Protein In Renal Cell Carcinoma Research Articles

The fission yeast FLCN/FNIP complex augments TORC1 repression or activation in response to amino acid (AA) availability

SummaryThe target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes.

DAB2IP Plays Important Clinical Significance and Correlates With Immune Infiltration in Renal Cell Carcinoma.

Background:Disabled homolog 2-interacting protein is a new member of the Ras GTPase superfamily involved in the regulation of cell proliferation, apoptosis, and metastasis. However, the expression of disabled homolog 2-interacting protein in renal cell carcinoma, its correlation with cancer prognosis, and tumor infiltrating lymphocytes remains unclear.Methods:The expression of disabled homolog 2-interacting protein was analyzed by UALCAN database, GEPIA database and the evaluation of disabled homolog 2-interacting protein effects on clinical prognosis. Prognostic factor analysis was used to identify the correlations between disabled homolog 2-interacting protein and cancer immune infiltration via the TIMER database. In addition, COXPRESdb database was used to analyze the enrichment of disabled homolog 2-interacting protein co-expression genes.Results:Compared to the normal tissues, the messenger RNA expression levels of DAB2IP are higher in 8 while lower in 15 types of tumor tissues. Furthermore, disabled homolog 2-interacting protein has high expression in kidney chromophobe and low expression in both kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. The messenger RNA expression levels of disabled homolog 2-interacting protein decrease gradually due to the increasing tumor staging which positively correlates with disease-free survival and overall survival in both kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. The expression levels of disabled homolog 2-interacting protein also positively correlate with the tumor purity of kidney chromophobe, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma samples. Besides, the expression of disabled homolog 2-interacting protein in renal cell carcinoma has negative correlation with the immune infiltration, and the immune infiltration of B cells and CD8+ T cells affects the prognosis of kidney renal papillary cell carcinoma. Enrichment analysis of disabled homolog 2-interacting protein co-expressed genes suggested that its biological role was mainly in regulating GTPase activity.Conclusions:These findings suggest that disabled homolog 2-interacting protein functions as a tumor suppressor in the progression of renal cell carcinoma, and the expression of disabled homolog 2-interacting protein is related to the immune infiltrating cells and affects the survival of renal cell carcinoma. Disabled homolog 2-interacting protein can be a novel clinical biomarker for patients with renal cell carcinoma, which also provides new insights for the future treatments of renal cell carcinoma.

PLK1 promotes proliferation and suppresses apoptosis of renal cell carcinoma cells by phosphorylating MCM3.

Minichromosome maintenance 3 (MCM3) protein has been widely studied due to its essential role in DNA replication. In addition, it is overexpressed in several human tumor types. However, the role of this protein in renal cell carcinoma (RCC) is not widely known. In this study, we demonstrated that polo-like kinase 1 (PLK1)-mediated MCM3 phosphorylation regulates proliferation and apoptosis in RCC. Our results confirm that PLK1 and phospho-MCM3 (p-MCM3) are highly expressed in renal cell carcinoma. The expression of PLK1 is closely related to the clinical characteristics of renal cell carcinoma. They play important roles in the proliferation and apoptosis of RCC. In vitro, after overexpression of PLK1 or MCM3, the proliferation of RCC cells was significantly enhanced and cell apoptosis was inhibited, while after knockout, the proliferation of RCC cells was weakened and cell apoptosis was promoted. In addition, Mn2+-Phos-tag SDS-PAGE, western blotting, and immunofluorescence were utilized to determine that MCM3 is a physiological substrate of PLK1, which is phosphorylated on serine 112 (Ser112) in a PLK1-dependent manner. PLK1-mediated MCM3 phosphorylation promotes RCC cell cycle proliferation and suppresses apoptosis in vitro. Moreover, we found that PLK1-mediated MCM3 phosphorylation induced cellular proliferation and decreased apoptosis, as well as tumor growth in mice. Overall, we conclude that PLK1-mediated MCM3 phosphorylation is a novel mechanism to regulate RCC proliferation and apoptosis.

Fibronectin protein expression in renal cell carcinoma in correlation with clinical stage of tumour

BackgroundCarcinogenesis is a multistep process which involves interplay between the tumour cells and the matrix proteins. This occurs by adherence between the tumour cells and proteins in the extracellular matrix. VHL mutation affects through the hypoxia inducible factor (HIF) and causes changes in various tissue proteins like VEGF, PDGF, TGF, Fibronectin and others. As not much literature is available, we aim to quantify the changes of fibronectin protein in renal cell carcinoma (RCC) tissue.MethodsThis Prospective unbalanced case control study was conducted over a period of 18 months from April 2016 to September 2017. The patients undergoing nephrectomy for the diagnosis of RCC were included in the study after obtaining written informed consent. Patients were excluded from study, if normal renal tissue could not be identified in the resected kidney and if the artery clamp time to retrieval of tissue was more than 30 min. Fibronectin protein is estimated in the tumour tissue by gel electrophoresis and western blotting which is compared with that of normal kidney tissue of the same kidney. Results have been expressed as absolute values with standard deviation and relative expression (RE).ResultsOf the 21 patients analysed 15 showed an increase in fibronectin expression in the renal tumour tissue while 6 did not. The mean expression of Fibronectin protein has increased 1.5 times in the tumour tissue when compared with the normal tissue. The increase was 1.54 times in early tumours compared to 1.37 times in advanced tumours of RCC.ConclusionsFibronectin showed a 1.5 times increase in the tumour compared to normal. This increase is more in Stage 1&2 tumours when compared to the Stage 3&4 tumours.

Mevalonate pathway blockage enhances the efficacy of mTOR inhibitors with the activation of retinoblastoma protein in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common malignancy of kidney and remains largely intractable once it recurs after resection. mTOR inhibitors have been one of the mainstays used against recurrent RCC; however, there has been a major problem of the resistance to mTOR inhibitors, and thus new combination treatments with mTOR inhibitors are required. We here retrospectively showed that regular use of antilipidemic drug statins could provide a longer progression free survival (PFS) in RCC patients prescribed with an mTOR inhibitor everolimus than without statins (median PFS, 7.5 months vs. 3.2 months, respectively; hazard ratio, 0.52; 95% CI, 0.22–1.11). In order to give a rationale for this finding, we used RCC cell lines and showed the combinatorial effects of an mTOR inhibitor with statins induced a robust activation of retinoblastoma protein, whose mechanisms were involved in statins-mediated hindrance of KRAS or Rac1 protein prenylation. Finally, statins treatment also enhanced the efficacy of an mTOR inhibitor in RCC xenograft models. Thus, we provide molecular and (pre)clinical data showing that statins use could be a drug repositioning for RCC patients to enhance the efficacy of mTOR inhibitors.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

AB054. The role of urine ErbB3 protein in early diagnosis and prognosis evaluation of renal cell carcinoma

BackgroundTo study the expression of ErbB3 protein in renal cell carcinoma patients’ urine and to explore the diagnostic value of ErbB3 protein in renal cell carcinoma.Methods(I) We collected 42 renal cell carcinoma patients’ urine (including 31 clear cell renal cell carcinoma patients, 4 chromophobe renal carcinoma patients, 3 papillary cell renal carcinoma patients, 2 MTT renal carcinoma patients, 1 sarcomatoid carcinoma patient and 1 neurogenic renal carcinoma patient), 19 urinary calculus patients’ urine, 40 urothelium carcinoma patients’ urine, 33 prostate cancer patients’ urine, 17 benign prostate hyperplasia patients’ urine and 50 normal people’s urine as control. ELISA was used to test the expression of ErbB3 protein in urine of different diseases. (II) We used SPSS 21.0 to analyze ErbB3 protein in urine of different diseases. Then we established the ROC curve of which diagnosing renal carcinoma and clear cell renal carcinoma by ErbB3 protein, respectively. Also, we analyzed the relation between ErbB3 protein in urine and the patients’ BMI, creatinine, tumor diameter and underlying diseases such as hypertension and hyperglycemia.ResultsThe content of ErbB3 protein was 18.9±26.4 pg/mL in renal cell carcinoma group, 17.8±26.6 pg/mL in clear cell renal carcinoma group, 3.1±37.4 pg/mL in urinary calculus group, 335.3±702.4 pg/mL in urothelium carcinoma group, 13.7±15.6 pg/mL in prostate cancer group, 40.4±52.4 pg/mL in BPH group and 59.0±54.7 pg/mL in normal group, respectively. The expression of ErbB3 protein in renal cell carcinoma group and clear cell renal cell carcinoma group was significantly lower than normal group(P<0.001). Comparing with normal group, ErbB3 protein of urothelium carcinoma group has a higher expression and prostate cancer group has a lower expression on the contrary. The contents of ErbB3 protein in urinary calculus group and BPH group had no significantly differences with normal group. When diagnosing renal carcinoma by ErbB3 protein, the AUC of ROC was 0.790 (P≤0.001. When setting the cutoff as 11.714 pg/mL, the max Youden index was 0.519., the sensitivity was 0.619 and the specificity was 0.900. The Kappa value of diagnostic test was 0.530 (P≤0.001. In the same way, when diagnosing clear cell renal carcinoma by ErbB3 protein, the AUC of ROC was 0.802 (P≤0.001. When setting the cutoff as 13.9804 pg/mL, the max Youden index was 0.525., the sensitivity was 0.645 and the specificity was 0.880. The Kappa value of diagnostic test was 0.542 (P≤0.001. According to the most appropriate cutoff of renal cell carcinoma and clear cell renal cell carcinoma diagnostic test, we divided the renal cell group into ErbB3 high-expression group and ErbB3 low-expression group. Comparing the patients’ BMI, tumor diameter and creatinine between two groups, there was no significant difference. There was no correlation between the ErbB3 content and patients’ BMI, tumor diameter and creatinine by correlation analysis. Basing on the preoperative data, we divided the renal cell group into hypertension and normotension group, there was no significant difference between two groups about the ErbB3 content. Also, we compared the expression of ErbB3 between hyperglycemia and euglycemia groups, there was no difference. We got similar results in analysis of clear cell renal cell carcinoma.Conclusions(I) The expression of urine ErbB3 protein in renal cell carcinoma, clear cell renal carcinoma and prostate cancer was lower than normal people. (II) The expression of urine ErbB3 protein had no significant difference between urinary calculus group, BPH group and normal control. (III) The expression of urine ErbB3 protein in urothelium carcinoma was higher than normal people. (IV) When diagnosing renal carcinoma by urine ErbB3 protein, the AUC of ROC was 0.790. The diagnostic cutoff was 11.714 pg/mL, the max Youden index was 0.519, the sensitivity was 0.619 and the specificity was 0.900. The Kappa value of diagnostic test was 0.530. When diagnosing clear cell renal carcinoma by urine ErbB3 protein, the AUC of ROC was 0.802. The diagnostic cutoff was 13.9804 pg/mL, the max Youden index was 0.525, the sensitivity was 0.645 and the specificity was 0.880. The Kappa value of diagnostic test was 0.542. (V) There was no relation between the expression level of urine ErbB3 protein and patients’ BMI, tumor diameters, creatinine, blood pressure and blood glucose in renal cell carcinoma and clear cell renal cell carcinoma.

Tumor Specific Epigenetic Silencing of Corticotropin Releasing Hormone -Binding Protein in Renal Cell Carcinoma: Association of Hypermethylation and Metastasis.

The relevance of Corticotropin Releasing Hormone (CRH)-system in human malignancies is a question of growing interest. Here we investigated hypermethylation and epigenetic silencing of the CRH-Binding Protein (CRHBP) gene in clear cell renal cell cancer (ccRCC). Relative methylation of the CRHBP CpG island (CGI) was determined in 17 tumor cell lines as well as 86 ccRCC samples and 66 paired normal tissues using pyrosequencing and quantitative methylation specific PCR of bisulfite converted DNA. Results were statistically compared with relative mRNA expression levels of CRHBP and clinicopathological parameters of patients. Re-expression of CRHBP following 5-aza-2´-deoxycytidine treatment was investigated by quantitative mRNA expression analysis. Real-time impedance analysis was applied for analysis of invasiveness of renal tumor cells following si-RNA knockdown of CRHBP expression or ectopic expression of CRHBP. We found the CRHBP CGI to be frequently methylated in tumor cell lines of renal, prostatic, and bladder cancer. Comparison of methylation in normal and paired renal cancer tissue specimens revealed hypermethylation of the CRHBP CGI in tumors (p<1*10−12). DNA methylation and decreased mRNA expression were correlated (R = 0.83, p<1*10−12). Tumor cell lines showed 5-aza-2´-deoxycytidine dependent reduction of methylation and re-expression of CRHBP was associated with altered cellular invasiveness of renal cancer cells in real-time impedance invasion assays. Hypermethylation and inverse relationship with mRNA expression were validated in silico using the TCGA network data. We describe for the first time tumor specific epigenetic silencing of CRHBP and statistical association with aggressive tumors thus suggesting the CRH system to contribute to the development of kidney cancer.

AB156. Seminal plasma protein in renal cell carcinoma: expression of semenogelin I is a predictor for cancer progression and prognosis

ObjectiveThe incidence of renal cell carcinoma (RCC) has been steadily rising each year. There are currently few recognized biomarkers for the diagnosis and prognosis of RCC.MethodsWe investigated semenogelin I (Sg I) expression and its clinical significance in patients with RCC. The expression levels of Sg I and its protein were measured by qPCR and Western blotting, respectively. Immunohistochemistry was used to investigate the protein expression of Sg I in RCC and normal renal tissue from 53 patients. The Kaplan-Meier method and log-rank test were used to evaluate the data.ResultsBy qRCR (P<0.01) and Western blot, the level of Sg I expression in benign tissues was higher than that in RCC tissues. Expression of Sg I was observed in 30 (57%) RCC cases, which was significantly lower than that observed in benign renal tissues from the same patients [Sg I positive in 53 (100%) cases (P<0.0001)] by immunohistochemistry. There was an inverse relation between Sg I expression and clinical stage (pT1-2 vs. pT3-4, P<0.0001). Patients with Sg I-negative tumor had a significantly higher risk of recurrence (Kaplan-Meier and log-rank tests, P<0.0001). There was low Sg I expression in RCC.ConclusionsSg I expression has potential value in predicting cancer progression and prognosis. These findings support the use of Sg I as a novel biomarker for RCC.

Editorial Comment

Editorial Comment

EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

ObjectivesTo investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.MethodsExpression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.ResultsIn all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.ConclusionsOur data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism.

During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the "von Hippel Lindau" (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL-/VHL(+) RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.

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.

Seminal plasma protein in renal cell carcinoma: expression of semenogelin I is a predictor for cancer progression and prognosis.

The incidence of renal cell carcinoma (RCC) has been steadily rising each year. There are currently few recognized biomarkers for the diagnosis and prognosis of RCC. We investigated semenogelin I (Sg I) expression and its clinical significance in patients with RCC. The expression levels of Sg I and its protein were measured by qPCR and Western blotting, respectively. Immunohistochemistry was used to investigate the protein expression of Sg I in RCC and normal renal tissue from 53 patients. The Kaplan-Meier method and log-rank test were used to evaluate the data. By qRCR (p < 0.01) and Western blot, the level of Sg I expression in benign tissues was higher than that in RCC tissues. Expression of Sg I was observed in 30 (57 %) RCC cases, which was significantly lower than that observed in benign renal tissues from the same patients [Sg I positive in 53 (100 %) cases (p < 0.0001)] by immunohistochemistry. There was an inverse relation between Sg I expression and clinical stage (pT1-2 vs pT3-4, p < 0.0001). Patients with Sg I-negative tumor had a significantly higher risk of recurrence (Kaplan-Meier and log-rank tests, p < 0.0001). There was low Sg I expression in RCC. Sg I expression has potential value in predicting cancer progression and prognosis. These findings support the use of Sg I as a novel biomarker for RCC.

Reduced L/B/K alkaline phosphatase gene expression in renal cell carcinoma: plausible role in tumorigenesis.

Renal cell carcinoma (RCC) is the most common kidney cancer in adults. Although several genes have been found to be involved in carcinogenesis of RCC, more great efforts are needed to identify new genes which are responsible for the process. Clear cell RCC, originates from proximal tubule cells, is the most common pathological type of RCC. Alkaline phosphatase (ALP) is a marker enzyme of brush border membrane of proximal tubular cells. Our previous studies showed a significant decreased activity of Liver/Bone/Kidney (L/B/K) alkaline phosphatase in RCC. In the present study, we explored the molecular basis of the decreased activity of ALP in RCC. Immunohistochemistry, immunofluorescence and flow cytometry analysis showed decreased ALP protein in RCC. Additionally, real time PCR documented significantly reduced ALP gene expression (P=0.009). Moreover, RCC cell lines (ACHN and A498) transfected with full length L/B/K cDNA showed decreased migratory property as well as viability of these cells as compared with controls (P=0.000). Further, L/B/K ALP cDNA transfected cells (ACHN and A498) showed significant increased apoptosis as compared to control (P=0.000). These findings suggest the new role of ALP in cell viability and apoptosis and involvement in RCC tumorigenesis. However, further studies are needed to explore the exact molecular mechanism.

Expression and significance of intratumor C-reactive protein in renal cell carcinoma

Objective To observe the expression and significance of intratumor C-reactive protein (CRP) in renal cell carcinoma (RCC).Methods Immunohistochemistry was used to detect the CRP expression in 40 eases of RCC and 20 cases of normal kidney tissue.Results The positive rate of CRP expression in RCC was 100％ (strongly positve expression rate of 22.50％),and that was 0 in the normal kidney tissue was 0 (0/20) with the difference being statistically significant (P ＜ 0.01).The expression of CRP in RCC was not related to the gender,age and histological classification (P ＞ 0.05),but to different histological grade,TNM stage and metastases of RCC.Conclusion Intratumor CRP testing can help determine tumor invasiveness and prodict the prognosis of RCC. Key words: C-reactive protein; Renal cell carcinoma; Biological markers

Underactivation of the adiponectin–adiponectin receptor 1 axis in clear cell renal cell carcinoma: implications for progression

Energy-sensing pathways, normally coordinated by 5' AMP-activated protein kinase (AMPK), are dysregulated in renal cell carcinoma (RCC). Obesity can accentuate the pre-existing pro-tumorigenic metabolic machinery in RCC cells through its associated obesogenic hormonal milieu, characterized by lower circulating levels of adiponectin. In RCC patients, low adiponectin levels associate clinically with more aggressive disease. We investigated the adiponectin signaling pathway in RCC, focusing on adiponectin receptor 1 (AdipoR1) and associated activation of AMPK. AdipoR1 protein in RCC and normal surrounding renal tissues was determined by Western blot analysis and immunohistochemistry. Anti-tumorigenic effects of adiponectin in RCC cells in vitro were investigated via VEGF and MMP ELISA and invasion assays. Using in vivo models of RCC, the effect of AdipoR1-knockdown (shRNA) on tumor latency, growth and dissemination were determined. AdipoR1 protein was significantly reduced in clear cell RCC specimens. Adiponectin treatment inhibited VEGF, MMP-2 and MMP-9 secretion and activity and invasive and migratory capacities of RCC cells. AMPKα1-knockdown (shRNA) attenuated adiponectin's effects. In cells stably expressing AdipoR1-specific shRNA, AMPK activation by adiponectin was significantly reduced compared to cells expressing control shRNA. In vivo, AdipoR1 knockdown increased the growth, dissemination and angiogenesis of RCC. These findings suggest that deficiencies in the entire adiponectin hormonal axis (the hormone and its receptor) result in underactivation of AMPK leading to increased angiogenic and invasive capacities of RCC. The established link between obesity and RCC can therefore be further explained by the adiponectin deficiency in obese individuals together with reduced AdipoR1 protein in RCC.

154 KNOCKDOWN OF INTEGRIN-LINKED KINASE REDUCES INVASIVE AND METASTATIC POTENTIAL OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2012154 KNOCKDOWN OF INTEGRIN-LINKED KINASE REDUCES INVASIVE AND METASTATIC POTENTIAL OF RENAL CELL CARCINOMA Kyung Seok Han, Peter Raven, Shannon Awrey, Estelle Li, Ladan Fazli, Martin Gleave, and Alan So Kyung Seok HanKyung Seok Han Vancouver, Canada More articles by this author , Peter RavenPeter Raven Vancouver, Canada More articles by this author , Shannon AwreyShannon Awrey Vancouver, Canada More articles by this author , Estelle LiEstelle Li Vancouver, Canada More articles by this author , Ladan FazliLadan Fazli Vancouver, Canada More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Alan SoAlan So Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.204AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES 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. 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 assess the effect of ILK on tumor growth and cell cycle. Confocal microscopy was used to evaluate changes in stress fibers and focal adhesions with phalloidin-rhodamine and vinculin antibody. Scratch assay and Matrigel invasion assay were performed to evaluate changes of EMT phenotypes after knockdown of ILK. RESULTS ILK is relatively 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 suppressed phenotypic EMT markers including cell migration and invasion in Caki-1 and UMRC-3 cells. 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. These results suggest the therapeutic potential of ILK inhibition on invasion and metastasis in advanced RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e63-e64 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyung Seok Han Vancouver, Canada More articles by this author Peter Raven Vancouver, Canada More articles by this author Shannon Awrey Vancouver, Canada More articles by this author Estelle Li Vancouver, Canada More articles by this author Ladan Fazli Vancouver, Canada More articles by this author Martin Gleave Vancouver, Canada More articles by this author Alan So Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression and clinical significance of T-lymphoma invasion and metastasis 1 protein in renal cell carcinoma

Objective To explore the expression of Tiaml in clear cell renal cell carcinoma and analyze its correlations to pathology of disease and prognosis.Methods The expressions of Tiam1 protein in 107 specimens of human clear cell renal cell carcinoma and 20 specimens of normal renal tissues were detected by immunohistochemical staining and its clinical significance was then analyzed.Results The expression of Tiam1 protein was higher in renal cancers than in the adjacent normal tissues ( P ＜ 0.01 ).Tiam1 protein expression rates were 47.6％ and 72.7％ in Ⅰ - Ⅱ and Ⅲ - Ⅳ tumors,while 49.3％ and 76.5％ in T1 - T2 and T3 - T4 tumors,respectively ( P ＜ 0.01 ).Expression of Tiam1 protein was higher in lymph node positive renal carcinoma tissues than in lymph node negative renal carcinoma tissues ( 71.7％ versus 47.5％,P ＜ 0.05 ).The expression of Tiam1 in carcinoma tissues showed a positive relationship with tumor vascular invasion (81.3％ versus 48.0％,P ＜ 0.01 ).In patients followed-up 5 - 8 years,Kaplan-meier analysis and the log-rank test showed that the 5-year survival was significantly different between the group of lower and higher Tiaml expression groups ( 84.4％ versus 46.8％,P ＜ 0.05 ).Conclusions The expression of Tiaml protein was higher in human primary renal carcinoma than in normal renal tissues.The positive rate of Tiam1 protein expression was related to classification,TNM stage,lymph node metastasis and vascular invasion.The detection of the expression of Tiaml protein may be helpful in the diagnosis and prognosis of renal carcinoma. Key words: Kidney neoplasms ; T-lymphoma invasion and metastasis 1 ; Immunohistochemistry

UP-2.071: Expression of Ezrin Protein in Renal Cell Carcinoma

UP-2.071: Expression of Ezrin Protein in Renal Cell Carcinoma