Potential Of Renal Cell Carcinoma Research Articles

Abstract 4828: Therapeutic targeting of P2 × 4 receptor and mitochondrial membrane potential in renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common lethal subtype of kidney cancer. Large-scale metabolomics data have associated metabolic alterations with the pathogenesis and progression of renal cell carcinoma and have correlated mitochondrial activity with poor patient survival. Our group has recently reported that the lysosome purinergic receptor P2 × 4 (P2 × 4R) regulates neoangiogenesis. As ccRCC is characterized by increased angiogenesis we hypothesized that P2 × 4R may play a role in this disease. Interestingly, TCGA data suggest that P2 × 4R over-expression correlates with poor overall ccRCC patient survival. Our preliminary results also suggest that oxophosphorylation is the main source of tumor-derived ATP, which exerts a critical impact on tumor energy metabolism and mitochondrial activity in ccRCC. P2 × 4 receptor expression appears to contribute to the homeostasis of intracellular calcium and integrity of mitochondrial membrane potential in different ccRCC models. Seahorse experiments showed that P2 × 4R inhibition by 5BDBD, a potent and selective antagonist, caused a rapid dose-dependent reduction of mitochondrial activity. Moreover, prolonged mitochondrial failure induced by 5BDBD was associated with increased radical oxygen species, changes in mitochondrial permeability (i.e. opening of the transition pore complex, dissipation of membrane potential and calcium overload), and cell death via both necrosis and apoptosis. P2 × 4R inhibition by 5BDBD was associated with a significant anti-tumor effect both in vitro and in vivo by utilizing several ccRCC cell lines and patient-derived organoids. Interestingly, higher mitochondrial activity was associated with greater sensitivity to 5BDBD. Overall, our results suggest that the perturbed balance between lysosomal integrity and mitochondrial activity induced by P2 × 4R inhibition may be a therapeutic strategy for a subset of renal carcinoma patients Citation Format: Christopher Rupert, Roberto Pili, Filomena DeNigris. Therapeutic targeting of P2 × 4 receptor and mitochondrial membrane potential in renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4828.

Advances in Imaging-Based Biomarkers in Renal Cell Carcinoma: A Critical Analysis of the Current Literature.

Cross-sectional imaging is the standard diagnostic tool to determine underlying biology in renal masses, which is crucial for subsequent treatment. Currently, standard CT imaging is limited in its ability to differentiate benign from malignant disease. Therefore, various modalities have been investigated to identify imaging-based parameters to improve the noninvasive diagnosis of renal masses and renal cell carcinoma (RCC) subtypes. MRI was reported to predict grading of RCC and to identify RCC subtypes, and has been shown in a small cohort to predict the response to targeted therapy. Dynamic imaging is promising for the staging and diagnosis of RCC. PET/CT radiotracers, such as 18F-fluorodeoxyglucose (FDG), 124I-cG250, radiolabeled prostate-specific membrane antigen (PSMA), and 11C-acetate, have been reported to improve the identification of histology, grading, detection of metastasis, and assessment of response to systemic therapy, and to predict oncological outcomes. Moreover, 99Tc-sestamibi and SPECT scans have shown promising results in distinguishing low-grade RCC from benign lesions. Radiomics has been used to further characterize renal masses based on semantic and textural analyses. In preliminary studies, integrated machine learning algorithms using radiomics proved to be more accurate in distinguishing benign from malignant renal masses compared to radiologists' interpretations. Radiomics and radiogenomics are used to complement risk classification models to predict oncological outcomes. Imaging-based biomarkers hold strong potential in RCC, but require standardization and external validation before integration into clinical routines.

POS-220 DEVELOPING RENAL CLEARABLE NANOPARTICLES FOR THE TREATMENT OF RENAL CELL CARCINOMA

Nanoparticles (NPs) have been anticipated to revolutionise health care based on their potential to improve drug delivery. However, the design of effective cancer nano-therapeutics remains a challenge, and only a few nanoparticles have entered clinical trial. The majority of current NPs when administered intravenously are known to accumulate in lungs, liver, spleen & kidney and cause organ damage. Metastatic Renal Cell Carcinoma (RCC) is incurable, with median survival time of only 18 months, and currently there are no effective NP-based therapeutics. To effectively treat RCC and eliminate the side effects caused by NP organ accumulation, we have developed renal clearable NPs based on carbon material (Carbon Dots, CD). The aim of this project is to develop novel NPs with size less than 5.5nm as a vehicle to carry chemotherapeutic drugs such as Doxorubicin (DOX). The nanoparticles should possess renal clearable properties to target RCC and avoid non-specific distribution of drug causing adverse side effects. The study includes NP design and characterisation, drug conjugation, uptake of NPs and outcome measures. The properties of NPs were characterised, and the drug loading capacity was determined. The effect of drug and its conjugates were assessed initially in vitro. The pH difference between cancer cells and normal cells has been used as the triggering mechanism for DOX release. The cell viability of CD NPs, free DOX and CD-DOX conjugate were investigated in Renca and Hela cells by the MTT assay after 24 and 48 hr treatment. The development of an in vivo tumour model is in progress. We have successfully developed a novel type CD Nanoparticles by one-step synthesis. The Transmission Electron microscopy images revealed the CDs are spherical with an average diameter of 4nm. These CDs were shown to have low cytotoxicity with cell viability of higher than 90% at concentrations up to 200ug/ml; possess high quantum yield (58%) with bright blue fluoresce observed under UV light; very high drug loading capacity (93%). The maximum emission wavelength is at 440 nm upon excitation at 340 nm. The excitation-dependent photoluminescence properties allowed visual monitoring of the cells in blue, green and red fluorescence channels. The effective delivery and uptake of CDs into the cell’s cytoplasm was observed within 2 hrs by confocal microscopy. The cell viability assay demonstrated that CD-DOX significantly reduced the viability of the Renca cells by 2-fold compared to free DOX (24% vs 50% respectively, P value=0.0167). In Hela cells, the cell viability was furthermore reduced by 10-fold with CD-DOX compared to free DOX (5.5% vs 57.5% respectively, P value=0.0001). We have developed Carbon Dot nanoparticles with size <5.5nm with low cytotoxicity. The carbon dots have high drug loading capacity and show better inhibition of growth in cancer cell lines compared to unbound chemotherapeutics, suggesting that these nanoparticles have therapeutic potential in renal cell carcinoma.

Screening of Urinary Renal Cancer Metabolic Biomarkers with Gold Nanoparticles-assisted Laser Desorption/Ionization Mass Spectrometry.

Renal cell carcinoma is a very aggressive and often fatal disease for which there are no specific biomarkers found to date. The purpose of this work was to find features that differentiate urine metabolic profiles of healthy people and cancer patients. Laser desorption/ionization mass spectrometry on gold nanostructures-based techniques were used for the metabolic analysis of urine of 50 patients with kidney cancer. Comparison with data from 50 healthy volunteers led to the discovery of several compounds that may be considered potential renal cell carcinoma (RCC) biomarkers. Statistical analysis of data allowed for the discovery of m/z values that had the greatest impact on group differentiation. A database search enabled the assignment of signals for the most promising 15 features among them: serine, heptanol, 3-methylene-indolenine, 2-methyl-3-hydroxy-5-formylpyridine-4-carboxylate, phosphodimethylethanolamine, 4-methoxyphenylacetic acid, N-acetylglutamine, 3,5-dihydroxyphenylvaleric acid, hydroxyhexanoylglycine, valyl-leucine, leucyl-histidine, oleamide, 9,12,13-trihydroxyoctadecenoic acid, stearidonyl carnitine and squalene. Differences of metabolite profiles of human urine could be identified by gold nanoparticle-enhanced target (AuNPET) LDI MS method and used for the detection of renal cancer.

Enhanced YB1/EphA2 axis signaling promotes acquired resistance to sunitinib and metastatic potential in renal cell carcinoma

VHL mutations are the most common tumorigenic lesions in clear cell renal cell carcinoma (ccRCC) and result in continued activation of the HIF/VEGF pathway and uncontrolled cancer progression. Receptor tyrosine kinase (RTK) inhibitors such as sunitinib have been demonstrated to target tumorigenic signaling pathways, delay tumor progression, and improve patient prognosis in metastatic renal cell carcinoma (mRCC). Although several mechanisms of sunitinib resistance have been reported, the solutions to overcome this resistance remain unclear. In our study, we found that increased expression of Y-box binding protein 1 (YB1, a multidrug resistance associated protein) and EphA2 (a member of the erythropoietin-producing hepatocellular (Eph) receptor family, belonging to the RTK family) mediated sunitinib resistance and mRCC exhibited a large phenotypic dependence on YB1 and EphA2. In addition, our findings confirm that YB1 promotes the invasion, metastasis and sunitinib resistance of ccRCC by regulating the EphA2 signaling pathway. Furthermore, pharmacological inhibition of EphA2 through the small molecule inhibitor ALW-II-41-27 reduced the proliferation of sunitinib-resistant tumor cells, suppressed tumor growth in vivo, and restored the sensitivity of sunitinib-resistant tumor cells to sunitinib in vitro and in vivo. Mechanistically, YB1 increases the protein levels of EphA2 by maintaining the protein stability of EphA2 through inhibition of the proteasomal degradation pathway. Collectively, our findings provide the theoretical rationale that ccRCC metastasis and RTK-directed therapeutic resistance could be prospectively and purposefully targeted.

Editorial Comment to Annexin A1 expression is correlated with malignant potential of renal cell carcinoma

None declared.

AnnexinA1 expression is correlated with malignant potential of renal cell carcinoma.

To evaluate the expression of annexinA1 protein in patients with renal cell carcinoma. AnnexinA1 expression was examined in renal cell carcinoma specimens from 27 patients, and their disease-free survival was analyzed using the log-rank test. AnnexinA1 knockdown in the human renal cell carcinoma cell line Caki-1 was carried out, and its proliferation, invasion, motility and adhesion were compared with those of control cells. In 13 out of 27 patients, annexin A1 was highly expressed in the membrane of renal cell carcinoma tumor cells, whereas in the rest of the patients, annexinA1 expression was weak or negligible in the membrane of those cells. Patients with high annexinA1 expression had significantly poorer disease-free survival than those with weak or negligible annexinA1 expression (P=0.031). In the renal cell carcinoma cell line, annexinA1 knockdown cells showed significantly decreased proliferation, invasion, motility and adhesion relative to control cells, and expressed lower relative levels of membrane-type1 matrix metalloproteinase and hypoxia-inducible factor1-alpha transcripts, showing a potential pathway regulated by annexinA1. Annexin A1 is associated with renal cell carcinoma malignant potential and could serve as a marker of poor prognosis.

Integrin α5 triggers the metastatic potential in renal cell carcinoma.

The therapy of advanced renal cell carcinoma (RCC) is still a major challenge. To intervene therapeutically a deeper comprehension of the particular steps of metastasis is necessary. In this context membrane bound receptors like integrins play a decisive role. We analyzed the integrin α5 expression in 141 clear cell RCC patients by Western blot. Patients with RCC expressed a significant higher level of integrin α5 in tumor than in normal tissue. The integrin α5 expression correlated with tumor grade, the development of distant metastases within five years after tumor nephrectomy and reduced survival. The RCC cell lines Caki-1 and CCF-RC1, which highly express integrin α5, were treated with fibronectin in combination with or without an inhibiting anti-integrin α5 antibody. Afterwards the migration, adhesion, viability and prominent signaling molecules were analyzed. Both cell lines showed a significant reduced migration potential as well as a decreased adhesion potential to fibronectin after treatment with an integrin α5 blocking antibody. A contribution of the AKT and ERK1/2 signaling pathways could be demonstrated. The results indicate integrin α5 as a potent marker to discriminate patients’ tumor prognosis. Consequently the integrin subunit α5 can be considered as a target for individual therapy of advanced RCC.

Abstract 5486: Identification of novel candidate genes associated with sorafenib cytotoxicity

Abstract Background: Sorafenib is an oral multikinase inhibitor that decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. Novel pathways and targets of sorafenib activity remain to be identified, and no predictive biomarkers of sorafenib activity exist to help guide clinicians. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains were genomically characterized previously (PMID: 21623374). MEFs were treated with ten concentrations (0-300 μM) of sorafenib, incubated for 72 h, and then fixed and stained. Cytochrome C was immunolabeled in the fixed cells and imaged using an automated fluorescence microscope. An image analysis software assessed sorafenib-induced cytochrome C release from mitochondria. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the EMMA algorithm, was performed on cytochrome C EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values ≤ 1.0×10−7 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 72 h (17.2-44.5 μM) sorafenib incubations. We identified one peak (chromosome 9 from 51-52 Mb; p = 1.0×10−8), which reached genome-wide significance and significantly associated with cytochrome C release. From this peak, we have identified candidate genes that may underlie variability in sorafenib-induced cytochrome C release from mitochondria. A total of nine genes expressed in MEF cells at mRNA level are present in this QTL. Of particular interest, the identified locus contains Rdx, a gene that encodes radixin. Radixin is a component of the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) complex, and is conserved between humans and mice. In humans, radixin, as part of the EBP50 complex, has been shown to act as a tumor suppressor and to promote apoptosis in hepatocellular carcinoma by modulating β-catenin/E-cadherin (PMID: 23483729). It has also been shown to potentially enhance the metastatic potential of renal cell carcinoma (PMID: 20395446). Conclusions: Our high-throughput cellular genetics approach has detected robust interstrain cellular differences in sorafenib activity, and identified one region, which reached genome-wide significance, that potentially associates with sorafenib-induced cytochrome C release from mitochondria. Functional validation of Rdx and other promising candidates is currently ongoing. Citation Format: Daniel James Crona, Oscar Suzuki, O. Joseph Trask, Bethany Parks, Amber Frick, Timothy Wiltshire, Federico Innocenti. Identification of novel candidate genes associated with sorafenib cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2015-5486

Renin-angiotensin system (RAS) blockade attenuates growth and metastatic potential of renal cell carcinoma in mice

ObjectivesRenal cell carcinoma (RCC) is the most frequent type of cancer among renal neoplasms in adults and responds poorly to radiotherapy and chemotherapy. There is evidence that blockade of the renin-angiotensin system (RAS) might have antineoplastic effects. The aim of this study was to investigate the effects of RAS blockade on RCC in a murine model. Methods and materialsMurine renal cancer cells (Renca) were injected (1×105) into the subcapsular space of the left kidney of BALB/c mice (8wk of age). The animals were divided into 4 groups: a control group (no treatment), angiotensin-receptor blockers group (losartan 100mg/kg/d), angiotensin-converting enzyme inhibitor group (captopril 10mg/kg/d), and angiotensin-receptor blockers +angiotensin-converting enzyme inhibitor group (losartan 100mg/kg/d +captopril 10mg/kg/d). The animals received the drugs by gavage for 21 days after inoculation, beginning 2 days before tumor induction, and were then euthanized. After killing the animals, the kidneys and lungs were removed, weighed, and processed for histopathological and immunohistochemical analyses. Angiogenesis and vascular microvessels were assessed with the antibodies anti–vascular endothelial growth factor and anti-CD34. ResultsAngiotensin II–inoculated animals developed renal tumors. Treated animals presented smaller tumors, regardless of the therapeutic regimen, and far fewer lung metastases in both quantity and dimension compared with the controls. The expression of vascular endothelial growth factor and CD34 were significantly decreased in renal tumors of treated animals compared with the controls. ConclusionsOur findings suggest that blockade of RAS decreases tumor proliferation and metastatic capacity of RCC in this experimental model.

Abstract A36: Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma

Abstract Introduction: Integrin-linked kinase (ILK) is a serine/threonine kinase implicated in the regulation of cell growth and survival, cell cycle progression, epithelial-mesenchymal transition (EMT), invasion and migration, angiogenesis. However, the role of ILK has not been evaluated in renal cell carcinoma (RCC). We investigated the role of ILK on cancer progression and metastasis and therapeutic potential of ILK inhibition in RCC. Methods: Baseline expression of ILK was evaluated by immunofluorescence and western blotting in non-cancerous renal tubular cells (HK-2) and RCC cells (UMRC-6, UMRC-3 and Caki-1). Molecular EMT markers were examined by Western blotting. RNAi using siRNA was used to knock down ILK in vitro. After transient transfection, crystal violet assay and cell cycle analysis using FACS were performed to check the effect of ILK on tumor growth and cell cycle. Confocal microscopic evaluation was performed to check changes of stress fibers and focal adhesions stained with phalloidin-rhodamine and vinculin antibody. Scratch assay and Matrigel invasion assay were performed to evaluate changes of EMT phenotypes after knockdown of ILK. To evaluate the effects of ILK knockdown in vivo, subcataneuous xenografts and orthotopic renal xenografts were used and tumor growth was traced. Results: ILK is less expressed in normal cells (HK-2) and low stage RCC cells (UMRC-6) but highly expressed in advanced and metastatic RCC cells (UMRC-3 and Caki-1). Caki-1, metastatic RCC cells showed higher expressions of molecular EMT markers including Snail, Zeb1 but decreased activity of GSK3β. Knockdown of ILK using si-ILK inhibited tumor proliferation but the inhibition rate was less than 10% and cell cycle progression was not significantly affected by ILK inhibition. However, ILK knockdown suppressed formation of stress fibers and focal adhesions in UMRC-3 and Caki-1 cells and also effectively impeded phenotypic EMT markers including cell migration and invasion in Caki-1 and UMRC-3 cells. In subcutaneous tumor xenografts with Caki-1-shILK or Caki-1-EV, ILK knockdown inhibited tumor growth and progression. In primery orthotopic animal model, ILK knockdown showed inhibitory effects of invasion and metastasis. Conclusions: ILK is highly expressed in advanced RCC and its high expression is related to EMT-related protein in RCC. Knockdown of ILK inhibited molecular EMT markers and suppressed cell migration and invasion in vitro and metastasis in orthotopic tumor model. These results suggest the therapeutic potential of ILK inhibition on invasion and metastasis in advanced RCC. Citation Format: Han Kyung Seok, Raven Peter, Awrey Shannon, Li Estelle, Fazli Ladan, Gleave Martin, So Alan. Knockdown of integrin-linked kinase reduces invasive and metastatic potential of renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A36.

158P - Renin Angiotensin System (Ras) Blockade Attenuates Growth and Metastasis Formation of Renal Cell Carcinoma in Mice

ABSTRACT Aim: Renal cell carcinoma (RCC) is the most frequent cancer among renal neoplasms in adults and poorly responsive to radio-, chemo- and immunotherapy. There is increasing evidence that blockade of RAS has antineoplasic effects. Objective: Investigate the effects of RAS blockade on renal cell carcinoma (RCC) in a murine model. Methods: Murine renal cancer cells (RENCA) were cultivated and injected (1x105) into subcapsular space of the left kidney of BALB/c mice (8 weeks). The animals were divided into 4 groups : control group (no treatment), Losartan (100 mg / kg / day), Captopril group (10 mg / kg / day) and Losartan + Captopril group (100 mg / kg / day + 10 mg / kg / day). The animals received the drugs by gavage 2 days before tumor induction and continued until euthanasia (21 days after inoculation). After sacrifice, kidneys and lungs were removed, weighed and processed for histopathological analysis. Angiogenesis and vascular microvessel were determined by immunohistochemical evaluation for VEGF and CD34. Results: All inoculated animals developed renal tumors. Treated animals presented smaller tumors, regardless of the therapeutic regimen, and far fewer lung metastases in both quantity and dimension in relation to controls. The expressions of VEGF and CD34 were significantly decreased in renal tumors of animals treated compared with controls. Conclusions: Our findings suggest that blockade of RAS decreases the tumor proliferation capacity and metastatic potential of renal cell carcinoma in this experimental model. Disclosure: All authors have declared no conflicts of interest.

MP23-13 MICRO-RNA DIFFERENTIATES TUMOR TYPE & METASTATIC POTENTIAL IN RCC

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-13 MICRO-RNA DIFFERENTIATES TUMOR TYPE & METASTATIC POTENTIAL IN RCC Michael Garcia-Roig, Nicolas Ortiz, and Vinata Lokeshwar Michael Garcia-RoigMichael Garcia-Roig More articles by this author , Nicolas OrtizNicolas Ortiz More articles by this author , and Vinata LokeshwarVinata Lokeshwar More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.881AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Small renal tumors (<4 cm) pose a challenge as 16-28% are benign, indistinguishable from malignant lesions on imaging & pose a metastatic risk. Percutaneous biopsy has regained popularity for small masses by using histology, immunology or molecular markers for diagnosis and prognosis. miRNAs are 22-nucleotide noncoding segments regulating gene expression by causing degradation of target mRNAs. We examined the differential expression of microRNAs (miRs) in renal cell carcinoma (RCC) & normal kidney specimens. Our objective was to examine whether miRNA expression can identify tumor type & predict metastasis. METHODS RCC & normal kidney specimens were collected from 86 patients at the time of nephrectomy. miR enriched total RNA was isolated from 46 RCC & 59 normal specimens. Clinical & pathologic data were collected including histology (oncocytoma 6, chromophobe 4, papillary 6, clear cell 25, sarcoma 4, & collecting duct 1), stage (T1a, 11; T1b, 9; T2, 9; T3, 10; T4, 1), renal vein involvement (+: 5; −: 41), & lymph node status (+: 4; −: 41). At 14 months median follow−up, 6 patients were positive for metastasis. Microarray (Thermo Fisher Scientific 10.1) was performed on 30 samples to characterize 554 miRNA targets. 7 miRs were chosen (mir−21, 150, 155, 142−3p, 142−5p, 192, & 194) based on the fold change in tumor samples. Their expression was quantified by quantitative PCR, & normalized to a housekeeping miR. Statistical analysis was performed using the Mann−Whiteny U−test & multivariate analysis. RESULTS miR microarray of RCC vs non-RCC specimens identified significant upregulation of 65 (mean fold change (FC) 2.13), & downregulation of 47 (mean FC 1.7) miRNAs in (p<0.05), respectively. Q-PCR of RCC specimens showed differential expression of miRs-21, 155, 192 & 194. miR-150 & miR-155 levels were 5.8-8.2-fold upregulated in chromophobe (25.8±21; 2.3±2.9) vs. oncocytomas (4.4±2.0; 0.28±0.36; P=0.038). miR-21, miR-1423P, miR-1425P & miR-155 were 3-10-fold upregulated in CC-RCC (1731±1145; 77±174; 4.2±5.2; 2.2±2.7) vs oncocytoma (553±523; 7.3±8.2; 0.43±0.42; 0.28±0.36), respectively (P ≤0.01). Only miR-155 was 5.5-fold upregulated in papillary tumors vs oncocytoma (P=0.015). In multivariate analysis including TNM stage, renal vein invasion, age, gender & miR levels, only mir−194 expression correlated with metastasis (p=0.034; Xˆ2 = 4.5). CONCLUSIONS miR−155, 192, 194, & 21 are differentially expressed in kidney cancer. miR−194 expression independently associates with metastasis. miR-155 was differentially expressed in all RCC types when compared to normal kidney & oncocytomas. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e248 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Michael Garcia-Roig More articles by this author Nicolas Ortiz More articles by this author Vinata Lokeshwar More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Germline PTEN Mutation Cowden Syndrome: An Underappreciated Form of Hereditary Kidney Cancer

Cowden syndrome is a hereditary cancer syndrome associated with a germline mutation in PTEN. Patients are predisposed to multiple malignancies including renal cell carcinoma. Patients with Cowden syndrome were evaluated as part of a clinical protocol. Those with a history of renal cell carcinoma underwent review of clinical features, tumor characteristics and family history. Renal tumors were evaluated for loss of heterozygosity. Among 24 patients with Cowden syndrome 4 were identified with renal cell carcinoma (16.7%). Three patients had solitary tumors, 2 with papillary type I histology and 1 with clear cell histology. The fourth patient had bilateral, synchronous chromophobe tumors. No patients had a prior family history of renal cell carcinoma. All patients with renal cell carcinoma had dermatologic manifestations of Cowden syndrome and had macrocephaly. Loss of heterozygosity at the PTEN mutation was identified in 4 tumors (80%). No genotype-phenotype association was found, as the same mutation was identified in different renal cell carcinoma histologies. Renal cell carcinoma is an underappreciated feature of Cowden syndrome. As most patients lack a prior family history or a distinctive renal cell carcinoma histology, recognition of the associated nonrenal features should target referral for genetic counseling. PTEN loss of heterozygosity is common in Cowden syndrome renal tumors. Because loss of PTEN can activate mTOR and mTOR inhibitors are Food and Drug Administration approved to treat renal cell carcinoma, these agents have clinical potential in renal cell carcinoma associated with Cowden syndrome.

Effect of host immunity on metastatic potential in renal cell carcinoma: the assessment of optimal in vivo models to study metastatic behavior of renal cancer cells

There has been little information about metastatic behavior of renal cell carcinoma (RCC) cells because human cancers metastasize only rarely in immunodeficient mice. Moreover, it is difficult to know the effect of host immunity on RCC metastasis due to lack of such RCC cells as transplantable in not only xenograft models but also counterparts with intact immunity. Therefore, we scrutinized in vivo metastasis of RCC cells to seek for the optimal preclinical model to study metastatic behavior. The luciferase-expressing three representative human RCC cell lines (Caki-1, A498, and 786-O) and rat ACI-RCC cell which were established in our laboratory were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice or immunocompetent ACI rats by intracardiac injection as well as orthotopic inoculation. Metastasis was monitored using a bioluminescent imaging technique. Metastasis was rare in the three human RCC cells even when they were directly disseminated into systemic circulation under the condition least susceptible to host immune attack in NOD/SCID mice. In contrast, ACI-RCC cells spontaneously metastasized to pulmonary tissue from orthotopic tumor sites and systemically spread via intracardiac route. Metastases were more extensive when the cells were inoculated into an immunodeficient host, implying suppressive effect of host immunity on colonization of RCC cells. These results suggest that the representative human RCC cells are not adequate resource to study metastasis but that the luciferase-labeled ACI-RCC cell characterized by its luminescent stability, enhanced tumorigenicity, and widespread metastatic potential provides a useful in vivo model for preclinical assessment of cancer progression and potential therapies against RCC.

Abstract 606: Expression and role of high mobility group protein AT-hook 1 (HMGA1) in human renal cell carcinoma

Abstract Even though molecular targeted therapy has improved the clinical outcome of metastatic renal cell carcinoma (mRCC) patients, complete response is rarely observed. Identification of molecules possessing significant molecular functions in mRCC is mandatory for developing new therapeutic modalities for mRCC. High mobility group protein AT-hook 1 (HMGA1) is overexpressed in many types of cancer cells, but is not or at very low levels expressed in normal adult tissues. Expression of HMGA1 has been reported to be associated with metastatic potential and progression of cancer. We have reported that transfection of HMGA1 into prostate cancer cell lines induces chromosomal rearrangement (Cancer Res, 2002) and expression of matrix metalloproteinase 2 (Prostate, 2004) suggesting that HMGA1 may play an important role in the progression of prostate cancer. In this study, we examined expression and function of HMGA1 in human renal cell carcinoma (RCC). Expression of HMGA1 in six human RCC cell lines, Caki-1, ACHN, NC 65, RCC-4, 786-O, and A498, was examined by immunoblot and immunohistochemistry. The expression level of HMGA1 detected by immunoblot was Caki-1=ACHN&amp;gt;NC65&amp;gt;786-O, while RCC-4 and A498 barely expressed HMGA1. Immunohistochemistry showed nuclear localization of HMGA1 in these cell lines. Expression of HMGA1 in renal tumor tissue and normal kidney tissue from the same patients who underwent radical nephrectomy was examined by immunoblot. Immunoblot of surgical samples of 41 cases of RCC revealed that HMGA1 is not expressed in normal kidney tissues from all the cases. On the other hand, immunoblot showed that expression of HMGA1 was observed in 1 out of 23 (4%) non-metastatic cases (M0) compared to 6 out of 18 (33%) metastatic cases (M1) (P=0.031), and 3 out of 29 (10%) G1-2 cases compared to 4 out of 9 (44%) G3 cases (P=0.041). Colony formation assay and flowcytometry were performed using Caki-1 after transfecting with siRNA to knock down HMGA1. Knock-down of HMGA1 expression in Caki-1 cells by siRNA remarkably suppressed colony formation (control siRNA 86.0±5.3 vs HMGA1 siRNA 7.3±1.5, P&amp;lt;0.001)and also induced apoptosis associated with increased subG1 fraction (control siRNA 1.9% vs HMGA1 siRNA 34.6%). These findings suggest that HMGA1 might be correlated with histological grade and metastatic potential of RCC associated with enhanced anti-apoptotic mechanism. No expression in normal kidney tissues, preferential expression in renal tumor tissues of metastatic cases, and remarkable suppression of colony formation by siRNA suggest that HMGA1 might be a potential target molecule for novel therapeutic modalities of mRCC. 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 606. doi:10.1158/1538-7445.AM2011-606

Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma

Inappropriate kinase expression and subsequent promiscuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Further, identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. Here, we report the identification of Ror2, a new tumor-associated kinase in RCC cell lines and primary tumors. Ror2 is an orphan receptor tyrosine kinase with physiological expression normally seen in the embryonic kidney. However, in RCC, Ror2 expression correlated with expression of genes involved at the extracellular matrix, including Twist and matrix metalloprotease-2 (MMP2). Expression of MMP2 in RCC cells was suppressed by Ror2 knockdown, placing Ror2 as a mediator of MMP2 regulation in RCC and a potential regulator of extracellular matrix remodeling. The suppression of Ror2 not only inhibited cell migration, but also inhibited anchorage independent growth in soft agar and growth in an orthotopic xenograft model. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within a subset of renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

Expression of apoptotic tumour necrosis factor receptor‐associated factor, caspase recruitment domain and cell death‐inducing DFF‐45 effector genes in therapy‐treated renal cell carcinoma

Dysfunction in apoptosis plays a role in development of renal cell carcinoma (RCC). This investigation aimed to identify expression of apoptosis-related genes not previously characterized in human RCC. The RCC ACHN cell line was treated with radiation plus interferon-alpha to induce significant apoptosis. Apoptosis RNA microarrays were used to compare control and treated RCC for apoptosis-regulatory genes with significantly altered expression (>or= twofold). Translational correlates were analysed using western blot. Immunohistochemistry of human RCC and non-cancerous kidney in tissue microarrays was also completed. Several gene families, not well characterized in RCC, were significantly upregulated in RNA microarray. These were the tumour necrosis factor receptor-associated factors (TRAF1, 3 and 4), caspase recruitment domain (NOL3 and PYCARD), and cell death-inducing DFF-45 effector domain (ICAD/CAD) genes. The protein expression patterns did not always increase similarly, perhaps indicating some post-transcriptional controls needing further investigation. TRAF1 had significantly increased expression for RNA and protein (P<0.01). NOL3 had significantly decreased whole-cell protein expression (P<0.05), but had strongly localized nuclear positivity in RCC in the immunohistochemistry. These newly identified RCC apoptosis genes have shown potential for improving outcome in other cancers and may prove to have the same potential in RCC with further study.

Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma

Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma

Genetic variation in platelet integrin αIIbβ3 (GPIIb/IIIa) and the metastatic potential of renal cell carcinoma

To explore whether genetic polymorphisms in platelet receptors known to be associated with platelet activity have any association with haematogenous metastases in renal cell carcinoma (RCC), as platelets and their fibrinogen receptors may be central to haematogenous cancer spread, in addition to various adhesive proteins on both platelets and tumour cells on themselves. The glycoprotein alpha(IIb)beta3 (GPIIb/IIIa) is the main fibrinogen receptor on platelets, with GPIb-IX-V and GPIa/IIa being the von Willebrand Factor and collagen receptors, respectively. In a cross-sectional survey of 128 men treated for RCC (55 with disseminated disease and 73 with a local disease), they were genotyped using DNA extracted from freshly drawn blood, and central clinical data were recorded. The frequency of possessing the GPIIIaPl(A2) allele among patients with metastatic RCC was 43.6%, and with local disease was 24.7% (P = 0.024). The frequencies of the different alleles of the GIB-IX-V, C3550T and GPIa/IIa C807T polymorphisms did not differ between the groups. In a stepwise logistic regression (metastatic vs local RCC) the Pl(A2) allele remained a significant risk factor, with an odds ratio of 2.7 (95% confidence interval, 1.1-6.5; P = 0.02). The prothrombotic Pl(A2) allele of platelet fibrinogen receptor GPIIb/IIIa increased the risk of metastases in RCC in men.