Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

Objective

To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.

Methods

Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC.

Results

A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis.

Conclusions

These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.     

Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2''-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.     

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.     

肾透明细胞癌预后相关miRNAs的生物信息学分析

目的寻找可作为肾透明细胞癌（ccRCC）生物标志物的miRNA,以及ccRCC与正常组织间miRNA差异表达情况。 方法利用TCGA数据库下载ccRCC中miRNA表达数据,分析肿瘤与正常组织间差异表达miRNA。使用Kaplan-Meier曲线对患者进行生存分析,筛选出表达情况与临床预后相关的miRNA。通过生物信息学对miRNA的靶基因进行预测,然后运用FunRich软件和ClueGO对靶基因进行GO和KEGG富集分析。 结果通过TCGA数据库分析发现,ccRCC较正常组织差异表达miRNA共54个,其中上调33个,下调21个。通过生存分析发现hsa-miR-21和hsa-miR-155与患者预后相关,P≤0.05。进一步通过Perl软件在Targetscan、miRDB、miRTarBase、miRPath这四个数据库中预测miRNA靶基因并将结果取交集,共发现129个靶基因。GO和KEGG分析结果表明,这些靶基因主要与转录因子活性、信号转导以及FoxO、TNF等信号通路密切相关。 结论通过生物信息学分析发现了ccRCC与正常组织的差异表达miRNA;其中hsa-miR-21和hsa-miR-155与患者总体生存率相关,并通过调控靶基因参与相关的信号通路进而影响ccRCC的发生发展进程,提示hsa-miR-21和hsa-miR-155可能是ccRCC潜在的生物标志物。     

Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1 (Kyoto University Renal Cancer 1) and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance in vivo when IL13RA2 was overexpressed. Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.     

Fifteen hub genes associated with progression and prognosis of clear cell renal cell carcinoma identified by coexpression analysis

Renal cell carcinoma (RCC) is the most common type of renal tumor, and the clear cell renal cell carcinoma (ccRCC) is the most frequent subtype. In this study, our aim is to identify potential biomarkers that could effectively predict the prognosis and progression of ccRCC. First, we used The Cancer Genome Atlas (TCGA) RNA-sequencing (RNA-seq) data of ccRCC to identify 2370 differentially expressed genes (DEGs). Second, the DEGs were used to construct a coexpression network by weighted gene coexpression network analysis (WGCNA). Moreover, we identified the yellow module, which was strongly related to the histologic grade and pathological stage of ccRCC. Then, the functional annotation of the yellow module and single-samples gene-set enrichment analysis of DEGs were performed and mainly enriched in cell cycle. Subsequently, 18 candidate hub genes were screened through WGCNA and protein–protein interaction (PPI) network analysis. After verification of TCGA’s ccRCC data set, Gene Expression Omnibus (GEO) data set (GSE73731) and tissue validation, we finally identified 15 hub genes that can actually predict the progression of ccRCC. In addition, by using survival analysis, we found that patients of ccRCC with high expression of each hub gene were more likely to have poor prognosis than those with low expression. The receiver operating characteristic curve showed that each hub gene could effectively distinguish between localized and advanced ccRCC. In summary, our study indicates that 15 hub genes have great predictive value for the prognosis and progression of ccRCC, and may contribute to the exploration of the pathogenesis of ccRCC.     

Identification of a four immune-related genes signature based on an immunogenomic landscape analysis of clear cell renal cell carcinoma

Renal clear cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which has strong immunogenicity. A comprehensive study of the role of immune-related genes (IRGs) in ccRCC is of great significance in finding ccRCC treatment targets and improving patient prognosis. In this study, we comprehensively analyzed the expression of IRGs in ccRCC based on The Cancer Genome Atlas datasets. The mechanism of differentially expressed IRGs in ccRCC was analyzed by bioinformatics. In addition, Cox regression analysis was used to screen prognostic related IRGs from differentially expressed IRGs. We also identified a four IRGs signature consisting of four IRGs (CXCL2, SEMA3G, PDGFD, and UCN) through lasso regression and multivariate Cox regression analysis. Further analysis results showed that the four IRGs signature could effectively predict the prognosis of patients with ccRCC, and its predictive power is independent of other clinical factors. In addition, the correlation analysis of immune cell infiltration showed that this four IRGs signature could effectively reflect the level of immune cell infiltration of ccRCC. We also found that the expression of immune checkpoint genes CTLA-4, LAG3, and PD-1 in the high-risk group was higher than that in the low-risk group. Our research revealed the role of IRGs in ccRCC, and developed a four IRGs signature that could be used to evaluate the prognosis of patients with ccRCC, which will help to develop personalized treatment strategies for patients with ccRCC and improve their prognosis. In addition, these four IRGs may be effective therapeutic targets for ccRCC.     

Expression of Ror2 Mediates Invasive Phenotypes in Renal Cell Carcinoma

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.     

Identification of a three-miRNA signature as a novel potential prognostic biomarker in patients with clear cell renal cell carcinoma

Current studies suggest that some microRNAs (miRNAs) are associated with prognosis in clear cell renal cell carcinoma (ccRCC). In this paper, we aimed to identify a miRNAs signature to improve prognostic prediction for ccRCC patients. Using ccRCC RNA-Seq data of The Cancer Genome Atlas (TCGA) database, we identified 177 differentially expressed miRNAs between ccRCC and paracancerous tissue. Then all the ccRCC tumor samples were divided into training set and validation set randomly. Three-miRNA signature including miR130b, miR-18a, and miR-223 were constructed by the least absolute shrinkage and selection operator (LASSO) Cox regression model in training set. According to optimal cut-off value of three-miRNA signature risk score, all the patients could be classified into high-risk group and low-risk group significantly. Survival of patients was significantly different between two groups (hazard ratio, 5.58, 95% confidence interval, 3.17-9.80; P < 0.0001), and three-miRNA signature performed favorably prognostic and predictive accuracy. The results were further validated in the validation set and total set. Multivariate Cox regression analyses and subgroup analyses showed that three-miRNA signature was an independent prognostic factor. Two nomograms that integrated three-miRNA signature and three clinicopathological risk factors were constructed to predict overall survival and disease-free survival after surgery for ccRCC patients. Functional enrichment analysis showed the possible roles of three-miRNA signature in some cancer-associated biological processes and pathways. In conclusion, we developed a novel three-miRNA signature that performed reliable prognostic for patient survival with ccRCC, it might facilitate ccRCC patients counseling and individualize management.     

FBXL6 depletion restrains clear cell renal cell carcinoma progression

BackgroundF-box proteins play important roles in cell cycle and tumorigenesis. However, its prognostic value and molecular function in clear cell renal cell carcinoma (ccRCC) remain unclear. In this study, we established a survival model to evaluate the prognosis of patients with ccRCC using the F-box gene signature and investigated the function of FBXL6 in ccRCC.MethodsComprehensive bioinformatics analyses were used to identify differentially expressed F-box and hub genes associated with ccRCC carcinogenesis. Based on the F-box gene signature, we constructed a risk model and nomogram to predict the overall survival (OS) of patients with ccRCC and assist clinicians in decision-making. Finally, we verified the function and underlying molecular mechanisms of FBXL6 in ccRCC using CCK-8 and EdU assays, flow cytometry, and subcutaneous xenografts.ResultsA risk model based on FBXO39, FBXL6, FBXO1, and FBXL16 was developed. In addition, we drew a nomogram based on the risk score and clinical features to assess the prognosis of patients with ccRCC. Subsequently, we identified FBXL6 as an independent prognostic marker that was highly expressed in ccRCC cell lines. In vivo and in vitro assays revealed that the depletion of FBXL6 inhibited cell proliferation and induced apoptosis. We also demonstrated that SP1 regulated the expression of FBXL6.ConclusionsFBXL6 was first identified as a diagnostic and prognostic marker in patients with ccRCC. Loss of FBXL6 attenuates proliferation and induces apoptosis in ccRCC cells. SP1 was also found to regulate the expression of FBXL6.