MiR-506 Is Down-Regulated in Clear Cell Renal Cell Carcinoma and Inhibits Cell Growth and Metastasis via Targeting FLOT1

Background

Some microRNAs (miRNAs) are abnormally expressed in cancer and contribute to tumorigenesis. In the present study, we investigated the role of miR-506 in clear cell renal cell carcinoma (ccRCC).

Methods

miR-506 expression was detected in renal cancer cell lines 786-O, ACHN, Caki-1, and Caki-2 and ccRCC specimens by quantitative real-time-PCR. We assessed the association of miR-506 expression with pathology and prognosis in ccRCC patients. We over-expressed and knocked-down miR-506 expression in two renal cancer cell lines, 786-O and ACHN, and assessed the impact on cell proliferation, migration and invasion. A luciferase reporter assay was conducted to confirm the target gene of miR-506 in renal cancer cell lines.

Results

miR-506 was significantly down-regulated in renal cancer cell lines and ccRCC specimens. Low miR-506 expression in ccRCC specimens was associated with an advanced clinical stage and poor prognosis. miR-506 expression was an independent prognostic marker of overall ccRCC patient survival in a multivariate analysis. Over-expression of miR-506 in renal cancer cells decreased cell growth and metastasis, In contrast, down-regulation of miR-506 expression promoted renal cancer cell growth and metastasis. FLOT1, a potential target gene of miR-506, was inversely correlated with miR-506 expression in ccRCC tissues. Consistent with the effect of miR-506, knockdown of FLOT1 by siRNA inhibited cell malignant behaviors. Rescue of FLOT1 expression partially restored the effects of miR-506.

Conclusions

miR-506 exerts its anti-cancer function by directly targeting FLOT1 in renal cancer, indicating a potential novel therapeutic role in renal cancer treatment.     

Impact of miR-21, miR-126 and miR-221 as Prognostic Factors of Clear Cell Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava

Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients.     

miR-30b,Down-Regulated in Gastric Cancer,Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1

Background

Gastric cancer is one of the most common malignant diseases worldwide. Emerging evidence has shown that microRNAs (miRNAs) are associated with tumor development and progression. Our previous studies have revealed that H. pylori infection was able to induce the altered expression of miR-30b in gastric epithelial cells. However, little is known about the potential role of miR-30b in gastric cancer.

Methods

We analyzed the expression of miR-30b in gastric cancer cell lines and human gastric cancer tissues. We examined the effect of miR-30b mimics on the apoptosis of gastric cancer cells in vitro by flow cytometry (FCM) and caspase-3/7 activity assays. Nude mouse xenograft model was used to determine whether miR-30b is involved in tumorigenesis of gastric cancer. The target of miR-30b was identified by bioinformatics analysis, luciferase assay and Western blot. Finally, we performed the correlation analysis between miR-30b and its target expression in gastric cancer.

Results

miR-30b was significantly down-regulated in gastric cancer cells and human gastric cancer tissues. Enforced expression of miR-30b promoted the apoptosis of gastric cancer cells in vitro, and miR-30b could significantly inhibit tumorigenicity of gastric cancer by increasing the apoptosis proportion of cancer cells in vivo. Moreover, plasminogen activator inhibitor-1 (PAI-1) was identified as the potential target of miR-30b, and miR-30b level was inversely correlated with PAI-1 expression in gastric cancer. In addition, silencing of PAI-1 was able to phenocopy the effect of miR-30b overexpression on apoptosis regulation of cancer cells, and overexpression of PAI-1 could suppressed the effect of promoting cell apoptosis by miR-30b, indicating PAI-1 is potentially involved in miR-30b-induced apoptosis on cancer cells.

Conclusion

miR-30b may function as a novel tumor suppressor gene in gastric cancer by targeting PAI-1 and regulating the apoptosis of cancer cells. miR-30b could serve as a potential biomarker and therapeutic target against gastric cancer.     

miR-26a Suppresses Tumor Growth and Metastasis by Targeting FGF9 in Gastric Cancer

The role of miR-26a in cancer cells seemed controversial in previous studies. Until now, the role of miR-26a in gastric cancer remains undefined. In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC. We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo. We further identified a novel mechanism of miR-26a to suppress GC growth and metastasis. FGF9 was proved to be a direct target of miR-26a, using luciferase assay and western blot. FGF9 overexpression in miR-26a-expressing cells could rescue invasion and growth defects of miR-26a. In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC. Taken together, our data suggest that miR-26a functions as a tumor suppressor in GC development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for GC.     

Circulating Tumor Cell Composition in Renal Cell Carcinoma

PurposeDue to their minimal-invasive yet potentially current character circulating tumor cells (CTC) might be useful as a “liquid biopsy” in solid tumors. However, successful application in metastatic renal cell carcinoma (mRCC) has been very limited so far. High plasticity and heterogeneity of CTC morphology challenges currently available enrichment and detection techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We recently described a method that enables us to identify and characterize non-hematopoietic cells in the peripheral blood stream with varying characteristics and define CTC subgroups that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize feasibility of this approach and its potential usage in clinical studies.ResultsWe detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics at different time-points during anti-angiogenic therapy. The presence and quantity of N-cadherin-positive or CD133-positive CTC was associated with inferior PFS. There was an inverse correlation between high expression of HIF1A, VEGFA, VEGFR and FGFR and the presence of N-cadherin-positive and CD133-positive CTC.ConclusionsPatients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeutic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic and predictive biomarker in mRCC is warranted.     

LncRNAs Expression Signatures of Renal Clear Cell Carcinoma Revealed by Microarray

Background

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. They are aberrantly expressed in many types of cancers. In this study, we described lncRNAs profiles in 6 pairs of human renal clear cell carcinoma (RCCC) and the corresponding adjacent nontumorous tissues (NT) by microarray.

Methodology/Principal Findings

With abundant and varied probes accounting 33,045 LncRNAs in our microarray, the number of lncRNAs that expressed at a certain level could be detected is 17157. From the data we found there were thousands of lncRNAs that differentially expressed (≥2 fold-change) in RCCC tissues compared with NT and 916 lncRNAs differentially expressed in five or more of six RCCC samples. Compared with NT, many lncRNAs were significantly up-regulated or down-regulated in RCCC. Our data showed that down-regulated lncRNAs were more common than up-regulated ones. ENST00000456816, {"type":"entrez-nucleotide","attrs":{"text":"X91348","term_id":"1418768","term_text":"X91348"}}X91348, {"type":"entrez-nucleotide","attrs":{"text":"BC029135","term_id":"23271405","term_text":"BC029135"}}BC029135, {"type":"entrez-nucleotide","attrs":{"text":"NR_024418","term_id":"212286160","term_text":"NR_024418"}}NR_024418 were evaluated by qPCR in sixty-three pairs of RCCC and NT samples. The four lncRNAs were aberrantly expressed in RCCC compared with matched histologically normal renal tissues.

Conclusions/Significance

Our study is the first one to determine genome-wide lncRNAs expression patterns in RCCC by microarray. The results displayed that clusters of lncRNAs were aberrantly expressed in RCCC compared with NT samples, which revealed that lncRNAs differentially expressed in tumor tissues and normal tissues may exert a partial or key role in tumor development. Taken together, this study may provide potential targets for future treatment of RCCC and novel insights into cancer biology.     

hepaCAM和VEGF基因表达与肾透明细胞癌侵袭转移关系的探讨

研究肾癌细胞株786-0,RC-2及肾透明细胞癌组织中肝细胞黏附分子(hepatocyte cell adhesion molecule,hepaC-AM)和血管内皮生长因子(VEGF)mRNA表达及其与肾透明细胞癌侵袭转移的关系。应用逆转录聚合酶链反应(RT-PCR)检测786-0、RC-2、正常肾组织hepaCAM和VEGFmRNA表达,73例肾透明细胞癌组织及相应癌旁组织中hepaCAMmRNA表达,43例肾透明细胞癌组织及相应癌旁组织VEGFmRNA表达,并比较它们之间的差异性和相关性。与正常肾组织比较786-0,RC-2的hepaCAMmRNA显著降低(P0.05);VEGFmRNA显著升高(P0.05)。肾透明细胞癌组织hepaCAMmRNA显著低于癌旁组织(P0.05);VEGFmRNA显著高于癌旁组织(P0.05)。在肾透明细胞癌组织中临床Ⅰ+Ⅱ期和Ⅲ+Ⅳ期两组VEGFmRNA表达差异具有统计学意义(P0.05),hepaCAM与VEGFmRNA呈负相关(r=-0.329,P0.05)。提示hepaC-AM基因缺失可能参与肾透明细胞癌侵袭转移,其机制可能与调节VFGF表达改变有关,hepaCAM有望成为一种新的肾癌基因治疗的靶分子。     

Computational Modelling of Metastasis Development in Renal Cell Carcinoma

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.     

Genetic Variants in RKIP Are Associated with Clear Cell Renal Cell Carcinoma Risk in a Chinese Population

Background

Raf-1 kinase inhibitor protein (RKIP) plays a critical role in tumor development by regulating cell functions such as invasion, apoptosis and differentiation. Down-regulation of RKIP expression has been implicated in the development and progression of renal cell carcinoma (RCC). Herein, we hypothesized that genetic polymorphisms in RKIP might be associated with susceptibility and progression of RCC.

Methods

A total of 5 tagging single-nucleotide polymorphisms (tSNPs) in RKIP were selected and genotyped by SNapShot method in a case-control study of 859 RCC patients and 1004 controls. The logistic regression was used to evaluate the genetic association with occurrence and progression of RCC. The functionality of the important SNP was preliminary examined by qRT-PCR.

Result

We found that the rs17512051 in the promoter region of RKIP was significantly associated with decreased clear cell RCC (ccRCC) risk (TA/AA vs. TT: P = 0.039, OR = 0.78, 95%CI = 0.62–0.99). Another SNP (rs1051470) in the 3′UTR region of RKIP was marginally associated with increased ccRCC risk (TT vs. CC+CT: OR = 1.45, 95%CI = 1.01–2.09). In the stratified analysis, the protective effect of rs17512051 was more predominant in the subgroups of male, non-smokers, non-drinkers as well as subjects without history of diabetes. Furthermore, we observed higher RKIP mRNA levels in the presence of the rs17512051A allele in normal renal tissues.

Conclusion

Our results suggest that the potentially functional RKIP rs17512051 polymorphism may affect ccRCC susceptibility through altering the endogenous RKIP expression level. Risk effects and the functional impact of this polymorphism need further validation.     

Blood Levels of Carbonic Anhydrase 9 Correlate with Clear Cell Renal Cell Carcinoma Activity

Introduction  Biomarkers for early detection of renal cell carcinoma (RCC) may help diagnose minimal residual disease in patients at risk for RCC, can guide anti-angiogenic therapy, or may help identify candidates for adjuvant treatment. In this study, we investigated whether blood levels of carbonic anhydrase 9 (CA9) correlate with RCC tumor burden and therefore disease activity. Methods  CA9 is a von Hippel–Lindau–hypoxia inducible factor target upregulated in clear cell RCC. We used an anti-CA9 antibody (M75)-based enzyme-linked immunosorbent assay test to measure CA9 levels in blood obtained before and after nephrectomy for clinically localized disease in patients with: (1) clear cell RCC, (2) papillary and chromophobe RCC or oncocytoma, or (3) benign kidney lesions, and we compared these samples to blood drawn from normal control individuals. Results  We observed a significant (p < 0.006) decrease in the blood levels of CA9, after nephrectomy for localized disease, in the majority of patients with clear cell RCC (57%). In contrast, patients with nonclear cell RCC, benign disease, or those having undergone debulking nephrectomy for metastatic disease did not have a decrease in CA9 blood levels after nephrectomy. Preliminary longitudinal follow up measurements of CA9 levels in a small group of patients indicated that rising CA9 levels may correlate with disease progression. Conclusions  Plasma CA9 levels correlate with disease activity in a subset of clear cell RCC patients and should be considered in future multiplex RCC biomarker development algorithms.     

Piwi-Interacting RNAs (piRNAs) Are Dysregulated in Renal Cell Carcinoma and Associated with Tumor Metastasis and Cancer-Specific Survival

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.     

The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

MicroRNAs (miRNAs) are a family of small RNA molecules that negatively regulate the expression of protein-coding genes and play critical roles in orchestrating diverse cellular processes. This regulatory mechanism is also exploited by viruses to direct their life cycle and evade the host immune system. Epstein-Barr virus (EBV) is an oncogenic virus that is closely associated with multiple human diseases, including nasopharyngeal carcinoma (NPC), which is a highly metastatic type of tumor and is frequently reported in South Asia. Several viral proteins have been found to promote the migration and invasiveness of NPC cells. However, not all tumor tissues express these viral oncoproteins, suggesting that other mechanisms may contribute to the aggressive behavior of NPC tumor cells. A previous sequencing study by our group revealed that the EBV miRNA miR-BART9 was expressed at high levels in all EBV-positive NPC tissues. In the present study, we used gain- and loss-of-function approaches to investigate the effect of miR-BART9 in EBV-negative and EBV-positive NPC cells. We discovered that miR-BART9 promotes the migration and invasiveness of cultured NPC cells. The promigratory activity observed in vitro was manifested as an enhanced metastatic ability in vivo. Computational analysis revealed that miR-BART9 may target E-cadherin, a membrane protein that is pivotal in preserving cell-cell junctions and the epithelial phenotype. Through biochemical assays and functional rescue analysis, we confirmed that miR-BART9 specifically inhibits E-cadherin to induce a mesenchymal-like phenotype and promote the migration of NPC cells. These results indicated that miR-BART9 is a prometastatic viral miRNA and suggested that high levels of miR-BART9 in EBV-positive NPC cells may contribute to the aggressiveness of tumor cells.     

Overexpression of Wip1 Is Associated with Biologic Behavior in Human Clear Cell Renal Cell Carcinoma

Wild-type p53-induced phosphatase (Wip1 or PPM1D) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of Wip1 in RCC remains unclear. The present study investigated its abnormal expression and dysfunctions in clear cell renal cell carcinoma (ccRCC) in vitro. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of Wip1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of Wip1 was significantly associated with depth of invasion (P<0.001), Distant metastasis (P = 0.001), lymph node status (P<0.001) and Fuhrman grade (P<0.001). Wip1 knockdown inhibited the proliferation, migration and invasion of 786-O and RLC-310 cells, whereas Wip1 overexpression promoted the growth and aggressive phenotype of 786-O and RLC-310 cells in vitro. The uni- and multivariate analyses indicated that expression of Wip1 was an independent predictor for survival of ccRCC patients (P = 0.003, P = 0.027 respectively). Wip1- negative patients had a higher tumor-free/overall survival rate than patients with high Wip1 expression (P = 0.001, P = 0.002 respectively). Overexpression of Wip1 is useful in the prediction of survival in ccRCC patients.     

Assessment of Intratumoral Micromorphology for Patients with Clear Cell Renal Cell Carcinoma Using Susceptibility-Weighted Imaging

Background

Multiple treatment options exist for the management of renal cell carcinomas. Preoperative evaluation of clear cell renal cell carcinoma (CRCC) grades is important for deciding upon the appropriate method of therapy. We hypothesize that susceptibility weighted imaging (SWI) is sensitive enough to detect intratumoral microvessles and microbleeding in renal cell carcinoma, which can be used to grade CRCC.

Material and Methods

Retrospective reviews of 37 patients with pathologically proven CRCCs were evaluated. All patients underwent SWI examinations. The characteristics of intratumoral susceptibility signal intensity (ITSS) includes the likelihood of the presence of ITSS, morphology of ITSS, dominant structure of ITSS and ratio of ITSS area to tumor area, which were all assessed on SWI. The results were compared using the nonparametric Mann-Whitney test.

Results

ITSS was seen in all patients except 4 patients with low-grade CRCCs. There was no significant difference between low and high-grade CRCCs when looking at the likelihood of the presence of ITSS. There was a significant difference in the mean score of dominant structures between low and high-grade CRCCs. Specifically, more dominant vascular structures and less hemorrhage were seen in low-grade tumors (2.15±1.05) compared to high-grade tumors (1.27±0.47) (P<0.005). The ratio of ITSS area to tumor area was also significantly higher for the high-grade group (1.55±0.52) than that for the low-grade group (0.88±0.43) on SWI (P<0.005).

Conclusion

SWI is useful for grading CRCCs.     

Regulation of Renal Cell Carcinoma Cell Proliferation,Invasion and Metastasis by Connexin 32 Gene

Gap junctions composed of connexin (Cx), a large protein family with a number of subtypes, are a main apparatus to maintain cellular homeostasis in many organs. Gap junctional intercellular communication (GJIC) is actively involved in all aspects of the cellular life cycle, ranging from cell growth to cell death. It is also known that the Cx gene acts as a tumor-suppressor due to the maintenance of cellular homeostasis via GJIC. In addition to this function, recent data show that the GJIC-independent function of Cx gene contributes to the tumor-suppressive effect of the gene with specificity to certain cells. With respect to the tumor-suppressive effects, Cx genes acts as tumor-suppressors in primary cancers, but the effects are still conflicting in invasive and metastatic cancers. We have previously reported that Cx32 is specifically downregulated in human renal cell carcinoma (RCC) cell lines as well as cancerous regions when compared to normal regions in kidneys. In recent studies, we have also reported that Cx32 suppresses growth, invasion and metastasis of RCC cells. In this minireview, we refer to a new aspect of Cx32-dependent functions against cell proliferation, invasion and metastasis in RCC cells, especially in a GJIC-independent manner.     

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.