Adjacent Normal Kidney Research Articles

PAX-8 expression in renal tumours and distant sites: A useful marker of primary and metastatic renal cell carcinoma?

AimsImmunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice,...

Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma

BackgroundGalectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC.MethodsTissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression.ResultsThe expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059).ConclusionThe mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent.

The Expression and Evaluation of Androgen Receptor in Human Renal Cell Carcinoma

To investigate the expression of androgen receptor (AR) with clinical and pathologic features in patients with renal cell carcinoma (RCC) and to explore the function of AR using human RCC cells. The expression of AR was detected by immunohistochemistry in 44 adjacent normal kidney tissues of 120 RCC patients and also in 16 metastatic RCC patients with their respective primary and metastatic tissue samples. The expression of AR was examined by western blot in commonly used human RCC cell lines and normal kidney epithelial cells, and the luciferase assay was performed in those AR-positive RCC cells. The expression rate of AR was higher in adjacent normal kidney (90.9%) than in RCC tissues (30.0%, P <.001), and it was negatively associated with pT stage and Fuhrman's grade. Specifically, there were 40.7% AR-positive cases in pT1 compared with 8.0% in pT3 (P = .013), and 50.0% of grade I cases were found to be AR positive compared with 12.9% in grade III (P = .008). AR expression was slightly higher in primary RCC tissues (12.5%) than their respective metastases (0%, P = .484). AR strongly expressed in CAKI-2 and OSRC-2 cells with little transactivation, which might indicate that AR in those 2 RCC cells has little function. Our results suggest that any attempt to investigate the roles of AR in RCC progression might need to combine the detection of AR expression in tissue samples with examining its function to make a correct correlation between AR and RCC progression.

ALS2CL gene expression in clear-cell renal cell carcinoma and the implication

Objective To study the differential expression of ALS2CL in clear cell renal cell carcinoma (ccRCC) and normal kidney tissue and the implication.Methods Real-time quantitative polymerase chain reaction (Real-time PCR),immunohistochemistry (IPH) and Western blotting were applied to examine the expression status of ALS2CL in 26 ccRCC tumor and adjacent normal kidney tissue samples as well as two kinds of ccRCC-derived cell lines (786/O,SN12-PM6).Results As compared with normal control group,the two ccRCC-derived cell lines (786/O,SN12-PM6) and 65.38％ (17/26) of ccRCC alls showed an obvious down-regulated expression of ALS2CL mRNA.Under the examination of ALS2CL protein by IPH,73.08％ (19/26) of ccRCC specimens were negative or weakly positive.Western blotting also showed a highly down-regulated rate-53.85％ (14/26) in ccRCC samples,and the low expression of ALS2CL was also found in 786/O and SN12-PM6 cells.All of them showed statistically significant difference between ccRCC group and corresponding normal control group (P ＜ 0.05).Conclusion The absence or downregulated expression of ALS2CL may be closely related to the occurrence of ccRCC. Key words: Clear cell renal cell carcinoma; Gene expression; Tumor suppressor genes

Abstract 4189: Tumor suppressive microRNA-218 regulates oncogenes of focal adhesion pathways in renal cell carcinoma.

Abstract INTRODUCTION&amp;OBJECTIVES: Our recent microRNA (miRNA) expression signature of renal cell carcinoma (RCC) revealed downregulation of miR-218 in RCC, suggesting that it might be a tumor suppressor. The aim of this study is to investigate the functional significance of miR-218 and to identifymiR-218-mediated cancer pathways in RCC. METHODS: We evaluated miR-218 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We performed gain-of-function studies (cell migration and cell invasion assays) by using miR-218 transfectants (TFs). Oligo-microarray analyses of the miR-218 TFs and the RCC specimens were carried out to identify molecular targets of miR-218. According to GENECODIS software, we focused on the “focal adhesion”pathway, in which caveolin-2 (CAV2) was a candidate target gene. A luciferase reporter assay was performed to determine miR-218binding sites with CAV2 3’UTR. Loss-of-function assays using si-CAV2 were performed to investigate the functional significance of CAV2. The expression of CAV2 mRNA and protein was evaluated by qRT-PCR and immunohistochemistry. Furthermore, to investigate signaling pathways regulated by CAV2, we performed gene expression analysis of si-CAV2TFs. RESULTS: The expression of miR-218 was significantly reduced in the RCC specimens. Significant inhibitions of cell migration and invasion were observed in the miR-218 TFs. Gene expression analysis showed that 615 genes were downregulated in the miR-218 TFs. The GENECODIS software revealed that miR-218 appeared to regulate 25 pathways (P&amp;lt;0.0001). In these pathways, we focused on ten genes in “focal adhesion” and seven genes in “tight junction”. CAV2 was the most upregulated gene among the genes related to these pathways by using available data sets (GSE36895 and GSE22541) in gene expression omnibus (GEO). Luciferase reporter assay showed that CAV2 was directly regulated by miR-218. Silencing study of CAV2 demonstrated significant inhibition of cell migration and invasion. The mRNA and protein expression of CAV2 were significantly up-regulated in RCC specimens. Gene expression analysis of si-CAV2 TFs revealed that CAV2 might control 15 pathways including “tight junction” pathway, containing pro-metastatic genes such as claudin-1, AKT3 and R-Ras2. CONCLUSION: Tumor suppressive miR-218contributes to inhibition of cell migration and invasion through regulating focal adhesion pathway, especially CAV2 in RCC. This is the first report that CAV2 contributes to metastatic processes in RCC. Citation Format: Hideki Enokida, Takeshi Yamasaki, Hirofumi Yoshino, Hideo Hidaka, Toshihiko Itesako, Masayuki Nakagawa, Naohiko Seki. Tumor suppressive microRNA-218 regulates oncogenes of focal adhesion pathways in renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4189. doi:10.1158/1538-7445.AM2013-4189

463 TUMOR SUPPRESSIVE MICRORNA-138 CONTRIBUTES TO INHIBITION OF BOTH CELL MIGRATION AND INVASION THROUGH TARGETING VIMENTIN IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research (III)1 Apr 2013463 TUMOR SUPPRESSIVE MICRORNA-138 CONTRIBUTES TO INHIBITION OF BOTH CELL MIGRATION AND INVASION THROUGH TARGETING VIMENTIN IN RENAL CELL CARCINOMA Takeshi Yamasaki, Naohiko Seki, Hirofumi Yoshino, Hideo Hidaka, Hideki Enokida, and Masayuki Nakagawa Takeshi YamasakiTakeshi Yamasaki Kagoshima, Japan More articles by this author , Naohiko SekiNaohiko Seki Chiba, Japan More articles by this author , Hirofumi YoshinoHirofumi Yoshino Kagoshima, Japan More articles by this author , Hideo HidakaHideo Hidaka Kagoshima, Japan More articles by this author , Hideki EnokidaHideki Enokida Kagoshima, Japan More articles by this author , and Masayuki NakagawaMasayuki Nakagawa Kagoshima, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1854AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our recent miRNA expression signature of renal cell carcinoma (RCC) revealed that microRNA-138 (miR-138) expression was significantly reduced in RCC, suggesting that miR-138 was a candidate tumor suppressor. Recent publications showed that miR-138 might regulate genes related to epithelial-mesenchymal transition (EMT) in several cancers. The aim of study is to investigate the functional significance of miR-138 and to identify its target genes in RCC. METHODS We evaluated miR-138 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We observed morphologic changes in the miR-138-transfected RCC cell lines (A498 and 786-O). We performed gain-of-function studies such as cell migration and cell invasion assays by using miR-138 transfectants (TFs). Gene expression analysis of the miR-138 TFs and RCC clinical specimens by oligo-microarray was carried out to identify molecular targets of miR-138. TargetScan database implied that vimentin (VIM), an EMT-related gene, was a promising candidate target gene of miR-138. We investigated whether VIM was regulated by miR-138 by real-time PCR and Western blotting. Loss-of-function studies using si-VIM were performed to investigate the functional significance of VIM. We evaluated the expression levels of both VIM mRNA and protein by qRT-PCR and immunohistochemistry. RESULTS The expression levels of miR-138 were significantly reduced in the RCC specimens compared with the normal tissues. Restoration of mature miR-138 in A498 and 786-O caused changes in the bleb-like cell morphology, as characteristics of EMT. Significant inhibitions of cell migration and cell invasion were observed in the miR-138 TFs. The expression levels of VIM expression was suppressed in the miR-138 TFs at both mRNA and protein levels. Loss-of-function studies demonstrated that significant inhibition of cell migration and invasion occurred in the si-VIM transfectants. In clinical RCC specimens, the expression level of VIM was significantly upregulated in comparison with adjacent normal tissues. Furthermore, immunohistochemistry showed that VIM expression levels in RCC specimens were significantly higher than those in the normal tissues. CONCLUSIONS These data suggest that VIM might function as an oncogene and might be regulated by tumor suppressive miR-138. Tumor suppressive miR-138-mediated EMT pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e189-e190 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Yamasaki Kagoshima, Japan More articles by this author Naohiko Seki Chiba, Japan More articles by this author Hirofumi Yoshino Kagoshima, Japan More articles by this author Hideo Hidaka Kagoshima, Japan More articles by this author Hideki Enokida Kagoshima, Japan More articles by this author Masayuki Nakagawa Kagoshima, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EphA2 protein expression in metastatic renal cell carcinoma treated with VEGF-inhibitors is predictive of patient survival

Background: EphA2 is a receptor tyrosine kinase of the Eph family, which regulates angiogenesis, cell growth, survival and migration. EphA2 targeting has been proposed as a novel therapeutic strategy for neoplasms that overexpress this protein, particularly in case of resistance to VEGF-targeted therapy. This work raised the question whether EphA2 expression would be a predictive factor in patients with metastatic renal cell carcinoma (RCC) treated with VEGF inhibitors. Methods: We analyzed the levels of EphA2 protein expression by immunohistochemistry in specimens of 23 patients with metastatic RCC, which were postsurgically treated with Anti-VEGF-therapy. The quantification of EphA2 protein expression was performed by automated digital image analysis using the open source software ImageJ. Statistical analysis using Cox proportional hazard modeling, Bayesian information criterion (BIC) statistic and Kendall’s tau ( τ ) correlation was done using the program R. Results: The expression of EphA2 correlated significantly with the histological grading (correlation coefficient Kendall’s τ =0.52; p <0.05 for grades II and III). High levels of EphA2 expression levels by the adjacent kidney tissue of RCC tumors had a positive predictive value for overall survival, suggesting a tumor suppressive effect of EphA2 expressed in the tumor surrounding tissue. We also found a negative correlation between the EphA2 expression level in the primary tumor and the matching metastasis. These findings implicate different dominating signaling pathways in the primary tumor and its metastasis, with consequently a need for a complex therapeutic approach. Conclusions: Our study suggests an association between EphA2 receptor expression and RCC carcinogenesis and metastasis. The differential effects of EphA2 signaling, from a tumor suppressive to a tumor promoting role, are greatly dependent upon its precise localization: In addition to its previously described tumor promoting role in EphA2 overexpressing tumors we observed an association between high levels of EphA2 in the adjacent normal kidney tissue and survival, suggesting a tumor suppressive role. An improved understanding of the different tasks of EphA2 signaling in the primary tumors, their surrounding tissues and metastases may be of benefit for a better targeting of metastatic RCC.

MicroRNA‐155 is a predictive marker for survival in patients with clear cell renal cell carcinoma

To investigate the clinical significance of micro-ribonucleic acid-155 in clear cell renal cell carcinoma, in particular focusing on the association of expression levels of micro-ribonucleic acid-155 with clinicopathological factors, cancer-specific survival and therapeutic outcomes in clear cell renal cell carcinoma patients. Quantitative reverse transcription polymerase chain reaction of micro-ribonucleic acid-155 was carried out on 137 clear cell renal cell carcinoma cases, containing 77 matched pairs of clear cell renal cell carcinoma and normal adjacent kidney tissues from the same patients. Significant overexpression of micro-ribonucleic acid-155 was found in clear cell renal cell carcinoma compared with normal kidney tissue. Expression of micro-ribonucleic acid-155 was not associated with prognosis in all stage groups. However, in 43 patients with stage III and IV clear cell renal cell carcinoma, low expression levels of micro-ribonucleic acid-155 correlated with a poor prognosis. Regarding cancer-free survival of 26 patients with stage III and IV clear cell renal cell carcinoma who received curative resection and cancer-specific survival of 31 patients who received postoperative therapy with interferon-α after radical nephrectomy, low expression levels of micro-ribonucleic acid-155 correlated with poor clinical outcomes in these two groups. Low expression of micro-ribonucleic acid-155 represents a valuable marker of poor clinical outcomes in patients with stage III and IV clear cell renal cell carcinoma.

Genetic determinants of long-term response to rapalog therapy in advanced renal cell carcinoma (RCC).

4604 Background: Temsirolimus (T) and everolimus (E) are rapalog inhibitors of the mammalian target of rapamycin (mTOR) with efficacy in advanced RCC [NEJM;356(22):227-81; Lancet;372(9637):449-56]. With reported median progression-free survival (PFS) of 5.5 and 4.9 months (mo), respectively, benefit is typically modest, yet a subset of patients (pts) achieves long-term disease control evident by extended PFS. The oncogenomic background for this is unclear. Methods: We analyzed frozen specimens of tumor and adjacent normal kidney from pts with advanced RCC and documented long-term response to T or E, defined as PFS of ≥ 20mo. Samples from pts with PFS ≤ 2mo served as comparators. Specimens were subjected to whole exome sequencing; a targeted next-generation sequencing platform was used for deep sequencing and investigation of copy number variations (CNV) in 230 genes of interest. Results: In 5 pts with long-term response to T [n=3] or E [n=2], histologic subtypes were clear cell [n=3] and unclassified [n=2] RCC. Median number of prior treatments was 2 (1-3). Two pts remain on therapy, 3 have stopped drug for disease progression; treatment duration was 20, 25+, 27, 28, and 28+ mo. Three control pts progressed after 1, 2, and 2 mo on therapy. Mean target coverage was 86x for whole exome and 443x for targeted sequencing. Three of 5 long-term responders harbored acquired somatic mutations and/or CNV in genes encoding for members of the Phosphoinositide 3-Kinase (PI3K) / mTOR pathway. Effects on amino acid sequence and gene function suggest a hyperactive pathway in all 3. For the other 2 long-term responders genomic changes with possible indirect link to the pathway, but no apparent determinants of treatment response were seen. In 3 control patients with rapid disease progression no genomic alterations suggesting hyperactivation of the pathway were seen. Conclusions: In this retrospective discovery set of RCC pts with unusually long response to rapalog therapy, next generation sequencing using pre-treatment tissue specimens revealed plausible oncogenomic determinants of treatment benefit in 3 of 5 cases. These findings provide basis for further biomarker development and studies in a larger sample set are ongoing.

433 SPERM ASSOCIATED ANTIGEN 4 IS A NOVEL BIOMARKER FOR RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2012433 SPERM ASSOCIATED ANTIGEN 4 IS A NOVEL BIOMARKER FOR RENAL CELL CARCINOMA Takumi Shiraishi, Naoki Terada, Yu Zeng, Steven Mooney, Sayuri Takahashi, Natsuki Takaha, Tsuneharu Miki, Robert Getzenberg, and Prakash Kulkarni Takumi ShiraishiTakumi Shiraishi Baltimore, MD More articles by this author , Naoki TeradaNaoki Terada Baltimore, MD More articles by this author , Yu ZengYu Zeng Baltimore, MD More articles by this author , Steven MooneySteven Mooney Baltimore, MD More articles by this author , Sayuri TakahashiSayuri Takahashi Baltimore, MD More articles by this author , Natsuki TakahaNatsuki Takaha Kyoto, Japan More articles by this author , Tsuneharu MikiTsuneharu Miki Kyoto, Japan More articles by this author , Robert GetzenbergRobert Getzenberg Baltimore, MD More articles by this author , and Prakash KulkarniPrakash Kulkarni Baltimore, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.500AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Loss of the von Hippel-Lindau (VHL) tumor suppressor gene is associated with a variety of proliferative disorders, especially renal cell carcinoma (RCC). In the absence of pVHL, hypoxia inducible factor α (HIFα) is stabilized under normoxic conditions and stimulates the expression of several genes associated with the survival of cancer cells and tumorigenesis. The Cancer/Testis Antigens (CTAs) are an important group of proteins that are typically restricted to the testis in the normal adult but are aberrantly expressed in several types of cancers. As a result of their restricted expression patterns, the CTAs have the potential to serve as unique biomarkers and therapeutic targets for cancer. In the present study, we investigated the association between the VHL-mediated gene pathway and the panel of CTAs expression. METHODS RCC4-EV (VHL null) and RCC4-VHL (restored wild type VHL) cells were used to identify CTAs which was associated with the VHL-mediated gene pathway. Deferoxamine mesylate (DFO) was used to mimic hypoxic conditions. The expression of sperm associated antigen 4 (SPAG4) was measured by quantitative real-time PCR using cancer and adjacent normal tissues obtained from 35 patients with RCC. RESULTS SPAG4 was dramatically down-regulated in RCC4-VHL cells compared to RCC4-EV cells. SPAG4 mRNA expression was induced by DFO and this induction was blocked by the knockdown of HIF1α in renal cancer cells expressing both wild type HIF1α and HIF2α. On the other hand, HIF2α played a predominant role in SPAG4 expression in HIF1α defective renal cancer cells. Over-expression of SPAG4 had no effect on cell growth but significantly increased invasion ability using a Matrigel invasion assay. Quantitative real time PCR showed that SPAG4 mRNA expression in cancer specimens from RCC patients was an average 15 times higher than adjacent normal kidney tissues from same patients. Furthermore, the SPAG4 mRNA expression was inversely correlated with tumor size, grade and stage. CONCLUSIONS These results indicate that SPAG4 mRNA expression could serve as a promising diagnostic and prognostic biomarker for RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e177-e178 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takumi Shiraishi Baltimore, MD More articles by this author Naoki Terada Baltimore, MD More articles by this author Yu Zeng Baltimore, MD More articles by this author Steven Mooney Baltimore, MD More articles by this author Sayuri Takahashi Baltimore, MD More articles by this author Natsuki Takaha Kyoto, Japan More articles by this author Tsuneharu Miki Kyoto, Japan More articles by this author Robert Getzenberg Baltimore, MD More articles by this author Prakash Kulkarni Baltimore, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Epigenetic inactivation of HOXA5 and MSH2 gene in clear cell renal cell carcinoma.

424 Background: The high-throughput method using microarray is easy and fast way to analyze the methylation status of hundreds of preselected genes and to screen them for signatures in methylation. The aim of our study is to detect hypermethylated genes and to analyze the association between methylation status and clinicopathological parameters of clear cell renal cell carcinoma. Methods: The genetic substrate included 62 cancer tissues and 62 matched adjacent normal kidney tissues. We adapted the GoldenGate genotyping assay to determine the methylation state of 1505 specific CpG sites in 807 genes. Results: We identified two genes (HOXA5 and MSH2) with β-value differences of more than 0.3 between cancer and normal tissues. High methylation group in HOXA5 had high Fuhrman’s nuclear grade (P=0.041). Other data in HOXA5 and MSH2 were not significant with methylation status (P&gt;0.05). Survival curve of high methylation group in HOXA5 was slightly lower than that of low methylation group. However, the statistical significances of overall survival in HOXA5 and MSH2 were low (P&gt;0.05). Conclusions: We report the hypermethylation of two genes in clear cell renal cell carcinoma. The data we obtained could provide the basis for a diagnostic test pathological assessment, or prognosis in clear cell renal cell carcinoma.

Protein profiling of microdomains purified from renal cell carcinoma and normal kidney tissue samples

Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery.

Full-Length Enrich c-DNA Libraries-Clear Cell-Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, is characterized by high metastasis potential and strong resistance to traditional therapies, resulting in a poor five-year survival rate of patients. Several therapies targeted to VEGF pathway for advanced RCC have been developed, however, it still needs to discover new therapeutic targets for treating RCC. Genome-wide gene expression analyses have been broadly used to identify unknown molecular mechanisms of cancer progression. Recently, we applied the oligo-capping method to construct the full-length cDNA libraries of ccRCC and adjacent normal kidney, and analyzed the gene expression profiles by high-throughput sequencing. This paper presents a review for recent findings on therapeutic potential of MYC pathway and nicotinamide N-methyltransferase for the treatment of RCC.

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.

Expression of endothelin 2 and localized clear cell renal cell carcinoma

Despite the rising incidence of clear cell renal cell carcinoma, the molecular events that support its development and progression remain unclear. Herein, we evaluate the association of endothelin 2 expression with both clear cell renal cell carcinoma development and progression-free survival. We conducted real-time polymerase chain reaction to determine endothelin 2 expression levels on 238 patients who underwent nephrectomy for localized clear cell renal cell carcinoma, 161 of whom also had adjacent normal kidney samples available for analysis. To evaluate associations with clear cell renal cell carcinoma development, linear mixed models were used to compare differential expression between tumor and a normal kidney as well as to explore interactions with clinicopathologic features. To evaluate associations with prognosis, Cox proportional hazards models were used to assess the association of progression-free survival and endothelin 2 expression in tumor tissue. Overall, endothelin 2 expression was higher in tumor samples versus patient-matched normal kidney samples, with an average fold change of 1.99 (95% confidence interval, 1.48-2.60; P < .0001). This overexpression in tumor versus normal kidney samples was more pronounced in low- compared with high-grade tumors (interaction, P = .0002), in early- compared with late-stage tumors (interaction, P = .001), and in tumors without compared with those with necrosis (interaction, P = .001). Moreover, an increasing endothelin 2 expression in tumors was associated with a longer progression-free survival (hazard ratio, 0.89; 95% confidence interval, 0.80-0.99; P = .03); however, after controlling for known clinicopathologic factors, this association was attenuated (hazard ratio, 0.99; 95% confidence interval, 0.89-1.09; P = .7). Up-regulation of endothelin 2 is a common and early event in localized clear cell renal cell carcinoma. Higher tumor expression of endothelin 2 is associated with a longer progression-free survival but not after adjustment for well-known pathologic indices. Thus, although endothelin 2 does not appear to be an independent prognostic marker, there is evidence of a putative role in clear cell renal cell carcinoma progression. If supportive mechanistic data can be produced, endothelin 2 could represent a potential target for chemopreventive or neoadjuvant therapeutics for clear cell renal cell carcinoma.

Abstract 3466: Tumor pericytes: A novel target for immune therapy in renal cell carcinoma

Abstract Tumor vasculature is classified by leaky, chaotic blood vessels consisting of several components including vascular endothelial cells (VEC) and mural cells (smooth muscle cells and pericytes), as well as immune regulatory cells, stem cells and vascular progenitors. Irregular tumor vasculature limits immune effector cell access to the tumor, and may in part be responsible for the modest success observed in many current anti-cancer immunotherapies. Certain drugs targeting tyrosine kinase receptors expressed on tumor stromal cells induce a Type-1 proinflammatory skewing of the tumor microenvironment (TME) with a coordinate reduction in the number of immune regulatory cell populations. A transient normalization of the vasculature may also occur in association with enhanced leukocyte recruitment. We have shown that vaccination against tumor-associated stromal antigens leads to the activation of VCAM-1 expression on tumor vascular endothelial cells and the enhanced recruitment of protective Type-1 T cells into the TME. Currently little is known regarding pericyte biology and their behavior in the TME, with no existing treatments specifically targeting pericytes. We hypothesize that pericytes residing in the TME differentially express antigens that distinguish them from pericytes in healthy tissue, and vaccination specifically targeting tumor pericyte antigens will result in vascular normalization and improved delivery of protective Type-1 T cells into the TME. To address these hypotheses, we examined mRNA expression of pericytes and VEC that have been flow-sorted from human renal cell carcinoma (RCC) tumors and patient-matched normal adjacent kidney (NAK) tissue. We have identified antigens that are differentially expressed by pericytes and/or by VEC in the TME versus NAK (such as RGS5). We are currently investigating the therapeutic efficacy of genetic (lentiviral) vaccines encoding tumor-associated stromal antigens in murine tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2011-3466

Abstract 5173: DKK4 activates non-canonical JNK signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

Abstract Introduction and Objective: Renal cell carcinoma (RCC) is the third leading cause of death among urological tumors. Wnt/beta-catenin signaling, which plays an important role in cancer, includes beta-catenin dependent and beta-catenin independent pathways. The functional significance of several wnt antagonists have been reported in renal cancer. However the functional significance of Wnt antagonist DICKKOPF-4 (DKK4) has not been investigated in RCC. Recently DKK4 function has been reported to have two opposing functions (oncogenic or tumor suppressor) in colon cancer. As far as we know, there have been no reports regarding DKK4 and renal cancer. Therefore we focused on the role of DKK4 in renal cancer. Methods: We analyzed the expression of DKK4 in renal cancer tissues and adjacent normal kidney tissues by immunohistochemistry. To assess the function of DKK4, we established stable DKK4 transfected A-498 cells and performed functional analyses including TCF/LEF reporter assay, cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound healing capability and in vivo tumor growth. Results: DKK4 expression was significantly higher in cancer compared to adjacent normal kidney tissues. The relative TCF/LEF activity was significantly lower in DKK4 transfected cells compared to empty vector, and nuclear beta-catenin expression was decreased in DKK4 transfectants. Also beta-catenin downstream effector proteins, cyclinD1 and c-Myc, showed decreased expression in DKK4 transfectants. This result suggests that DKK4 may be involved in the beta-catenin dependent pathway and that DKK4 is a beta-catenin dependent pathway inhibitor. However, higher invasiveness and migration was observed in stably transfected DKK4 cells and DKK4 promoted tumor growth in vitro and in vivo. To investigate the oncogenic function in DKK4 transfectants, we focused on the Wnt non-canonical/JNK signaling pathway and phosphorylation of JNK. We found that expression and phosphorylation of c-Jun were increased in DKK4 transfected A-498 cells. MMP-2 expression was also significantly increased in DKK4 stable trasnfectants. These results suggest that DKK4 promotes the non-canonical pathway by activating JNK, resulting in up-regulation of c-Jun expression and phosphorylation, and increasing MMP-2 expression. Conclusions: This is the first report documenting that DKK4 expression is higher in renal cancer tissues compared to matched normal kidney tissues. We examined the role of DKK4 in the canonical and non-canonical Wnt pathways and found that DKK4 inhibited the canonical pathway. Though DKK4 did not induce apoptosis it promoted renal cancer cell proliferation, invasion, and migration via the non-canonical JNK pathway, which also increased MMP-2 expression. Thus the current findings contribute important information about the role of DKK4 in renal cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2011-5173

109 DKK4 ACTIVATES NON-CANONICAL JNK SIGNALING PATHWAY WHILE INHIBITING THE WNT-CANONICAL PATHWAY IN HUMAN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011109 DKK4 ACTIVATES NON-CANONICAL JNK SIGNALING PATHWAY WHILE INHIBITING THE WNT-CANONICAL PATHWAY IN HUMAN RENAL CELL CARCINOMA Hiroshi Hirata, Koji Ueno, Koichi Nakajima, Laura Tabatabai, Yuji Hinoda, Nobuhisa Ishii, Peter Carroll, and Rajvir Dahiya Hiroshi HirataHiroshi Hirata San Francisco, CA More articles by this author , Koji UenoKoji Ueno San Francisco, CA More articles by this author , Koichi NakajimaKoichi Nakajima Ohtaku, Japan More articles by this author , Laura TabatabaiLaura Tabatabai San Francisco, CA More articles by this author , Yuji HinodaYuji Hinoda Ube, Japan More articles by this author , Nobuhisa IshiiNobuhisa Ishii Ohtaku, Japan More articles by this author , Peter CarrollPeter Carroll San Francisco, CA More articles by this author , and Rajvir DahiyaRajvir Dahiya San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.175AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal cell carcinoma (RCC) is the third leading cause of death among urological tumors. Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions are still found at diagnosis in about 30% of RCC patients.Wnt/beta-catenin signaling, which is one of important cancer pathways, includes beta-catenin dependent and independent pathways. The functional significance of some Wnt antagonists has been reported in renal cancer. However the functional significance of Wnt antagonist DKK4 has not been investigated in RCC. Recently DKK4 function has been reported to have two opposing functions (oncogenic /tumor suppressor) in colon cancer by different research groups. As far as we know, there have been no reports regarding DKK4 and RCC. METHODS We initially found that the expression of DKK4 was significantly higher in renal cancer tissues compared to adjacent normal kidney tissues. To assess the function of DKK4, we established stable DKK4 transfected cells and performed functional analyses including TCF/LEF reporter assay. RESULTS The relative TCF/LEF activity was significantly lower in DKK4 transfected cells compared to empty vector, and nuclear beta-catenin expression was decreased in DKK4 transfectants. Also beta-catenin downstream effector proteins, cyclinD1 and c-Myc, showed decreased expression in DKK4 transfectants. This result indicates that DKK4 is a beta-catenin dependent pathway inhibitor. However, higher invasiveness and migration was observed in stably transfected DKK4 cells and DKK4 promoted tumor growth in vitro and in vivo. To investigate the oncogenic function in DKK4 transfectants, we focused on the Wnt non-canonical/JNK signaling pathway. We found that phosphorylation of JNK, expression and phosphorylation of c-Jun were increased in DKK4 transfected A-498 cells. MMP-2 expression was also significantly increased in DKK4 stable transfectants. CONCLUSIONS This is the first report documenting that DKK4 expression is higher in renal cancer tissues compared to matched normal kidney tissues. DKK4 inhibited the canonical pathway. Though DKK4 did not induce apoptosis it promoted renal cancer cell proliferation, invasion, and migration via the non-canonical JNK pathway, which also increased MMP-2 expression. Thus the current findings contribute significant information about the role of DKK4 in renal cancer cells. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e46 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiroshi Hirata San Francisco, CA More articles by this author Koji Ueno San Francisco, CA More articles by this author Koichi Nakajima Ohtaku, Japan More articles by this author Laura Tabatabai San Francisco, CA More articles by this author Yuji Hinoda Ube, Japan More articles by this author Nobuhisa Ishii Ohtaku, Japan More articles by this author Peter Carroll San Francisco, CA More articles by this author Rajvir Dahiya San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Differential Proteomic Analysis of Renal Cell Carcinoma Tissue Interstitial Fluid

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.

Regulatory Effects of microRNA-92 (miR-92) on VHL Gene Expression and the Hypoxic Activation of miR-210 in Clear Cell Renal Cell Carcinoma

In order to understand the role of miRNAs in renal tumorigenesis, we undertook a stepwise approach that included a comprehensive differential miRNA expression analysis for the most common histological subtypes of human renal neoplasms appearing in either sporadic or hereditary forms. We also aimed to test the hypothesis that microRNAs can act as an alternative mechanism of VHL gene inactivation and therefore might be correlated with tumorigenesis in ccRCC. Finally, we wanted to explore whether the well-known hypoxic activation of ccRCC is followed by a specific pattern of miRNA expression. Tumor and normal adjacent kidney parenchyma from patients with RCC were tested for microRNA expression. Twenty cases of different histologies were used for profiling by PCR miRNA arrays. For validation, a separate cohort of samples used to test specifically miR92a expression and its involvement in VHL gene mRNA silencing. Finally, miR210 as a marker of hypoxia was evaluated. Expression values were correlated with important clinicopathologic features from the patients. We identified unique miRNA expression signatures for each histologic subtype of kidney tumors. Expression values for downregulated miRNAs ranged from 0.3-fold (in VHL-clear cell RCC) up to 0.393 fold (in papillary type II (HLRCC) tumors). For the upregulated miRNAs, fold-changes ranged from 2.1 up to 290-fold. Specific patterns together with type-specific profiles were observed. Twenty-three miRNAs were found to be differentially expressed in both sporadic and VHL-dependent ccRCC. Sporadic clear cell tumors showed a unique pattern of 14-miRNA that were absent from the VHL-dependent tumors. These also showed 15 miRNAs specific to the hereditary type. Common miRNAs to both sporadic and hereditary forms included miR-92a and miR-210. For miR-92a, and a striking inverse correlation with VHL mRNA levels was found. For the hypoxia-regulated miR-210, clear cell tumors showed significantly higher expression levels when compared to tumor of non-clear cell histology (9.90-fold vs. 1.36, p<0.001). microRNA expression seems to be involved in every step of RCC pathogenesis: both as an element for tumor development as well as a consequence of or in response to the initial malignant transformation and part of tumor progression. Our data show consistent disregulation of miRNAs in human kidney cancer, some of which are potentially involved in critical gene silencing in RCC and others that are activated as part of the pathophysiological response in these tumors.