Human Clear Cell Renal Carcinoma Research Articles

MiR-425 involves in the development and progression of renal cell carcinoma by inhibiting E2F6.

To investigate the effect of miR-425 on the proliferation and apoptosis of clear cell renal carcinoma (ccRCA) cells, and to explore the underlying mechanism. A total of 80 pairs of human clear cell renal carcinoma (ccRCA) and cancer-adjacent normal tissue samples were collected in this study. Human ccRCA cell line (786-O) and normal human kidney cell line (HK-2) were used in cellular research. The expression level of miR-425 was detected in ccRCA tissues and cells, respectively. Target genes of miR-425 were predicted by bioinformatics and verified by luciferase reporter gene assay. Moreover, the role of miR-425 in regulating E2F6 as well as its effect on the proliferation and apoptosis of ccRCA cells were detected. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) results showed that the expression of miR-425 was significantly decreased in ccRCA tissues and cells. The proliferation ability and cell cycle of 786-O cells were significantly inhibited after miR-425 overexpression. The percentage of cells in G0/G1 phase was remarkably increased, while the percentage of cells in S and G2/M phases was significantly decreased. Besides, the number of apoptotic cells was significantly increased in the miR-425 intervention group. On-line target gene prediction software indicated that E2F6 was the potential downstream target gene of miR-425. RT-PCR, Western blotting and luciferase reporter gene assay demonstrated that the expression of E2F6 was negatively regulated by miR-425. In addition, subsequent experiments showed that the up-regulation of E2F6 could suppress the inhibitory effect of miR-425 on the proliferation and apoptosis of ccRCA cells. Our research demonstrated the inhibitory function of miR-425 in ccRCA. Therefore, the miR-425/E2F6 axis was expected to be one of the targets of ccRCA targeted therapy.

Distinct non-invasive evaluation values of tumor-derived cell-free microRNAs, circulating microvesicles and exosomal microRNAs after renal carcinoma cryoablation

The detection of peripheral circulating tumor-derived components, such as cell-free microRNAs, circulating microvesicles, and exosomal microRNAs, has been shown as a promising noninvasive strategy. However, the different roles of these components in tumor therapy evaluations have remained largely undefined. In this paper, we employed an in vivo model of the human clear cell renal cell carcinoma line Caki-1-bearing mice to evaluate the therapeutic effects of cryoablation, which is a new minimally invasive treatment for renal cell carcinoma. At different times after cryoablation, we found that the levels of the cell-free microRNAs miR-122, miR-155 and miR-210 were first increased and then decreased. Additionally, the number of large-sized microvesicles was increased after cryoablation, but the number of small-sized circulating microvesicles did not change. Furthermore, the exosomal microRNAs miR-126–3p, miR-17–5p, and miR-21–3p rapidly decreased one day after cryoablation, an effect that was well correlated with the treatment degree. Therefore, we suggest that these circulating components may have different levels of importance in the evaluation of the efficacy of renal cell cryoablation, furthermore, exosomal microRNAs may be more suitable for the early postoperative judgment of tumor elimination effects, which are worth further exploration in clinical practice.

DKC1 serves as a potential prognostic biomarker for human clear cell renal cell carcinoma and promotes its proliferation, migration and invasion via the NF‑κB pathway.

DKC1, an X‑linked gene encoding dyskerin at Xq28, is a crucial component of the telomerase complex and is indispensable for normal telomere function and the post‑-transcriptional modification of precursor rRNA. It has been revealed to exert diverse biological functions and prognostic values in numerous types of cancers. Our present study was aimed at examining DKC1 expression in normal renal tissues and clear cell renal cell carcinoma (ccRCC) samples and the prognostic value of DKC1 in ccRCC. We examined DKC1 protein expression levels in tissue microarrays including 307 cases of ccRCC tissues and in 75 pairs of ccRCC and paracancerous tissues with immunohistochemistry. The percentage of DKC1 expression in ccRCC (61.3%) was markedly higher than that in paracancerous tissues (34.7%) (P=0.001). Positive DKC1 expression tends to significantly be associated with unfavorable clinicopathological characteristics such as tumor diameter >7cm (P=0.002) and tumor‑node‑metastasis (TNM) stage III or IV (P<0.001). Multivariate COX analysis revealed that positive DKC1 expression was an independent unfavorable factor for prognosis of ccRCC patients [hazard ratio (HR)=1.932, 95% CI, 1.290‑2.893, P=0.001 for 5‑year overall survival; HR=1.778, 95% CI,1.150‑2.748, P=0.010 for disease‑free survival]. In the PROGgeneV2 platform, we also found that ccRCC patients with high DKC1 mRNA expression had a poorer prognosis than patients with low DKC1 expression in The Cancer Genome Atlas (TCGA). Furthermore, we found that knockdown of DKC1 inhibited proliferation, migration and invasion of ccRCC through regulation of the NF‑κB/MMP‑2 signaling pathway invitro. We also demonstrated that DKC1 regulated ccRCC proliferation and the expression of NF‑κB‑p65 and MMP‑2 invivo. In summary, the expression of DKC1 was upregulated in ccRCC, which was associated with unfavorable clinicopathological characteristics and DKC1 may act as an independent prognostic indicator of ccRCC patients.

Superiority of rat over murine model for studies on the evolution of cancer genome

Evolution of the species and carcinogenesis are similar in that genomic alterations are the key events. Oxidative stress derived from various etiologies is one of the major causes of carcinogenesis by inducing mutations in the genome. Persistent oxidative stress in the renal proximal tubules through Fenton reaction catalysed by ferric nitrilotriacetate (Fe-NTA) generates renal cell carcinoma (RCC) in mice and rats. Here, in order to observe the species difference in oxidative stress-induced carcinogenesis and to obtain an insight regarding the characteristics of each species, we compared the genomic alterations using array-based comparative genome hybridisation among RCCs in Mutyh knockout/wild-type mice (C57BL/6 background) induced by Fe-NTA, RCCs in F1 hybrids of Brown-Norway/Fischer-344 wild-type rats and clear cell renal cell carcinoma (CCRCC)/papillary renal cell carcinoma (PRCC) of humans. The average deviated fraction of genomic segments, either loss or gain, from the standard biallelic position was 0.220 (N = 4), 0.304 (N = 11), 0.283 (N = 12), and 0.261 (N = 5), respectively, for murine RCC, rat RCC, human CCRCC, and human PRCC. Notably, gain/loss ratio was remarkably different as indicated by 0.0820, 0.161, 0.821, and 4.44, respectively. These data suggest that higher species require more genomic alterations with amplification preference for renal carcinogenesis. Further studies are necessary to identify the molecular mechanisms whether the present results depend on cellular functional differences, etiology of carcinogenesis or the target cells in carcinogenesis.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system (“ccRCC-on-a-chip”). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell–derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies.

Bioinformatics and functional analyses of key genes and pathways in human clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. The present study was conducted to explore the mechanisms and identify the potential target genes for ccRCC using bioinformatics analysis. The microarray data of GSE15641 were screened on Gene-Cloud of Biotechnology Information (GCBI). A total of 32 ccRCC samples and 23 normal kidney samples were used to identify differentially expressed genes (DEGs) between them. Subsequently, the clustering analysis and functional enrichment analysis of these DEGs were performed, followed by protein-protein interaction (PPI) network, and pathway relation network. Additionally, the most significant module based on PPI network was selected, and the genes in the module were identified as hub genes. Furthermore, transcriptional level, translational level and survival analyses of hub genes were performed to verify the results. A total of 805 genes, 403 upregulated and 402 downregulated, were differentially expressed in ccRCC samples compared with normal controls. The subsequent bioinformatics analysis indicated that the small molecule metabolic process and the metabolic pathway were significantly enriched. A total of 7 genes, including membrane metallo-endopeptidase (MME), albumin (ALB), cadherin 1 (CDH1), prominin 1 (ROM1), chemokine (C-X-C motif) ligand 12 (CXCL12), protein tyrosine phosphatase receptor type C (PTPRC) and intercellular adhesion molecule 1 (ICAM1) were identified as hub genes. In brief, the present study indicated that these candidate genes and pathways may aid in deciphering the molecular mechanisms underlying the development of ccRCC, and may be used as therapeutic targets and diagnostic biomarkers of ccRCC.

A heterometallic ruthenium-gold complex displays antiproliferative, antimigratory, and antiangiogenic properties and inhibits metastasis and angiogenesis-associated proteases in renal cancer.

Heterobimetallic compounds are designed to harness chemotherapeutic traits of distinct metal species into a single molecule. The ruthenium-gold (Ru-Au) family of compounds based on Au-N-heterocyclic carbene (NHC) fragments [Cl2(p-cymene)Ru(μ-dppm)Au(NHC)]ClO4 was conceived to combine the known antiproliferative and cytotoxic properties of Au-NHC-based compounds and the antimigratory, antimetastatic, and antiangiogenic characteristic of specific Ru-based compounds. Following recent studies of the anticancer efficacies of these Ru-Au-NHC complexes with promising potential as chemotherapeutics against colorectal, and renal cancers in vitro, we report here on the mechanism of a selected compound, [Cl2(p-cymene)Ru(μ-dppm)Au(IMes)]ClO4 (RANCE-1, 1). The studies were carried out in vitro using a human clear cell renal carcinoma cell line (Caki-1). These studies indicate that bimetallic compound RANCE-1 (1) is significantly more cytotoxic than the Ru (2) or Au (3) monometallic derivatives. RANCE-1 significantly inhibits migration, invasion, and angiogenesis, which are essential for metastasis. RANCE-1 was found to disturb pericellular proteolysis by inhibiting cathepsins, and the metalloproteases MMP and ADAM which play key roles in the etiopathogenesis of cancer. RANCE-1 also inhibits the mitochondrial protein TrxR that is often overexpressed in cancer cells and facilitates apoptosis evasion. We found that while auranofin perturbed migration and invasion to similar degrees as RANCE-1 (1) in Caki-1 renal cancer cells, RANCE-1 (1) inhibited antiangiogenic formation and VEGF expression. We found that auranofin and RANCE-1 (1) have distinct proteolytic profiles. In summary, RANCE-1 constitutes a very promising candidate for further preclinical evaluations in renal cancer.

Combined Treatment with Valproic Acid and 5-Aza-2'-Deoxycytidine Synergistically Inhibits Human Clear Cell Renal Cell Carcinoma Growth and Migration.

BackgroundHistone acetylation and DNA methylation are important mammalian epigenetic modifications that participate in the regulation of gene expression. Because dysregulation of histone deacetylase and DNA methyltransferases are hallmarks of malignancy, they have become promising therapeutic targets. In this study, we explored the anti-tumor activity of valproic acid (VPA), a histone deacetylase inhibitor (HDACi) and 5-Aza-2′-deoxycytidine (5-Aza), an inhibitor of DNA methyltransferases, on renal cell carcinoma (RCC) cell lines 786-O and 769-P.Material/MethodsThe cell proliferation was detected by xCELLigence RTCA DP Instrument, viability by CCK8 assay, cell apoptosis and cell cycle by flow cytometry, and cell migration by wound healing assay, Transwell assay and xCELLigence RTCA DP Instrument.ResultsWe discovered that VPA and 5-Aza could individually induce decreased viability and have an inhibitory effect on the proliferation of 786-O and 769-P cells. This anti-growth effect was more pronounced when the cells were treated with both VPA and 5-Aza. The combination of VPA and 5-Aza also elicited more apoptosis and produced more cell cycle arrest in the G1 phase for both cell lines. On the other hand, treatment of RCC cells with VPA, 5-Aza, or a combination of both resulted in slow wound healing and impaired migration.ConclusionsThese findings clearly demonstrated that VPA combined with 5-Aza could significantly increase anti-RCC effects by inhibiting cellular proliferation, inducing apoptosis, promoting cell cycle arrest and prohibiting the migration of human RCC cells.

Identification of key genes and pathways in human clear cell renal cell carcinoma (ccRCC) by co-expression analysis.

Human clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidney, and its prognostic is influenced by the progression covering a complex network of gene interactions. In our study, we screened differential expressed genes, and constructed protein-protein interaction (PPI) network and a weighted gene co-expression network to identify key genes and pathways associated with the progression of ccRCC (n = 56). Functional and pathway enrichment analysis demonstrated that upregulated differentially expressed genes (DEGs) were significantly enriched in response to wounding, positive regulation of immune system process, leukocyte activation, immune response and cell activation. Downregulated DEGs were significantly enriched in oxidation reduction, monovalent inorganic cation transport, ion transport, excretion and anion transport. In the PPI network, top 10 hub genes were identified (TOP2A, MYC, ALB, CDK1, VEGFA, MMP9, PTPRC, CASR, EGFR and PTGS2). In co-expression network, 6 ccRCC-related modules were identified. They were associated with immune response, metabolic process, cell cycle regulation, angiogenesis and ion transport. In conclusion, our study illustrated the hub genes and pathways involved in the progress of ccRCC, and further molecular biological experiments are needed to confirm the function of the candidate biomarkers in human ccRCC.

α-1,2-Mannosidase MAN1C1 Inhibits Proliferation and Invasion of Clear Cell Renal Cell Carcinoma.

Background: This study investigated the biological function of the gene MAN1C1 α-mannosidase in renal cell carcinoma. It has been reported that MAN1C1 is probably a potential tumor suppressor gene in Wilms. However, the role of MAN1C1 in human clear cell renal cell carcinoma (ccRCC) has not been reported.Methods: In this study, MAN1C1 gene over-expression was used to transfect human renal cancer cell lines 786-O and OS-RC-2 to study apoptosis and the underlying mechanisms which influence epithelial-mesenchymal transition.Results: MAN1C1 was down-regulated in ccRCC and related to the clinicopathological factors and prognosis of ccRCC. We revealed that over-expression MAN1C1 showed anti-tumor effect by inducing apoptosis, as determined by Cell Counting Kit-8 (CCK-8) assay, cell cycle analysis, and western blot analysis. What's more, MAN1C1 over-expression remarkably increased the ratio of Bax/Bcl-2 and inhibited epithelial-mesenchymal transition by increasing the expression of E-CA. In addition, the ratio of Bax/Bcl-2 and E-CA were also increased in MAN1C1 gene over-expression renal cancer cells compared with the control cells.Conclusion: We find that re-expression of silenced MAN1C1 in ccRCC cell lines inhibited cell viability, colony formation, induced apoptosis, suppressed cell invasion and migration. In conclusion, MAN1C1 is a novel functional tumor suppressor in renal carcinogenesis. This is the first time that the function of MAN1C1 gene has been verified in the renal tumor tissue so far.

Suppression of homology-dependent DNA double-strand break repair induces PARP inhibitor sensitivity in VHL-deficient human renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.

A novel correlation between ATP5A1 gene expression and progression of human clear cell renal cell carcinoma identified by co‑expression analysis.

Clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidneys, and its prognostic is influenced by the progression covering a complex network of gene interactions. In our study, a weighted gene co-expression network was constructed to identify gene modules associated with the progression of ccRCC (n=35). In the significant module (R2 = −0.53), a total of 13 network hub genes were identified, and 2 of them were hub nodes in the protein-protein interaction network as well. In validation, ATP5A1 showed a higher correlation with the disease progression than any other hub gene in the hub module (P=0.001219). In the test set (n=202), ATP5A1 was also highly expressed in normal kidney than ccRCC tissues of each grade (P<0.001). Functional and pathway enrichment analysis demonstrated that ATP5A1 is overrepresented in pathway of oxidative phosphorylation, which associated with tumorigenesis and tumor progression. Gene set enrichment analysis (GSEA) also demonstrated that the gene set of ‘oxidative phosphorylation’ and metabolic pathways were enriched in ccRCC samples with ATP5A1 highly expressed (P<0.05). In conclusion, based on the co-expression analysis, ATP5A1 was validated to be associated with progression of ccRCC, probably by regulating tumor-related phosphorylation.

XRCC1 serves as a potential prognostic indicator for clear cell renal cell carcinoma and inhibits its invasion and metastasis through suppressing MMP-2 and MMP-9.

X-ray repair cross-complementing group 1 (XRCC1) is a major DNA repair gene that is responsible for fixing DNA base damage and single-strand breaks by interacting with DNA components at the damage site. This study explored the clinical significance of XRCC1 in human clear cell renal cell carcinoma (ccRCC) and further examined the mechanism of the role of XRCC1 in ccRCC. The clinical relevance of XRCC1 in ccRCC was evaluated using tissue microarrays and immunohistochemical staining of two independent human ccRCC cohorts. Our data demonstrated that XRCC1 expression was dramatically decreased in ccRCC tissues compared with that in normal renal tissues and paired adjacent non-tumor tissues. Low XRCC1 expression was significantly correlated with lymph node metastasis and with worse overall and disease-specific survival in patients, as determined by log-rank tests. However, Cox regression analysis revealed that XRCC1 expression was not an independent prognostic factor in ccRCC patients. Furthermore, XRCC1 suppressed ccRCC migration and invasion by inhibiting MMP-2 and MMP-9 expression through the regulation of TIMP-2 and TIMP-1. These findings indicated that decreased XRCC1 expression was associated with lymph node metastasis but was not an independent prognostic factor in ccRCC patients. XRCC1 may serve as a potential therapeutic target for inhibiting ccRCC metastasis but cannot be used as an independent prognostic factor.

PBRM1 and VHL expression correlate in human clear cell renal cell carcinoma with differential association with patient’s overall survival

ObjectiveTo identify the clinicopathological association of PBRM1 (Polybromo-1 gene) and VHL (von Hippel-Lindau gene) expression at mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) and its role in tumor progression. Patients and methodsImmunohistochemical analysis, Western blotting and qPCR analysis of PBRM1 and VHL were performed on fresh-frozen ccRCC and adjacent normal tissue obtained from 70 patients who underwent radical nephrectomy. In addition, a tissue microarray (TMA) from specimens of 326 ccRCC patients was used to evaluate the effect of loss of PBRM1 and VHL immunohistological expression on clinicopathological features as well as patient survival. ResultsIn frozen tissue, PBRM1 and VHL mRNA were significantly down-regulated in most ccRCC tumors (77.6%/80.6%). Simultaneous weak PBRM1 and VHL protein expression was observed in 21.4% of frozen tumors. In the TMA samples, weak PBRM1 and VHL immunohistochemical staining was observed in 60.4% of the cases and was correlated (P<0.001). The association of PBRM1 and VHL immunohistochemical expression with clinicopathological parameters depicts a variable picture: predominantly weak PBRM1 and VHL expression were significantly associated with higher Fuhrman grade (P = 0.012 and 0.024, respectively) but only weak VHL expression was associated with a higher pT stage (P = 0.023). PBRM1 expression did not affect the overall survival, whereas weak VHL expression was associated with decreased patient overall survival (P = 0.013). ConclusionsOur data suggest that reduced expression of PBRM1 and VHL is correlated with an increased tumor aggressiveness. Low VHL expression was identified as a risk factor for worse patient overall survival, independently from PBRM1 expression pattern.

The role of CRP and ATG9B expression in clear cell renal cell carcinoma.

The purpose of the study is to investigate the correlation between the expression of C-reactive protein (CRP) and autophagy-related 9B (ATG9B) and pathological features of clear cell renal cell carcinoma (CCRCC) patients. We also intended to explore the effects of manipulated expression of CRP and ATG9B on the apoptosis and cell cycle progression of CCRCC cell line. ATG9B expression in CCRCC tissues and adjacent renal tissues was analyzed by immunohistochemistry (IHC). Gene expression was determined at transcription and translational levels using real-time quantitative PCR (RT-qPCR) and Western blot. The association between CRP/ATG9B expression and clinical-pathological parameters including age, gender, pathological grades, TNM stage and distant metastasis of the patients was assessed by correlation analysis. siRNA and overexpression plasmids construction were used to manipulate the expression of CRP in human CCRCC cell line 786-O. Cell apoptosis and cell cycle progression were determined using flow cytometry (FCM) and Hoechst 33258 staining. CRP expression correlates with ATG9B expression. The expression of CRP and ATG9B are significantly correlated with TNM staging, distant metastasis, and survival time of CCRCC patients. A high-level of CRP indicates a poor overall survival (OS). In addition, CRP expression influences cell cycle and apoptosis of CCRCC cells. The study reveals that CRP might be a CCRCC development promoter. In addition, there is a close relationship between CRP and ATG9B in CCRCC carcinogenesis.

RETRACTED ARTICLE: Hispidulin mediates apoptosis in human renal cell carcinoma by inducing ceramide accumulation

Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin-induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 μmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg-1·d-1, ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Co-expression network analysis identified six hub genes in association with progression and prognosis in human clear cell renal cell carcinoma (ccRCC)

Human clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignant adult kidney tumors. We constructed a weighted gene co-expression network to identify gene modules associated with clinical features of ccRCC (n=97). Six hub genes (CCNB2, CDC20, CEP55, KIF20A, TOP2A and UBE2C) were identified in both co-expression and protein-protein interaction (PPI) networks, which were highly correlated with pathologic stage. The significance of expression of the hub genes in ccRCC was ranked top 4 among all cancers and correlated with poor prognosis. Functional analysis revealed that the hub genes were significantly enriched in cell cycle regulation and cell division. Gene set enrichment analysis suggested that the samples with highly expressed hub gene were correlated with cell cycle and p53 signaling pathway. Taken together, six hub genes were identified to be associated with progression and prognosis of ccRCC, and they might lead to poor prognosis by regulating p53 signaling pathway.

Differential expression of c-Met between primary and metastatic sites in clear-cell renal cell carcinoma and its association with PD-L1 expression.

In preclinical models, c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). However, this relationship in human clear cell renal cell carcinoma (ccRCC) is not well characterized. We evaluated c-Met expression in ccRCC patients using paired primary and metastatic samples and assessed the association with PD-L1 expression and other clinical features. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab) and calculated by a combined score (CS, 0–300): intensity of c-Met staining (0–3) x % of positive cells (0–100). PD-L1 expression in tumor cells was previously assessed by IHC and PD-L1+ was defined as PD-L1 > 0% positive cells. Our cohort consisted of 45 pairs of primary and metastatic ccRCC samples. Overall, c-Met expression was higher in metastatic sites compared to primary sites (average c-Met CS: 55 vs. 28, p = 0.0003). Higher c-Met expression was associated with higher FNG (4 vs. 3) in primary tumors (average c-Met CS: 52 vs. 20, p = 0.04). c-Met expression was numerically greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression in metastatic sites compared to primary tumors suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. While higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination clinical trials.

Promoter methylation inhibits expression of tumor suppressor KIBRA in human clear cell renal cell carcinoma

BackgroundKIBRA has been suggested as a key regulator of the Hippo signaling pathway, regulating organ size, cell contact inhibition, tissue regeneration as well as tumorigenesis and cystogenesis. We recently reported that human KIBRA expression depends on a complex alternative CpG-rich promoter system. Our current study aimed at the identification of epigenetic mechanisms associated with alterations in KIBRA expression regulation.ResultsWe identified two separated methylation-sensitive CpG islands located to independent KIBRA promoter regions. In vitro promoter methylation analysis using human neuroblastoma (SH-SY5Y) and immortalized kidney cells (IHKE) revealed that total promoter methylation by CpG methyltransferase SssI resulted in complete abrogation of transcriptional activity (p < 0.001), while partial methylation by HpaII selectively repressed KIBRA core promoter activity in kidney cells (p < 0.001). Cell culture-based experiments demonstrated that 5-azacitidine may be used to restore KIBRA mRNA and protein levels, while overexpression of transcription factor SP1 also induced KIBRA upregulation (all p < 0.001). Furthermore, SP1 transactivation of KIBRA transcription was largely prevented by methylation of KIBRA regulatory elements (p < 0.001). Analysis of human kidney biopsies revealed that KIBRA promoter methylation was associated with human clear cell renal cell carcinoma (ccRCC; n = 8 vs 16 controls, OR = 1.921, [CI 95% = 1.369–2.695]). The subsequent determination of KIBRA mRNA levels by real-time PCR in a larger patient sample confirmed significantly reduced KIBRA expression in ccRCC (n = 32) compared to non-neoplastic human kidney tissue samples (controls, n = 32, p < 0.001).ConclusionWe conclude that epigenetic downregulation of tumor suppressor KIBRA may involve impaired SP1 binding to functional methylation-sensitive KIBRA promoter elements as observed in human kidney clear cell carcinoma. Our findings provide a pathophysiological basis for future studies on altered KIBRA regulation in clinical disease entities such as renal cancer.

MLN4924 (Pevonedistat), a protein neddylation inhibitor, suppresses proliferation and migration of human clear cell renal cell carcinoma

Neddylation is a post-translational protein modification associated with cancer development. MLN4924 is a neddylation inhibitor currently under investigation in multiple phase I studies on various malignancies, and its clincal name is Pevonedistat. It has been documented that MLN4924 blocks Cullins neddylation and inactivates CRLs and, in turn, triggers cell-cycle arrest, apoptosis, senescence and autophagy in many cancer cells. In this study, we investigated the anti-tumor effect of MLN4924 in human clear cell renal carcinoma (ccRCC). Levels of both Nedd8 activating enzyme E1 and Nedd8-conjugating enzyme E2 were higher in ccRCC tissues and RCC cancer cells than in normal. Moreover, MLN4924 treatment led to rapid inhibition of Cullin1 neddylation and notably suppressed growth and survival as well as migration in a dose-and time-dependent manner. Mechanistic studies revealed that MLN4924 induced the accumulation of a number of CRL substrates, including p21, p27 and Wee1 to trigger DNA damage and induce growth arrest at the G2/M phase. MLN4924 also induced anti-migration and anti-invasion by activating E-cadherin and repressing Vimentin. Taken together, this study provides the first evidence that neddylation pathway is overactive in ccRCC and that MLN4924 induces dose-dependent anti-proliferation, anti-migration, anti-invasion in ccRCC cells. The study thus indicates that MLN4924 has potential therapeutic value for the clinical treatment of renal cancer.