Renal Cell Carcinoma Tumor Tissues Research Articles

Abstract 5305: Phosphoproteomics analysis reveals differential signaling pathways among clear cell and papillary renal cell carcinoma tumors

Abstract Introduction: The use of kinase inhibitors with targets such as VEGFR has heralded a new era of treatment for metastatic renal cell carcinoma (RCC) patients. However, patients develop resistance with time. Therefore, novel targets must be identified and a systems level description of RCC produced. To this end, we used mass spectrometry based phosphoproteomics to nominate targets as well as compare RCC subtypes. Methods: We developed an integrative strategy combining RCC phosphoproteomics and functional interrogation using drug screening. Tyrosine phosphorylated peptide immunoprecipitation and purification was performed in clear cell RCC (ccRCC) and normal renal cortex cell lines and analyzed via nano-liquid chromatography/tandem mass spectrometry interfaced with an electrospray hybrid ion-trap mass spectrometer. Eight ccRCC and five papillary RCC (pRCC) tumor tissues were also analyzed. We assembled a library of 180 small molecules, including 60 tyrosine kinase inhibitors (TKIs), and screened them at multiple concentrations in 10 clear cell RCC (ccRCC) cell lines. Results: Among the ccRCC cell lines, 153 phosphosites (114 proteins) were identified. Receptor tyrosine kinases included EGFR, MET, ERBB4, and EPHB2 and nonreceptor tyrosine kinases included ABL2, JAK2, KALRN, and PTK2. Serine/threonine kinases were the most abundant kinases and included, among others, several CDK and MAPK members. Other frequently observed phosphosites corresponded to cytoskeleton (10%) and cell adhesion (6%) proteins. Among the ccRCC tumors, 119 phosphosites (99 proteins) were identified. In contrast to the cell lines, the ccRCC tumors demonstrated increased Src family kinase signaling but less RTK signaling. Among the pRCC tumors, 113 phosphosites (98 proteins) were identified, including 37 not identified in the ccRCC tumors. This data, coupled with the TKI screen, offered some interesting observations. MET phosphotyrosine peptide intensity was relatively low in ccRCC compared to pRCC and among the eight MET inhibitors tested, seven failed to significantly inhibit cell viability. These results suggest that MET signaling may be more active in pRCC than ccRCC. Other divergent patterns of phosphotyrosine signaling emerged. For example, certain tyrosine phosphatases that could impact signaling displayed higher phosphorylation patterns in ccRCC. Other kinases currently not targeted by RCC therapeutics were more highly phosphorylated in pRCC relative to ccRCC tumors. The functional impact of these findings is currently undergoing evaluation. Conclusions: Integrating global phosphoproteomics of cell lines and tumors with functional analyses can shed light on key survival and growth pathways in clear cell and papillary RCC. Further studies in human tissues are ongoing and will be presented. Citation Format: Scott M. Haake, Jiannong Li, Yun Bai, Bin Fang, Julio Pow-Sang, John Koomen, Mayer Fishman, Eric B. Haura. Phosphoproteomics analysis reveals differential signaling pathways among clear cell and papillary renal cell carcinoma tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2014-5305

Important role of CYP2J2 in protein kinase inhibitor degradation: a possible role in intratumor drug disposition and resistance.

We have investigated in vitro the metabolic capability of 3 extrahepatic cytochromes P-450, CYP1A1, 1B1 and 2J2, known to be over-expressed in various tumors, to biotransform 5 tyrosine kinase inhibitors (TKI): dasatinib, imatinib, nilotinib, sorafenib and sunitinib. Moreover, mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in 6 hepatocellular and 14 renal cell carcinoma tumor tissues and their surrounding healthy tissues, was determined.Our results show that CYP1A1, 1B1 and especially 2J2 can rapidly biotransform the studied TKIs with a metabolic efficiency similar to that of CYP3A4. The mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in tumor biopsies has shown i) the strong variability of CYP expression and ii) distinct outliers showing high expression levels (esp. CYP2J2) that are compatible with high intratumoral CYP activity and tumor-specific TKI degradation.CYP2J2 inhibition could be a novel clinical strategy to specifically increase the intratumoral rather than plasma TKI levels, improving TKI efficacy and extending the duration before relapse. Such an approach would be akin to beta-lactamase inhibition, a classical strategy to avoid antibiotic degradation and resistance.

Antitumor and antiangiogenic activity of Schisandra chinensis polysaccharide in a renal cell carcinoma model

The aim of this study was to determine the antitumor and antiangiogenic effects of the Schisandra chinensis polysaccharides (SCP) in selected renal cell carcinoma (RCC) cells and evaluate its potential mechanism of action. In vitro, endothelial growth factor (VEGF) secretion by Caki-1 was blockaded in response to SCP treatment for 48h. In vivo, a significant tumor growth inhibition effect was observed after SCP administration for 4 weeks. Moreover, SCP treatment decreased the level of VEGF, CD31 and CD34 in RCC tumor tissues. Further analysis of the tumor inhibition mechanism indicated that the number of apoptotic tumor cells increased significantly; the expression of Bax and p53 increased; and the expression of Bcl-2 decreased dramatically in transplanted tumor tissues following SCP administration. These results indicated that the potential mechanisms involved by which SCP exerted its antitumor and antiangiogenic activity might be associated with the up-regulation of Bax and p53, downregulation of Bcl-2, as well as the reduction of VEGF, CD31 and CD34 in xenografted tumors. These findings demonstrated that the SCP is a potential antitumor agent for RCC treatment.

Renal carcinoma related factor is up-regulated in high Fuhrman grade renal cell carcinoma tissues and affects prognosis of patients

Objective To identify differentially expressed long non-coding RNAs( lncRNAs) in different Fuhrman grade renal cell carcinoma( RCC) tissues,and to investigate the association of one of these lncRNAs-renal carcinoma related factor( RCRF) with the clinicopathologic features of RCC patients,helping the prognosis of RCC patients. Methods A total of 6 RCC tumor tissues were used for lncRNA chip screening,with 3 being FuhrmanⅠgrade and the rest being Fuhrman Ⅲ-Ⅳ grade. Then another 40 RCC tumor tissues( 22 FuhrmanⅠ-Ⅱgrade and 18 Fuhrman Ⅲ-Ⅳ grade) and the matched adjacent noncancerous tissues were subjected to qPCR examination for RCRF expression,and the relationship of RCRF expression with the clinical stage,tumor grade,lymph node metastasis and distant metastasis was analyzed. Results A total of 320 differentially expressed lncRNAs were screened out from RCC tumor tissues. Compared with the adjacent normal tissues,RCC tissues,especially the high grade ones,had significantly up-regulated RCRF expression( P 0. 01). It was also found that RCRF expression was associated with the pathological grade,lymph node metastasis and distant metastasis of RCC( P 0. 05). Conclusion RCRF is up-regulated in high Fuhrman grade tissues and may affect the prognosis of RCC patients.

MiR-210 expression in tumor tissue and in vitro effects of its silencing in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3% of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P < 0.003)). Increased expression levels of miR-210 in RCC tumor tissue were independently validated. We observed decreased viability of ACHN and CAKI-2 cells and accumulation of CAKI-2 in G2 phase of cell cycle after silencing of miR-210 expression. Downregulation of miR-210 also reduced the migratory and invasive potential of ACHN metastatic RCC cells. Moreover, we showed downregulation of HIF1a protein in both cell lines after miR-210 silencing indicating participation of miR-210 in hypoxic processes of RCC not only through regulation of its target mRNAs but also by indirect regulation of HIF1a. To our knowledge, this is the first report to show miR-210 regulatory effects on cell migration, invasive potential, and HIF1a protein in RCC cells.

TNF-α Induces Epithelial–Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC.

Abstract 4213: Gene expression of hsp27 in renal cell carcinoma and the correlation with tumor progression

Abstract Background Overexpression of Hsp27 protein has been correlated with tumor progression in a variety of cancers including Renal Cell Carcinoma (RCC). Various phosphorylation patterns of Hsp27 correlate with the aggressiveness of tumor phenotype. Increased Hsp27 phosphorylation was reported to correlate with increased tumor progression in RCC. Since the phosphorylation status of Hsp27 is dependent on phosphorylation by kinases and dephosphorylation by phosphatases, a better understanding of these factors in the tumor microenvironment needed to be analyzed at the gene expression level. Gene expression studies of tumor tissues using microarray routinely fail to identify hsp27 because large amount of transcripts that microarrays analyze may mask the changes observed with hsp27 and its associated genes. Hence, we used quantitative RT-PCR to investigate the relative gene expressions of hsp27 in RCC tumor and normal adjacent tissues (NAT). We examined the gene expression levels of MAP kinases such as Mitogen Activated Protein Kinase 1 (mapk1) and MAPK-activated protein Kinase 5 (mk5) along with a major phosphatase, Protein Phosphatase 2 A (pp2a). Methods RCC tumor and normal control samples were frozen in liquid nitrogen within one hour post surgical excision. Frozen samples were pulverized in Covaris TissueTubes using the Covaris CryoPrep™ system. The pulverized tissue was homogenized for RNA extraction using the Covaris Adaptive Focused Acoustics (AFA) S220 system. Total RNA was extracted using a Qiagen RNeasy Plus kit. The gene expressions for Hsp27, MAPK1, MK5, and PP2A were analyzed by RT-PCR using validated TaqMan assays on an Applied Biosystems 7500 Fast Real-Time PCR System. A house keeping gene, beta actin, was measured in parallel to normalize the differences between samples. Results In RCC, in comparison to NAT, the relative gene expressions of hsp27 increased to 224.44% suggesting an upregulation of hsp27 in cancer tissue. Adding to this microenvironment, the phosphorylating enzymes were also upregulated in the RCC tissue. Expression levels of kinases, mapk1 and mk5 were increased to 22.11% and 37.18% respectively. Interestingly, the level of phosphatase (pp2a) was decreased 67.25 %. These data suggests that the combination of the upregulation of kinases along with down regulation of phosphatase may be a major factor creating the environment of enhanced phosphorylation in overexpressed hsp27. Conclusion Our data shows that the gene expression level increase of hsp27 in RCC is consistent with the predicted increase in protein level expression of Hsp27. However, the dramatic combined effect of the increase in the gene expression of the kinases, along with decrease in the gene expression of the phosphatase may result in a striking increase in phosphorylation. The enhanced phosphorylation will activate Hsp27 in the tumor microenvironment resulting increased tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4213. doi:1538-7445.AM2012-4213

Enhancer of zeste homolog 2 (EZH2) expression is an independent prognostic factor in renal cell carcinoma

BackgroundThe enhancer of zeste homolog 2 (EZH2) gene exerts oncogene-like activities and its (over)expression has been linked to several human malignancies. Here, we studied a possible association between EZH2 expression and prognosis in patients with renal cell carcinoma (RCC).MethodsEZH2 protein expression in RCC specimens was analyzed by immunohistochemistry using a tissue microarray (TMA) containing RCC tumor tissue and corresponding normal tissue samples of 520 patients. For immunohistochemical assessment of EZH2 expression, nuclear staining quantity was evaluated using a semiquantitative score. The effect of EZH2 expression on cancer specific survival (CSS) was assessed by univariate and multivariate Cox regression analyses.ResultsDuring follow-up, 147 patients (28%) had died of their disease, median follow-up of patients still alive was 6.0 years (range 0-16.1 years). EZH2 nuclear staining was present in tumor cores of 411 (79%) patients. A multivariate Cox regression analysis revealed that high nuclear EZH2 expression was an independent predictor of poor CSS (> 25-50% vs. 0%: HR 2.72, p = 0.025) in patients suffering from non-metastatic RCC. Apart from high nuclear EZH2 expression, tumor stage and Fuhrman's grading emerged as significant prognostic markers. In metastatic disease, nuclear EZH2 expression and histopathological subtype were independent predictive parameters of poor CSS (EZH2: 1-5%: HR 2.63, p = 0.043, >5-25%: HR 3.35, p = 0.013, >25%-50%: HR 4.92, p = 0.003, all compared to 0%: HR 0.36, p = 0.025, respectively).ConclusionsThis study defines EZH2 as a powerful independent unfavourable prognostic marker of CSS in patients with metastatic and non-metastatic RCC.

Panel of Candidate Biomarkers for Renal Cell Carcinoma

The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by the lack of specific biomarkers. To identify candidate RCC biomarkers, we used 2-DE gel electrophoresis with mass spectrometry and 2-DE spot intensity-based ROC analysis to analyze 18 sets of paired normal and RCC tumor tissue including conventional, papillary, and chromophobe subtypes. Validation was performed with RCC patient plasma samples and confirmed by clustergram, shRNA, and immunohistochemistry assays. Cardinal candidates were evaluated by ELISA. The leading candidate biomarker that was upregulated in RCC samples according to the clustergram and validation analysis was nicotinamide N-methyltransferase (NNMT) (13/15, P < 0.0001). Other upregulated candidate biomarkers that were identified by this method include ferritin, hNSE, NM23, secretagogin, and L-plastin. The upregulation of NNMT in RCC was confirmed by immunoblotting and immunohistochemistry. Analysis of fractionated membrane-associated proteins identified CAP-G, mitofillin, tubulin alpha, RBBP7, and HSP27. Of these, RBBP7 and HSP27 were highly expressed in the chromophobe subtype of RCC (3/3) but were absent from conventional RCC (0/3). The triple combination of the NNMT, FTL, and hNSE biomarkers had the highest predictive capacity of 0.993, while NNMT was the single, most powerful candidate diagnostic biomarker for all types of RCC.

207 EXPRESSION OF PYRUVATE DEHYDROGENASE KINASE-1 (PDK-1) AND DJ-1/PARK7 IN RENAL CELL CARCINOMA (RCC) AND ITS ASSOCIATION WITH PATHOLOGY AND OUTCOME

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2010207 EXPRESSION OF PYRUVATE DEHYDROGENASE KINASE-1 (PDK-1) AND DJ-1/PARK7 IN RENAL CELL CARCINOMA (RCC) AND ITS ASSOCIATION WITH PATHOLOGY AND OUTCOME Daniel Baumunk, Uta Reichelt, Jan Ebbing, Florian Jentzmik, Kurt Miller, Martin Schostak, and Steffen Weikert Daniel BaumunkDaniel Baumunk More articles by this author , Uta ReicheltUta Reichelt More articles by this author , Jan EbbingJan Ebbing More articles by this author , Florian JentzmikFlorian Jentzmik More articles by this author , Kurt MillerKurt Miller More articles by this author , Martin SchostakMartin Schostak More articles by this author , and Steffen WeikertSteffen Weikert More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.264AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metabolic adaptations, such as increases in glucose and energy metabolism, are thought to play a pivotal role in the biology of renal cell carcinoma (RCC). Pyruvate dehydrogenase kinase-1 (PDK-1) and the pTEN regulator DJ-1/PARK7 are thought to control metabolic pathways in cancer. In this study we investigated the expression of PDK-1 and PARK in RCC. METHODS RCC tumor tissue samples and corresponding normal parenchyma specimens were obtained from 126 patients with RCC during surgical removal of the primary tumor. Expression of PDK-1 and PARK were determined on the mRNA and protein levels using quantitative RT-PCR and immunohistochemistry. Relative mRNA expression and tumor expression relative to normal tissue were determined and analyzed for associations with pathological stage and grade categories (Kruskal-Wallis ANOVA and Chi-Square test, as appropriate). Potential associations of expression with progression-free and overall survival were analyzed using Cox regression models. RESULTS PDK-1 mRNA expression was up-regulated in RCC as compared to normal tissue (p<0.001). Significant differences were observed by tumor stage (p<0.05) with a significant trend towards lower expression with increasing tumor stage (p>0.01). The expression ratio tumor/normal also showed significant differences by tumor stage with the lowest ratio observed in pT3-disease. Expression data on the mRNA level were confirmed on the protein level with the lowest protein expression determined in pT3 tumors. PDK-1 expression was not associated with Fuhrman grade. No associations were observed for PARK expression. In an exploratory analysis, the PDK-1 expression ratio tumor/normal was inversely associated with outcome after adjustment for tumor stage and grade (HR: 0.54; 95 % CI: 0.31-0.94). CONCLUSIONS PDK-1 is up-regulated in RCC, but down-regulation may be associated with progression towards an aggressive metastasizing behaviour. Our findings further data on a key role for altered glucose metabolism in the biology of RCC, in particular in tumors with an aggressive phenotype. Berlin, Germany© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e82 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel Baumunk More articles by this author Uta Reichelt More articles by this author Jan Ebbing More articles by this author Florian Jentzmik More articles by this author Kurt Miller More articles by this author Martin Schostak More articles by this author Steffen Weikert More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Carbamylated albumin stimulates microRNA-146, which is increased in human renal cell carcinoma.

Carbamylation is a post-translational modification, the pathophysiological consequences of which remain poorly understood. MicroRNAs (miRNAs) are endogenous non-coding small ribonucleic acids that have emerged as one of the central players in gene expression regulation. This study was designed to determine the effect of carbamylated albumin (cAlb) on the expression of miRNAs. Albumin was carbamylated, and the extent of carbamylation was monitored using trinitrobenzenesulphonic acid. Albumin or cAlb were added to rat mesangial cells (RMCs), and RNA was extracted. miRNA microarray analysis was performed. The expression of microRNA-146a (miR-146a) and microRNA-146b (miR-146b) was analyzed by real-time RT-PCR. Of 365 miRNAs analyzed, the expression of miR-146a/b was found to be markedly induced by cAlb (miR-146a, 12.75-fold increase; miR-146b, 5.88-fold increase). Real-time RT-PCR analysis confirmed the increased levels of miR-146a/b by cAlb (p<0.05). It was also found that expression levels of miR-146a/b were increased in renal cell carcinoma tumor tissues compared to corresponding non-tumor tissues (p<0.05). Our data suggest that cAlb stimulates miR-146a/b in RMCs, the levels of which are increased in renal cell carcinoma. Further studies on the function of cAlb may provide new insights into the pathophysiology of renal cell carcinoma.

Dual Degradation of Aurora A and B Kinases by the Histone Deacetylase Inhibitor LBH589 Induces G2-M Arrest and Apoptosis of Renal Cancer Cells

This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6. The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G2-M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G2-M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

Dysfunctional DC subsets in RCC patients: Ex vivo correction to yield an effective anti-cancer vaccine

Dendritic cells (DCs) are potent antigen-presenting cells responsible for the activation and functional polarization of specific T cells. In patients with renal cell carcinoma (RCC) and other cancers, coordinate DC and T cell defects have been reported. In particular, DC and T cell functional subsets that are not conducive to tumor clearance are hypothesized to predominate in patients with advanced-stage disease. Two major peripheral blood DC subsets have been identified in humans: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) that are believed to mediate contrasting effects on cancer immunity. Given the lack of information regarding DC subsets in patients with RCC, in the present study we have investigated the comparative frequencies and activation states of mDC and pDC in peripheral blood, cancer tissues and lymph nodes of patients with RCC using flow cytometry and immunohistochemistry. Three monoclonal antibodies (mAbs) reactive against specific DC subsets (BDCA-2 or BDCA-4 for pDC and BDCA-1 and BDCA-3 which represent two distinct subsets of mDC, mDC1 and mDC2, respectively) were employed. We observed a significant reduction of both DC subsets in the peripheral blood of patients as compared to normal donors. Similarly, both mDC and pDC were recruited in large numbers into RCC tumor tissues, where they displayed an immature phenotype (DC-LAMP −) and appeared unable to differentiate into mature DC (CD83 +) that were competent to migrate to draining lymph nodes. However, we were readily able to generate ex vivo mDC from RCC patients. These DC stimulated robust anti-tumor CTL in vitro and would be envisioned for use in DC-based vaccines applied in patients with RCC whose existing immune system is judged dysfunctional, anergic or prone to undergo apoptosis.

Identification and expression analysis of an N-terminally truncated isoform of human PDGF-C

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF family that plays an important role in developmental and physiological processes, and in human diseases. Here, we report a novel splice variant of human PDGF-C (PDGF-Cb), which encodes an N-terminally truncated protein, lacking the signal peptide and CUB domain. This variant is coexpressed with PDGF-C in all normal tissues analyzed. PDGF-Cb is produced as a cytoplasmic protein, and has a similar intracellular localization to PDGF-C, but is not secreted from transfected cells. Further, we show that PDGF-Cb can form heterodimers (PDGF-CCb) with PDGF-C, which is thereby retained and degraded within cells. In primary renal cell carcinoma (RCC), expression of the two alternatively spliced transcripts was different. Generally, expression of the full-length PDGF-C transcript was increased in RCC tumors, whereas expression of PDGF-Cb was not in the 30 analyzed cases with paired RCC tumor tissues and normal renal tissues. Based on these findings, we suggest that PDGF-Cb might act as a dominant negative molecule regulating the secretion of PDGF-C, and that deregulation of full-length PDGF-C is involved in RCC tumorigenesis.

High expression of Ran GTPase is associated with local invasion and metastasis of human clear cell renal cell carcinoma

The Ran small GTPase (Ran) is involved in the regulation of nuclear transport, microtubule nucleation and dynamics, and spindle assembly. To address the question of whether Ran protein is associated with the progression of renal cell carcinoma (RCC), we compared by Western blotting the Ran protein levels in surgical RCC specimens from 180 consecutive Japanese patients with those in the corresponding nontumor tissue from the same patient. We also examined the Ran protein levels in tumors of different grades and stages. Ran proteins were more abundant in RCC tumor tissues than in nontumor tissues (p < 0.0001). High Ran expression was associated with higher grade, local invasion, and metastasis (p < 0.0001). Kaplan-Meier plots linked high Ran protein expression to a shorter overall survival in all cases (p < 0.0001) and a shorter disease-free survival in those without metastasis at radical or partial nephrectomy (M0; 131 cases, p < 0.0001). Ran protein expression was an independent factor influencing overall survival univariate analysis (p < 0.0001) and disease-free survival by multivariate analysis (p < 0.05). Our findings suggest that Ran is associated with the progression of RCC.

Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1-38 (20 microgram/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with (125)I-labeled GHRH antagonist JV-1-42, we demonstrated the presence of specific high-affinity (K(d) = 0.25 +/- 0.03 nM) binding sites for GHRH with a maximal binding capacity (B(max)) of 70.2 +/- 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of (125)I-JV-1-42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with (125)I-VIP (K(d) = 0.89 +/- 0.14 nM; B(max) = 183.5 +/- 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His(1),(125)I-Tyr(10), Nle(27)]hGHRH(1-32)NH(2). Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of (125)I-JV-1-42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

IDENTIFICATION OF NUCLEAR MATRIX PROTEIN ALTERATIONS ASSOCIATED WITH RENAL CELL CARCINOMA

Purpose Neoplastic transformation, including renal cell carcinoma (RCC), is always accompanied by changes in nuclear morphology. Nuclear grading of RCC is based on characteristic alterations in nuclear shape, size, area and other morphologic parameters. The nuclear matrix, which forms the skeleton of the nucleus, determines nuclear morphology. Alterations in nuclear matrix protein (NMP) composition specific to tissue and cancer type have been described in a variety of human cancers. We conducted a study to analyze the nuclear matrix protein composition of renal cell carcinoma and compare it to that of normal renal tissue and renal cell carcinoma cells grown in culture. Materials and Methods We analyzed the nuclear matrix protein composition of RCC tumor tissue and that of normal kidney tissue obtained from seventeen patients undergoing radical nephrectomy for RCC. We also analyzed the NMP composition of two renal cancer cell lines (A-498 and 769-P). Results We were able to identify five different and unique NMPs which were present only in the human RCC tumor samples and were absent in all normal kidney tissue. One NMP was found specifically in the normal kidney tissue. All five RCC specific NMPs were also identified in the nuclear matrix of the two cell lines analyzed. Conclusions Five nuclear matrix proteins specific and unique to RCC were identified. These NMPs are different from those previously identified in other tissues and neoplasms. The RCC specific NMPs identified in this study can potentially be used as diagnostic markers for renal cell carcinoma and for therapeutic tumor targeting.

Platelet endothelial cell adhesion molecule-1 (PECAM-1): a potential prognostic marker involved in leukocyte infiltration of renal cell carcinoma.

We investigated immunohistochemically the leukocyte infiltrate [CD3, CD4, CD8, CD11a, CD11b, CD14, CD56, VLA-4 and platelet endothelial cell adhesion molecule-1 (PECAM-1)] and the endothelial expression of cell adhesion molecules (PECAM-1, VCAM-1, ICAM-1 and ICAM-2) in 23 renal cell carcinoma tumor tissues. Tumors with a moderate or high density of PECAM-1 positive endothelia showed a stronger infiltration with PECAM-1-positive leukocytes as compared to tumors with a low density of positive endothelia (p<0.0085). Additionally, overall survival of patients who presented with tumors exhibiting a moderate or high density of PECAM-1 endothelia alone or in combination with a PECAM-1-positive infiltrate was extended (median survival: 23.5 months) as compared to patients without these tumor characteristics (median survival: 6.5 months). These results suggest an involvement of PECAM-1 in the process of leukocyte migration and a potential role as a prognostic marker in renal cell carcinoma.