells, but the presence of these receptors on lymphocytes was not confirmed. The objective of this study was to investigate the expression of tyrosine kinases receptors on lymphocyte subpopulations in healthy volunteers and RCC patients before and after removal of the primary tumor.Materials and methods: the study included 19 patients with pT1‑T3N0 / N+M0 / M+ RCC, subjected to nephrectomy, and 10 healthy volunteers. Blood samples were collected once from healthy donors and twice from RCC patients, immediately before and 180 days after surgery. Isolation of lymphocytes and flow cytometry were carried out using standard methods. A comparative analysis of RTKs expression levels in peripheral lymphocytes from healthy volunteers and RCC patients, as well as in RCC patients before and after the operation, was carried out. A search was performed for correlations between the initial RTKs expression on lymphocytes from RCC patients and characteristics of the tumor development, as well as the disease prognosis.Results: VEGFR-1, -2, -3, FGFR2, PDGFRα, β RTKs are expressed on CD45+ peripheral blood mononuclear cells, as well as subpopulations of CD3+ and CD8+ lymphocytes in healthy volunteers and untreated patients with RCC. No differences in the expression levels of all studied RTKs between subpopulations of lymphocytes were found in RCC patients (p &gt; 0.05 for all). The level of RTKs expression on CD45+ peripheral cells in RCC patients before treatment is significantly lower than in healthy volunteers (p &lt; 0.05 for all). The degree of a decrease in RTKs expression correlated with the pT status and the presence of tumor-associated venous thrombosis. A significant increase in the expression levels of VEGFR1 (on CB45+) and VEGFR2 (on CD8+, CD3+) (p &lt; 0.05 for all) was noted 180 days after the removal of the primary tumor in patients with RCC. No other significant changes in RTKs production were identified. We were not able to determine the effect of RTKs expression on the RCC outcome.Conclusions: lymphocytes express RTKs, their expression is more pronounced in healthy people than in patients with RCC. After surgical treatment, the RTKs expression becomes restored.

Modulators (Ms) are proteins that modify the activity of transcription factors (TFs) and influence expression of their target genes (TGs). To discover modulators of NF-κB/RelA, we first identified 365 NF-κB/RelA-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We used a probabilistic model to infer 8349 (M, NF-κB/RelA, TG) triplets and their modes of modulatory action from our combined LC-MS/MS and ChIP-Seq (ChIP followed by next generation sequencing) data, published RelA modulators and TGs, and a compendium of gene expression profiles. Hierarchical clustering of the derived modulatory network revealed functional subnetworks and suggested new pathways modulating RelA transcriptional activity. The modulators with the highest number of TGs and most non-random distribution of action modes (measured by Shannon entropy) are consistent with published reports. Our results provide a repertoire of testable hypotheses for experimental validation. One of the NF-κB/RelA modulators we identified is STAT1. The inferred (STAT1, NF-κB/RelA, TG) triplets were validated by LC-selected reaction monitoring-MS and the results of STAT1 deletion in human fibrosarcoma cells. Overall, we have identified 562 NF-κB/RelA modulators, which are potential drug targets, and clarified mechanisms of achieving NF-κB/RelA multiple functions through modulators. Our approach can be readily applied to other TFs.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and has been described as one of the deadliest of cancers affecting the genitourinary tract. Tropomyosin is a two-stranded α-helical coiled coil protein found in cell cytoskeletons. One of its isoforms, tropomyosin-1 (TPM1) has been reported as a novel tumor-suppressor gene and is downregulated in many solid tumors. However the expression level and function of TPM1 in RCC have not yet been determined. In the present study, we evaluated the TPM1-4 mRNA and TPM1 protein levels in RCC tissue samples. TPM1-overexpressing OSRC-2 and 786-O cell lines were also used to investigate the impact of TPM1 on RCC cells. We found that TPM1 was significantly and specifically downregulated in the RCC tissues. TPM1 expression was associated with tumor size, smoking status, Fuhrman grade and the prognosis of RCC patients. After TPM1 transfection, the migratory and invasive abilities of the OSRC-2 and 786-O cell lines were both reduced when compared to the control groups. Meanwhile, apoptosis was also enhanced in these two RCC cell lines following TPM1 transfection. Taken together, TPM1 exhibits characteristics of a tumor-suppressor gene while being overexpressed in RCC cell lines.

Background: The von Hippel-Lindau (VHL) gene is a tumor suppressor gene. Although VHL gene alterations play a key role in the pathogenesis of renal cell carcinoma (RCC), and several therapies targeting this molecular pathway have been studied, such as sunitinib, pazopanib and axitinib. However, it is not clear whether VHL mutation is a prognostic factor for immunotherapy in renal cell carcinoma. Methods: The impact of VHL mutations on survival outcomes in RCC patients received immunotherapy or non-immunotherapy were verified in the Memorial Sloan Kettering Cancer Center (MSKCC) trials and The Cancer Genome Atlas (TCGA) RCC cohort, respectively. An in-house Chinese RCC cohort (n=950) was used for the analysis of immune-related markers. The relationship between clinical pathologic features and VHL were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: In the MSKCC cohort, a total of 143 RCC patients receiving immunotherapy were enrolled, of which 117 (81.8%) were clear cell RCC and 143 (100%) microsatellite-stable RCC. VHL mutation was significantly associated with high TMB score (P =0.017). Kaplan-Meier survival analysis showed that patients with VHL mutation obtained better OS compared to VHL wild-type (50 months vs. 23 months, HR, 0.36; 95% CI 0.21-0.64; P&amp;lt;0.001). In the TCGA cohort, all RCC patients received non- immunotherapy. No association was observed between VHL and OS (HR, 1.00; 95% CI 0.73-1.38; P=0.981). VHL mutations were significantly associated with higher TMB levels (P=0.006) and MSI-H (P=0.004) in the in-house Chinese RCC cohorts. Conclusions: Our results suggest that VHL mutation may be associated with better OS in RCC patients receiving immunotherapy. The exact mechanisms underlying VHL are needed to be further evaluated. Citation Format: Dong Shen, Yufeng Huang, Zhiwei Wu, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. Prognosis of renal cell carcinoma patients with VHL mutations to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5090.

The Tale of Two Hypoxia-Inducible Factors in Renal Cell Carcinoma

This study explores the role of circular RNA derived from the Multidrug Resistance Protein 4 (MRP4/ABCC4) gene, which is markedly elevated in renal cell carcinoma (RCC). Our objective is to clarify how this circular RNA contributes to the progression and development of RCC. We quantified the presence of circular ABCC4 RNA in tissue samples, plasma and urine from patients diagnosed with RCC. In addition, the impact of this circular RNA on RCC tumour growth was assessed through studies in RCC cell lines and in animal models mimicking the disease. Our findings reveal that circular ABCC4 RNA, specifically the variant containing exons 25-29 (circABCC4e), is upregulated in RCC cell lines and tissues. This upregulation correlates with advanced tumor stages in RCC patients, suggesting circABCC4e's potential as a biomarker for RCC progression. Furthermore, the reduction in circABCC4e levels following tumor resection indicates its potential utility in monitoring treatment response. The mechanism by which circABCC4e promotes RCC tumor growth through the antagonism of tumor-suppressive microRNAs highlights its significance in RCC pathogenesis. These insights may inform the development of diagnostic and therapeutic strategies for RCC. This study demonstrates that circABCC4e accelerates RCC progression by inhibiting tumor-suppressive microRNAs. Its role as a diagnostic and prognostic biomarker for RCC underscores its potential value in improving RCC management.

BackgroundThere is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive.MethodBioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes.ResultsClinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor.ConclusionOur data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

Renal cell carcinoma (RCC) is a common malignancy of the urinary system. Although traditional therapies, such as surgery assisted with chemotherapy have improved the quality of life and survival time of patients with RCC, patients with metastasis or recurrence benefit little from such therapies. At present, little is known about the underlying mechanisms of RCC, rendering treatment selection and implementation challenging. Therefore, investigating the cause and underlying mechanisms of RCC remain of importance to explore potential new avenues for its treatment. Inter-α-trypsin inhibitor heavy chain 1 (ITIH1) is an inflammation-associated gene reported to suppress the progression of liver cancer. However, its role in RCC remains poorly understood. Therefore, the present study aimed to investigate the role and mechanism of ITIH1 in RCC. Based on data obtained from The Cancer Genome Atlas database, ITIH1 expression was demonstrated to be significantly higher in tumor tissues compared with normal tissues, which was in turn negatively associated with the survival of patients with RCC. However, in RCC cells, ITIH1 was shown to be expressed at significantly lower levels compared with those in HK-2 cells. The discrepancy between tissues and cell lines might be due to the different environment of cell growth. ITIH1 knockdown in RCC cells significantly increased cell proliferation and invasion whilst significantly decreasing the apoptosis rate, compared with those in control cells (without ITIH1 knockdown). By contrast, overexpression of ITIH1 significantly inhibited cell proliferation and invasion in RCC cells. In terms of western blotting results, the phosphorylation levels of NF-κB were significantly increased following ITIH1 knockdown. The protein expression level of IκB significantly decreased whereas that of IKK, Cyclin D1, proliferating cell nuclear antigen and α-smooth muscle actin were significantly increased in ITIH1-knockdown cells, compared with those in the control cells (without ITIH1 knockdown). This suggests that the NF-κB pathway may be activated after ITIH1 knockdown. Following treatment with the NF-κB pathway inhibitor JSH-23 in combination with ITIH1 knockdown, RCC cell proliferation and invasion were significantly reduced compared with those after ITIH1 knockdown alone. In summary, results from the present study suggest that ITIH1 can serve an inhibitory role in the progression of RCC, which could potentially be inhibited through the NF-κB signaling pathway.

Natural products are chemical compounds produced by living organisms. They are isolated and purified to determine their function and can potentially be used as therapeutic agents. The ability of some bioactive natural products to modify the course of cancer is fascinating and promising. In the past 50years, there have been advancements in cancer therapy that have increased survival rates for localized tumors. However, there has been little progress in treating advanced renal cell carcinoma (RCC), which is resistant to radiation and chemotherapy. Oncogenes and tumor suppressors are two roles played by microRNAs (miRNAs). They are involved in important pathogenetic mechanisms like hypoxia and epithelial-mesenchymal transition (EMT); they control apoptosis, cell growth, migration, invasion, angiogenesis, and proliferation through target proteins involved in various signaling pathways. Depending on their expression pattern, miRNAs may identify certain subtypes of RCC or distinguish tumor tissue from healthy renal tissue. As diagnostic biomarkers of RCC, circulating miRNAs show promise. There is a correlation between the expression patterns of several miRNAs and the prognosis and diagnosis of patients with RCC. Potentially high-risk primary tumors may be identified by comparing original tumor tissue with metastases. Variations in miRNA expression between treatment-sensitive and therapy-resistant patients' tissues and serum allow for the estimation of responsiveness to target therapy. Our knowledge of miRNAs' function in RCC etiology has a tremendous uptick. Finding and validating their gene targets could have an immediate effect on creating anticancer treatments based on miRNAs. Several miRNAs have the potential to be used as biomarkers for diagnosis and prognosis. This review provides an in-depth analysis of the current knowledge regarding natural compounds and their modes of action in combating cancer. Also, this study aims to give information about the diagnostic and prognostic value of miRNAs as cancer biomarkers and their involvement in the pathogenesis of RCC.

Changes in circulating adiponectin have been related to the risks of various cancers. However, the association between circulating adiponectin and the risk of renal cell carcinoma has not been fully determined. A meta-analysis was performed to evaluate the relationship between circulating adiponectin and renal cell carcinoma risk. Observational studies that evaluated the association between circulating adiponectin and renal cell carcinoma risk were identified via a systematic search of PubMed and Embase databases. The difference between circulating adiponectin in renal cell carcinoma cases and healthy controls, and the multivariable adjusted association between circulating adiponectin and renal cell carcinoma risk were evaluated. A random effects model was used if significant heterogeneity existed; otherwise a fixed effects model was applied. Eight case-control studies with 2624 renal cell carcinoma cases and 2904 healthy controls were included. Pooled results showed that circulating adiponectin was significantly lower in renal cell carcinoma cases than in healthy controls (mean difference = -1.08 ug/mL; 95% confidence interval (CI) -1.62, -0.54; P < 0.001). Higher circulating adiponectin was independently associated with a significantly lowered risk of renal cell carcinoma (adjusted odds ratio for 1 SD increment of adiponectin = 0.78; 95% CI: 0.63, 0.96; P = 0.02). Subgroup analyses according to characteristics including study design, ethnics of participants, blood samples, numbers of participants, mean ages of participants, and study quality showed consistent results. Lower circulating adiponectin is associated with increased risk of renal cell carcinoma. The potential pathophysiological mechanisms underlying the role of circulating adiponectin in the pathogenesis of renal cell carcinoma deserve further investigation.