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Specificity of TFE3 antibody across TFE3 oncoproteins

A complete list of targeted genes in the PI3K/AKT signaling pathway with their associated miRNA from cluster 3.

Comprehensive panels of 287 KEGG pathways associated identified by bioinformatics analysis of TFE3 ChIP-seq peaks in RP-R07 cells.

Comprehensive panels of 96 PANTHER pathways identified by bioinformatics analysis of TFE3 ChIP-seq peaks in RP-R07 cells.

Quality control for ChIP-seq

Purpose:Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease.Experimental Design:We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models.Results:The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3–tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation.Conclusions:These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3–tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3–tRCC.

Pie chart for TFE3 binding sites

Lists of statistically enriched pathways targeted by differential expression of miRNA in cluster 3.

Effect of different rapamycin concentrations

Enriched KEGG PI3K/AKT signaling pathway visualization

Pie chart for TFE3 binding sites

Comprehensive panels of 403 WIKI tools pathways identified by bioinformatics analysis of TFE3 ChIP-seq peaks in RP-R07 cells.

ChiP-seq for TFE3 in UOK-146

Schema for multimodal inhibition in tRCC

Purpose: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. Experimental Design: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. Results: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3–tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3–tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3–tRCC.

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors the most prevalent of which are clear cell, papillary, and chromophobe RCC. Accurate distinction between the typically benign-behaving renal oncocytoma and RCC subtypes is a frequent challenge for pathologists. This is critical for clinical decision making. Subtypes also have different survival outcomes and responses to therapy. We extracted RNA from ninety formalin-fixed paraffin-embedded (FFPE) tissues (27 clear cell, 29 papillary, 19 chromophobe, 4 unclassified RCC and 11 oncocytomas). We quantified the expression of six miRNAs (miR-221, miR-222, miR-126, miR-182, miR-200b and miR-200c) by qRT-PCR, and by in situ hybridization in an independent set of tumors. We developed a two-step classifier. In the first step, it uses expression of either miR-221 or miR-222 to distinguish the clear cell and papillary subtypes from chromophobe RCC and oncocytoma (miR-221 AUC: 0.96, 95% CI: 0.9132-1.014, p < 0.0001 and miR-222 AUC: 0.91, 95% CI: 0.8478-0.9772, p < 0.0001). In the second step, it uses miR-126 to discriminate clear cell from papillary RCC (AUC: 1, p < 0.0001) and miR-200b to discriminate chromophobe RCC from oncocytoma (AUC: 0.95, 95% CI: 0.8933-1.021, p < 0.0001). In situ hybridization showed a nuclear staining pattern. miR-126, miR-222 and miR-200b were significantly differentially expressed between the subtypes by in situ hybridization. miRNA expression could distinguish RCC subtypes and oncocytoma. miRNA expression assessed by either PCR or in situ hybridization can be a clinically useful diagnostic tool to complement morphologic renal tumor classification, improving diagnosis and patient management.

Objectives microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by mRNA cleavage or translational repression. The miR-200 family is involved in the regulation of various tumor biologic processes including apoptosis, proliferation, invasion, and metastasis. They function mainly as tumor suppressors. In this study, we aim to validate the prognostic significance of miR-200 family using large cohort of primary clear cell renal cell carcinoma (ccRCC) and matched normal tissue and to explore the role of miR-200 family in RCC pathogenesis and progression. Materials and Methods We analyzed the expression of 3 members of the miR-200 family; miR-141, miR-200b, and miR-200c, between primary ccRCC, matched normal renal tissues, and nonmatched metastatic RCC. We compared clinicopathologic parameter including disease-free survival to miR-200 family expression. Additionally, we validated our results using The Cancer Genome Atlas dataset. We explored functional role of these miRNAs by bioinformatics analyses. Results and Conclusions Expression of miR-200 family significantly decreased in cancer compared to non-neoplastic tissues. miR-141 and miR-200b were significantly down-regulated in metastatic than primary tumors. There was statistically significant negative association between all 3 miRNAs and tumor size and stage. As binary variables, univariate analyses revealed that miR-141, miR-200b, and miR-200c-positive ccRCC patients have a statistically significant lower chance of disease-recurrence or relapse and multivariate analyses showed miR-200b and miR-200c-positive patients have longer disease-free survival. We could predict disease-free survival better when 2 or more miRNAs were used as a combination. Overall survival analysis using The Cancer Genome Atlas data revealed that miR-200b-positive patients have significantly better survival. These results suggest that miR-141, miR-200b, and miR-200c are independent prognostic markers for ccRCC. Targets of these miRNAs are associated with pathways related to cancer invasion and metastasis, including TRAIL pathway, VEGF and VEGFR signaling network, and epithelial-mesenchymal transition.

Apart from an invasive biopsy, currently no tools are available to confirm the diagnosis of clear cell renal cell carcinoma (ccRCC); this resulted in approximately 30% of patients being diagnosed with metastatic disease.To determine whether urinary microRNAs (miRNAs) can serve as biomarkers to confirm the diagnosis of ccRCC.Global miRNA expression was assessed in 28 preoperative urine samples from patients with ccRCC and 18 healthy participants. The independent validation set consisted of 81 ccRCC patients, 24 patients with benign lesions, and 33 healthy participants. We extracted both cell-free and exosomal RNA for miRNA expression analysis using miRNA-specific polymerase chain reaction assays. We also investigated exosomal miRNA secretion in cell line models and performed exosome transfer between RCC and endothelial cell types.Receiver operating characteristic analysis was applied to identify the discrimination power of miRNAs.Overall, miR-126-3p combined with miR-449a or with miR-34b-5p could significantly distinguish ccRCC patients from healthy participants (miR-126-3p-miR-449a: area under the curve [AUC]: 0.84; 95% confidence interval [CI], 0.7620-0.9151; p0.001; miR-126-3p-miR-34b-5p: AUC: 0.79; 95% CI, 0.7013-0.8815; p0.001). The combination of miR-126-3p and miR-34b-5p was also able to distinguish small renal masses (pT1a, ≤4cm) from healthy controls (AUC: 0.79; 95% CI, 0.6848-0.8980; p0.001). Using miR-126-3p and miR-486-5p in combination, we were able to differentiate between benign lesions and ccRCC (AUC: 0.85; 95% CI, 0.7295-0.9615; p0.01). The expression of a number of miRNAs returned to a level comparable with health after surgery. Kidney cancer cell lines were found to secrete exosomal miR-126-3p, miR-17-5p, miR-21-3p, and miR-25-3p, and these miRNAs were found to be internalized by other cell types.We identified exosomal miRNAs as potential noninvasive diagnostic urinary biomarkers for ccRCC and provided evidence that miRNAs are secreted by the tumor and can function as a tool for intercellular communication.We identified urinary microRNAs that can serve as diagnostic biomarkers for clear cell renal cell carcinoma.

Sunitinib is a multitargeting tyrosine kinase inhibitor used for metastatic renal cancer. There are no biomarkers that can predict sunitinib response. Such markers are needed to avoid administration of costly medication with side effects to patients who would not benefit from it. We compared global miRNA expression between patients with a short (≤12 months) versus prolonged (>12 months) progression-free survival (PFS) under sunitinib as first-line therapy for metastatic renal cell carcinoma. We identified a number of differentially expressed miRNAs and developed miRNA statistical models that can accurately distinguish between the two groups. We validated our models in the discovery set and an independent set of 57 patients. Target prediction and pathway analysis showed that these miRNAs are involved in vascular endothelial growth factor (VEGF), TGFβ, and mammalian target of rapamycin (mTOR)-mediated signaling and cell–cell communication. We tested the effect of these miRNAs on cellular proliferation and angiogenesis. We validated the negative correlation between miR-221 and its target, VEGFR2.miR-221 overexpression was associated with a poor PFS while its target, VEGFR2 was associated with longer survival. Gain of function experiments showed that miR-221 and miR-222 decreased angiogenesis and cellular proliferation in human umbilical vein endothelial cells (HUVEC) while increasing cellular proliferation in ACHN cells. miRNAs represent potential predictive markers for sunitinib response.

// Henriett Butz 1,2 , Peter M. Szabo 3 , Heba W.Z. Khella 1,2 , Roy Nofech-Mozes 1 , Attila Patocs 4 and George M. Yousef 1,2 1 Department of Laboratory Medicine and The Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, Toronto, Canada 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 3 Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA 4 HAS-SE “Lendulet” Hereditary Endocrine Tumors Research Group, Hungarian Academy of Sciences, Hungary Correspondence to: George M. Yousef, email: // Keywords : renal cell carcinoma, miR-124-3p, integrated analysis, CAV1, FLOT1 Received : February 18, 2015 Accepted : March 11, 2015 Published : April 14, 2015 Abstract Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with frequent metastatic rate and poor survival. Integrated analyses allow understanding the interplay between different levels of molecular alterations. We integrated miRNA and gene expression data from 458 ccRCC and 254 normal kidney specimens to construct a miRNA-target interaction network. We identified the downregulated miR-124-3p, -30a-5p and -200c-3p as the most influential miRNAs in RCC pathogenesis.miR-124-3p and miR-200c-3p expression showed association with patient survival, miR-30a-5p was downregulated in metastases compared to primary tumors. We used an independent set of 87 matched samples for validation. We confirmed the functional impact of these miRNAs by in vitro assays. Restoration of these miRNAs reduced migration, invasion and proliferation. miR-124-3p decreased the S phase of cell cycle, as well. We compared transcriptome profiling before and after miRNA overexpression, and validated CAV1 and FLOT1 as miR-124-3p targets. Patients with higher CAV1 and FLOT1 had lower miR-124-3p expression and shorter overall survival. We hypothesize that these three miRNAs are fundamental contributing to ccRCC aggressive/metastatic behavior; and miR-124-3p especially has a key role through regulating CAV1 and FLOT1 expression. Restoration of the levels of these miRNAs could be considered as a potential therapeutic strategy for ccRCC.

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with unpredictable behavior. Clinical parameters are not always accurate for predicting prognosis. miR-126 is differentially expressed in many cancers, including RCC, and is down-regulated in metastatic versus primary ccRCC. We assessed the prognostic significance of miR-126 in 264 primary ccRCCs. We also compared its expression in normal kidney, primary and metastatic ccRCC, and RCC subtypes. We validated our results on an independent set of 481 ccRCCs. miR-126 was down-regulated in metastatic versus primary tumors and in tumors of higher stage (P = 0.005) or higher grade (P = 0.002). miR-126 up-regulation was associated with significantly prolonged disease-free survival (P 0.001) and overall survival (P = 0.015). For larger tumors (4 cm), patients with higher miR-126 expression had significantly longer survival. Restoration of miR-126 expression decreased cellular migration and proliferation in RCC cell lines. The ccRCCs exhibited the highest miR-126 expression, and papillary RCCs exhibited the lowest expression. We identified a number of miR-126 targets and pathways that are involved in carcinogenesis, including the apoptosis signaling pathway. miR-126 is a promising prognostic marker in ccRCC that can distinguish between clear cell and papillary subtypes. In addition, miR-126 has potential therapeutic applications.

MicroRNAs (miRNAs) are short noncoding RNA molecules that function by negatively regulating the expression of their target genes in a tightly controlled manner. Accumulating evidence, based in part on effects seen after miRNA overexpression and/or knockdown, points to the critical involvement of miRNAs in kidney function in health and disease. In this review, we provide a quick overview of the biogenesis of miRNAs and their potential involvement in kidney development and normal function. We also discuss the current literature that has begun to uncover the role of miRNAs in the pathogenesis of kidney diseases, including diabetic nephropathy, hypertension, glomerulonephritis, and cancer. As such, miRNAs have potential utility in the clinical realm as disease biomarkers. Moreover, miRNAs represent an attractive therapeutic target for a number of kidney diseases. We close by discussing a number of potential challenges that face the field of miRNA research and clinical use.

Clear cell renal cell carcinoma (ccRCC) is the most common adult kidney cancer. It is an aggressive tumour with unpredictable outcome. The currently used clinical parameters are not always accurate for predicting disease behaviour. miR-10b is dysregulated in different malignancies including RCC.We assessed the clinical utility of miR-10b as a prognostic marker in 250 patients with primary ccRCC. We examined the correlation between miR-10b and clinicopathological parameters. We compared miR-10b expression among different RCC subtypes and normal kidney tissue.We observed a stepwise decrease of miR-10b expression from normal kidney to primary ccRCC and a further decrease from primary to metastatic RCC. miR-10b expression was significantly lower in stages III/IV compared with stages I/II (p=0.038). Using a binary cut-off, miR-10b-positive patients had significantly longer disease-free survival (HR=0.47, CI 0.28 to 0.79, p=0.004). In the subgroup of patients with tumour size4 cm, higher miR-10b expression was associated with significant longer disease-free and overall survival (p=0.001 and p=0.036, respectively). miR-10b was significantly downregulated in ccRCC compared with normal kidney (p0.0001), and oncocytoma (p=0.031). It was also downregulated in chromophobe RCC. In addition, we identified a number of miR-10b-predicted targets and pathways that are involved in tumourigenesis.Our data point to miR-10b as a promising prognostic marker in ccRCC with potential therapeutic applications.

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. Increasing evidence suggests that microRNAs are dysregulated in RCC and are important factors in RCC pathogenesis. miR-21 is a known oncogene with tumor-promoting effects in many types of cancer. In this study, we analyzed miR-21 in 121 cases of healthy kidney and different RCC subtypes, including clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), and oncocytoma. Total RNA was extracted, and the expression of miR-21 was measured with real-time quantitative RT-PCR using miR-21-specific probes. The expression of miR-21 was significantly up-regulated in RCC compared with healthy kidney. There was a significant difference in the expression levels between RCC subtypes, with the highest levels of expression in ccRCC and pRCC subtypes. miR-21 expression distinguished ccRCC and pRCC from chRCC and oncocytoma with 90% specificity (95% CI, 63.9% to 98.1%) and 83% sensitivity (95% CI, 53.5% to 97.6%). Significantly higher miR-21 levels were associated with higher stage and grade. Patients who were miR-21 positive had statistically significant shorter disease-free and overall survival rates. Thus, miR-21 is up-regulated in RCC, and its expression levels can be used as a diagnostic marker to distinguish ccRCC and pRCC from chRCC and oncocytoma. Moreover, it has potential as a prognostic marker in RCC, although it is not independent of tumor stage and grade.

Background: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic RCC is ⩽10%. Recently, microRNAs (miRNAs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer. Method: We performed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared with primary RCCs. We validated our results by quantitative real-time PCR. We performed experimental and bioinformatic analyses to explore the involvement of miR-215 in RCC progression and metastasis. Results: We identified 65 miRNAs that were significantly altered in metastatic compared with primary RCCs. We validated our results by examining the expression of miR-10b, miR-126, miR-196a, miR-204 and miR-215, in two independent cohorts of patients. We showed that overexpression of miR-215 decreased cellular migration and invasion in an RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumour metastasis. Conclusion: Our analysis showed that miRNAs are altered in metastatic RCCs and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent adult kidney cancer. Prognostic markers are needed to guide patient management toward aggressive versus more conservative approaches, especially for small tumors ≤4 cm. miR‐194 was reported to be downregulated in several cancers and is involved in epithelial to mesenchymal transition. We evaluated miR‐194 as a prognostic marker in ccRCC. In a cohort of 234 patients with primary ccRCC, we correlated miR‐194 expression level with multiple clinicopathological features including disease‐free and overall survival, tumor size, clinical stage, and histological grade. Our results shows a stepwise decrease in miR‐194 expression from normal kidney to primary ccRCC (P = 0.0032) and a subsequent decrease from primary to metastatic lesions. Additionally, patients with higher miR‐194 expression has significantly longer disease‐free survival (P = 0.041) and overall survival (P = 0.031) compared to those with lower expression. In multivariate analysis, miR‐194‐positive tumors retain significance in disease‐free survival and overall survival, suggesting miR‐194 is an independent marker for good prognosis in ccRCC. Moreover, miR‐194 is a marker for good prognosis for patients with small renal masses (P = 0.014). These findings were validated on an independent data set from The Cancer Genome Atlas. We also compared miR‐194 expression between RCC subtypes. ccRCC had the highest levels, whereas chromophobe RCC and oncocytoma had comparable lower levels. Target prediction coupled with pathway analysis show that miR‐194 is predicted to target key molecules and pathways involved in RCC progression. miR‐194 represents a prognostic biomarker in ccRCC.

Kallikrein-related peptidase 5 (KLK5) displays aberrant expression in cancer. Recently, we showed KLK5 reconstitution in breast cancer cell lines suppresses malignancy. Present study aims to investigate the functional KLK5 mediated miRNA network on breast cancer progression, molecular subtype and survival. 28 miRNAs were up-regulated and 62 miRNAs were down-regulated upon KLK5 expression. Extracellular matrix (ECM) molecules and cell-adhesion pathways were the most significant KLK5-induced miRNA-mediated regulatory targets. Validation from The Cancer Genome Atlas (TCGA) database indicated KLK5 was specifically down-regulated in luminal B and basal-like breast cancer subtypes. There was a correlation between KLK5, miRNAs and their downstream ECM gene targets. Long-term patient survival correlated with dysregulation of KLK5 and interacting ECM target genes. It suggests biological differences between breast cancer molecular subtypes, patient survival, and their propensity for invasion and metastasis can be explained in part by altered miRNA networks induced by KLK5 dysregulation. We provide the first evidence that KLK5 can affect miRNA networks, which regulate MMPs and other novel ECM targets and a new compelling hypothesis of interplay between serine proteases and miRNAs. We developed a combined KLK5-(ITGB1+COL12A1) predictive score for recurrence-free survival that could be exploited in clinical applications.

Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. The results were validated with an independent data set from The Cancer Genome Atlas. miR-210 was significantly overexpressed in ccRCC compared with normal kidney. miR-210(+) patients had a statistically higher chance of disease recurrence [hazard ratio (HR), 1.82; P = 0.018] and shorter overall survival (HR, 2.46; P = 0.014). In multivariate analysis, miR-210 lost its statistically significant association with shorter disease-free survival and overall survival after adjusting for tumor size and tumor, node, metastasis stage. Papillary RCC showed comparable miR-210 overexpression, whereas decreased up-regulation was seen in chromophobe RCC and oncocytoma. A number of predicted targets that might be involved in carcinogenesis and aggressive tumor behavior were identified. miR-210 is a potential therapeutic target and independent marker of poor prognosis of ccRCC.

Metastasis results in most of the cancer deaths in clear cell renal cell carcinoma (ccRCC). MicroRNAs (miRNAs) regulate many important cell functions and play important roles in tumor development, metastasis and progression. In our previous study, we identified a miRNA signature for metastatic RCC. In this study, we validated the top differentially expressed miRNAs on matched primary and metastatic ccRCC pairs by quantitative polymerase chain reaction. We performed bioinformatics analyses including target prediction and combinatorial analysis of previously reported miRNAs involved in tumour progression and metastasis. We also examined the co-expression of the miRNAs clusters and compared expression of intronic miRNAs and their host genes. We observed significant dysregulation between primary and metastatic tumours from the same patient. This indicates that, at least in part, the metastatic signature develops gradually during tumour progression. We identified metastasis-dysregulated miRNAs that can target a number of genes previously found to be involved in metastasis of kidney cancer as well as other malignancies. In addition, we found a negative correlation of expression of miR-126 and its target vascular endothelial growth factor (VEGF)-A. Cluster analysis showed that members of the same miRNA cluster follow the same expression pattern, suggesting the presence of a locus control regulation. We also observed a positive correlation of expression between intronic miRNAs and their host genes, thus revealing another potential control mechanism for miRNAs. Many of the significantly dysregulated miRNAs in metastatic ccRCC are highly conserved among species. Our analysis suggests that miRNAs are involved in ccRCC metastasis and may represent potential biomarkers.

385 Background: miRNAs play a crucial rule in tumor progression and metastasis. We previously identified miR-192, miR-194 and miR-215 to be down-regulated in metastatic compared to primary clear cell renal cell carcinoma (ccRCC). In this work, we examine the role of miR-192, miR-194, and miR-215 in RCC progression and aggressiveness. Methods: We examined the role of these three miRNAs on tumor cell migration and invasion abilities using RCC cell line models. We performed target prediction analysis and experimentally validated the targets using independent approaches. In addition, we examined the clinical utility of miR-215 as a potential prognostic marker in RCC by measuring miR-215 expression using qRT-PCR in 61 formalin-fixed paraffin-embedded tissues from primary ccRCC and correlated the expression levels with clinical outcome. Results: Restoration of miR-192, miR-194, and miR-215 expression decreased cell migration and invasion in RCC cell lines. Target prediction analysis identified three potential targets of these miRNAs; MDM2, TYMS, and SIP1/ZEB2. We validated the miRNA-target interaction experimentally using three approaches. First by measuring the effect of miRNA overexpression on mRNA and protein levels of the predicted target, then by measuring the effect of miRNA overexpression on a luciferase signal of a vector containing the 3’UTR of the predicted target, and finally, by validating these interactions in vivoby examining the presence of an inverse correlation between miRNA changes and the expression levels of their targets on clinical specimens. In 61 patients with resected ccRCC tumors, we found that low miR-215 expression in the primary was associated with a significantly reduced recurrence-free survival. (26.4 vs. 49.2 months, respectively, p = 0.0320). Conclusions: Our analysis showed that miR-192, miR-194, and miR-215 are involved in RCC metastasis and that miR-215 predicts for recurrence in patients with resected RCC. Our findings pave the way to the clinical use of miRNAs as prognostic markers and potential therapeutic targets.

4633 Background: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The five-year survival rate for metastatic RCC is

392 Background: Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The five-year survival rate for metastatic RCC is Methods: We preformed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared to primary RCC. Results were validated by quantitative real time PCR. Target prediction analysis and gene expression profiling identified many of the dysregulated miRNAs could target genes involved in tumor metastasis. The effect of miR-215 on cellular migration and invasion was shown in a RCC cell line model. Results: We identified 65 miRNAs that were significantly altered in metastatic when compared to primary RCC. Nine (14%) miRNAs had increased expression while 56 (86%) miRNAs showed decreased expression. miR-10b, miR-196a, and miR-27b were the most downregulated while miR-638, miR-1915, and miR-149* were the most upregulated. A non-supervised 2D-cluster analysis showed that a sub-group of the primary tumors clustered under the metastatic arm with a group of miRNAs that follow the same pattern of expression suggesting they have an inherited aggressive signature. We validated our results by examining the expressions of miR-10b, miR-126, miR-196a, miR-204, and miR-215, in two independent cohorts of patients. We also showed that overexpression of miR-215 decreased cellular migration and invasion in a RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumor metastasis. Conclusions: Our analysis showed that miRNAs are altered in metastatic RCC and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.