Human Renal Cell Carcinoma Specimens Research Articles

MP88-16 THE DOWNREGULATION AND TUMOR SUPPRESSIVE FUNCTION OF RASAL2 IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2018MP88-16 THE DOWNREGULATION AND TUMOR SUPPRESSIVE FUNCTION OF RASAL2 IN RENAL CELL CARCINOMA Ke Hui, Kaijie Wu, Jer-Tsong Hsieh, and Dalin He Ke HuiKe Hui More articles by this author , Kaijie WuKaijie Wu More articles by this author , Jer-Tsong HsiehJer-Tsong Hsieh More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2936AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients with renal cell carcinoma (RCC) often develop resistance to anti-vascular drug and eventually succumb to disease. However, the underlying molecular mechanism of drug resistance remains poorly understood. In this study, we demonstrated that RASAL2, a RAS GTPase activating protein (RASGAP), played suppressive role in in RCC by targeting tumor angiogenesis. METHODS RCC and its paired normal kidney tissues were immunohistochemically stained for RASAL2 expression. Databases downloaded from TCGA or GEO were used to analyze the methylation status of RASAL2 and the correlation between RASAL2 and c-FOS or VEGFA in RCC tissues. In vitro, RASAL2 gain-function in 786-O cells or loss-function in ACHN cells were measured for the recruitment and tube formation of human umbilical vein endothelial cell (HUVEC) by HUVEC migration assay and tube formation assay, ELISA assay and western blot analysis. In vivo, subcutaneous xenografts in nude mice were used to detect tunorigenicity. Moreover, the expression of RASAL2, VEGFA and CD31 in RCC tissues and xenografts were detect by immunohistochemical (IHC) staining. RESULTS The expression of RASAL2 was downregulated in RCC tissues, and RASAL2 mRNA was inversely correlated with RASAL2 methylation or c-FOS mRNA or VEGFA mRNA in RCC tissues. Overexpression of RASAL2 in 786-O cells could inhibit the recruitment and tube formation of HUVECs, while RASAL2 knockdown (KD) in ACHN cells enhanced the recruitment and tube formation of HUVECs in vitro. Also, overexpression of RASAL2 could inhibit tumorigenecity of xenografts. Mechanistically, RASAL2 KD could enhance the phosphorylation of GSK3 and upregulate the expression of c-FOS and VEGFA. Furthermore, RASAL2 was inversely correlated with VEGFA and CD31 in tissues from human RCC specimens and xenografts. CONCLUSIONS RASAL2 was downregulated in RCC tissues, which could lead to tumor angiogenesis via p-GSK3/c-FOS/VEGFA signaling pathway. Therefore, RASAL2 could be a potential target to prevent patients with RCC from resistance to anti-vascular therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1203-e1204 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ke Hui More articles by this author Kaijie Wu More articles by this author Jer-Tsong Hsieh More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Overexpression of CAPN2 promotes cell metastasis and proliferation via AKT/mTOR signaling in renal cell carcinoma.

The calpain 2 (CAPN2) is upregulated in various malignant carcinomas. Previous studies have reported that CAPN2 functioned as an oncogenic factor in human cancers. However, its clinical role and potential effects on cell metastasis and proliferation in renal cell carcinoma (RCC) remain unknown. In this study, we evaluated the mRNA and protein levels of CAPN2 in human RCC specimens, matched normal specimens, and RCC cell lines using quantitative Real-time PCR (RT-PCR) and western blot. Immunohistochemistry of 74 RCC tissues in a tissue microarrays (TMAs) and normal kidney tissues were performed. Kaplan-Meier survival curve analyses were conducted to measure the correlation between CAPN2 and tumor prognosis. Cell migration, invasion and proliferation were detected by transwell assays and Cell Counting Kit-8 (CCK-8) assays. CAPN2 exhibited a significant overexpression in human RCC tissues and cell lines compared with adjacent non-tumor tissues and normal human proximal tubule epithelial cell line HK-2. Strong staining of CAPN2 was associated with higher clinical stage and histological grade. In addition, sh-CAPN2 could significantly inhibit migration, invasion and proliferation of 769-P and CAKI-1 cells. Conversely, increased cell biological behaviors were observed in CAPN2-OV CAKI-2 cells. Moreover, the subsequent mechanism investigation suggested that CAPN2 promoted tumor progression by activating AKT/mTOR signaling, enhancing epithelial mesenchymal transition (EMT) and MMP9 levels. The present study indicates that CAPN2 may act as a prominent indicator for RCC progression and a novel therapeutic target for RCC patients.

OSR1 is a novel epigenetic silenced tumor suppressor regulating invasion and proliferation in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most malignant tumors in human. Here, we found that odd-skipped related transcription factor 1 (OSR1) was downregulated in 769-P and 786-O cells due to promoter CpG methylation. OSR1 expression could be restored by pharmacological demethylation treatment in silenced cell lines. Knockdown of OSR1 in two normal expressed cell lines- A498 and ACHN promoted cell invasion and cellular proliferation. RNA-Sequencing analysis showed that expression profile of genes involved in multiple cancer-related pathways was changed when OSR1 was downregulated. By quantitative real-time PCR, we confirmed that depletion of OSR1 repressed the expression of several tumor suppresor genes involved in p53 pathway, such as p53, p21, p27, p57 and RB in A498 and ACHN. Moreover, knockdown of OSR1 suppressed the transcriptional activity of p53. Of note, OSR1 depletion also led to increased expression of a few oncogenic genes. We further evaluated the clinical significance of OSR1 in primary human RCC specimens by immunohistochemical staining and found that OSR1 expression was downregulated in primary RCC and negatively correlated with histological grade. Thus, our data indicate that OSR1 is a novel tumor suppressor gene in RCC. Downregulation of OSR1 might represent a potentially prognostic marker and therapeutic target for RCC.

Downregulation of tNASP inhibits proliferation through regulating cell cycle-related proteins and inactive ERK/MAPK signal pathway in renal cell carcinoma cells.

Nuclear auto-antigenic sperm protein (NASP), initially described as a highly auto-immunogenic testis and sperm-specific protein, is a histone chaperone that is proved to present in all dividing cells. NASP has two splice variants: testicular NASP (tNASP) and somatic form of NASP (sNASP). Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP. Up to now, little has been known about tNASP in renal cell carcinoma (RCC). In the present study, the molecular mechanism of tNASP in RCC was explored. The expression level of tNASP in 16 paired human RCC specimens was determined. Downregulation of tNASP by small interfering RNA (siRNA) was transfected in RCC cell lines. The effect of downregulation of tNASP by siRNA on cell colony formation and proliferation was examined by colony formation assay and CCK-8 assay, cell cycle was analyzed by flow cytometry, and the expression of cyclin D1 and P21 were detected by Western blotting. ERK/MAPK signaling was also analyzed. tNASP has a relative high expression level in human RCC tissues. Via upregulation of P21 and downregulation of cyclinD1, silence of tNASP can inhibit cell proliferation, which induces cell cycle arrest. Furthermore, ERK signaling pathway is confirmed to mediate the regulation of cell cycle-related proteins caused by silence of tNASP. Our research demonstrates that knockdown of tNASP effectively inhibits the proliferation and causes G1 phase arrest through ERK/MAPK signal pathway.

Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells

Targeting tumor angiogenesis is an established strategy for cancer therapy. Because angiogenesis is not limited to pathological conditions such as cancer, molecular markers that can distinguish between physiological and pathological angiogenesis are required to develop more effective and safer approaches for cancer treatment. To identify such molecules, we determined the gene expression profiles of murine tumor endothelial cells (mTEC) and murine normal endothelial cells using DNA microarray analysis followed by quantitative reverse transcription–polymerase chain reaction analysis. We identified 131 genes that were differentially upregulated in mTEC. Functional analysis using siRNA-mediated gene silencing revealed five novel tumor endothelial cell markers that were involved in the proliferation or migration of mTEC. The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma. Comparative gene expression analysis revealed molecular differences between tumor endothelial cells and normal endothelial cells and identified novel tumor endothelial cell markers that may be exploited to target tumor angiogenesis for cancer treatment.

The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma

BackgroundOverexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown.MethodsModulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens.ResultsHere, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).ConclusionsTaken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

BMP-6 in Renal Cell Carcinoma Promotes Tumor Proliferation through IL-10–Dependent M2 Polarization of Tumor-Associated Macrophages

Dysregulated bone morphogenetic proteins (BMP) may contribute to the development and progression of renal cell carcinoma (RCC). Herein, we report that BMP-6 promotes the growth of RCC by interleukin (IL)-10-mediated M2 polarization of tumor-associated macrophages (TAM). BMP-6-mediated IL-10 expression in macrophages required Smad5 and STAT3. In human RCC specimens, the three-marker signature BMP-6/IL-10/CD68 was associated with a poor prognosis. Furthermore, patients with elevated IL-10 serum levels had worse outcome after surgery. Together, our results suggest that BMP-6/macrophage/IL-10 regulates M2 polarization of TAMs in RCC.

310 MTOR PATHWAY REGULATES THE EXPRESSION OF ANGIOTENSIN 2 TYPE 1 RECEPTOR IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research (II)1 Apr 2013310 MTOR PATHWAY REGULATES THE EXPRESSION OF ANGIOTENSIN 2 TYPE 1 RECEPTOR IN RENAL CELL CARCINOMA Gou Kaneko, Akira Miyajima, Takeo Kosaka, Ryuichi Mizuno, Eiji Kikuchi, and Mototsugu Oya Gou KanekoGou Kaneko Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Ryuichi MizunoRyuichi Mizuno Tokyo, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1694AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The potential role of renin-angiotensin system in angiogenesis and promotion of tumor growth has been growing. We previously showed that angiotensin 2- angiotensin 2 type 1 receptor (AT1R) signaling led to potent induction of vascular endothelial growth factor in urogenital cancers. In addition, we reported that up-regulation of phosphorylated Akt contributed to elevated AT1R expression in castration-resistant prostate cancer. In renal cell carcinoma (RCC), AT1R expression was recently reported as a useful predictor of survival, however, the regulatory mechanism of AT1R expression has not yet been elucidated. The aim of the present study was to evaluate the regulatory mechanism of AT1R expression in RCC. METHODS AT1R expression was evaluated using protein lysates from human RCC specimens by western blot analysis, and analyzed a relationship between tumor grade and AT1R expression. AT1R and phosphorylated (pS6) expression in 6 human RCC cell lines (ACHN, A498, 786-O, 769-P, Caki-1, Caki-2) was evaluated by western blot analysis. We evaluated whether mammalian target of rapamycin (mTOR) inhibitor (RAD001) regulates AT1R expression in vitro and in vivo. RESULTS AT1R expression of a total of 80 human RCC specimens tended to express higher in high grade tumors. Western blot analysis revealed AT1R and pS6 were expressed at various levels in RCC cell lines. In Caki-1 cells, pS6 was strongly expressed than other cell lines, thus, we evaluated the regulation of AT1R expression by mTOR inhibitor using Caki-1 cells. In vitro, AT1R expression in Caki-1 cells was significantly inhibited by the treatment with RAD001 in a time and dose dependent manner (Figure 1). In vivo, Caki-1 cells were implanted subcutaneously in nude mice, and RAD001 (10 mg kg−1 per day) was administered by oral gavage, beginning at the tumor volume about 100mm3. RAD001 significantly inhibited tumor growth, accompanying reduced AT1R expression (Figure 2). CONCLUSIONS These findings suggested that mTOR pathway regulated AT1R expression in RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e126 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gou Kaneko Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Ryuichi Mizuno Tokyo, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-21 Downregulated TCF21 to Inhibit KISS1 in Renal Cancer

To investigate whether the microRNA-21 gene (miR-21) could regulate renal cancer cells invasion by downregulation of TFC21 and KISS1. Quantitative real-time polymerase chain reaction was applied to evaluate the expression level of miRNA-21 in renal cancer and normal renal cell samples. The regulated effects of miR-21 to TCF21 were detected by Western blot after pre/anti-miR-21 was transfected to Caki-1 cells. The luciferase activity assay was used to reveal the predicted target gene of miR-21 was direct and specific. Small interfering RNA-TCF21 was transfected to Caki-1 cells to inhibit the expression of the TCF21 gene. Next, the expression of the KISS1 gene was detected by Western blot in Caki-1 cells with TCF21 gene silencing. The expression vector, pcDNA3.1-KISS1, was transfected to Caki-1 cells to upregulate the expression of the KISS1 gene. The invasion ability of Caki-1 cells with KISS1 overexpression was analyzed using the Transwell assay. Our study showed that miR-21 was upregulated in human renal cell carcinoma specimens compared with its expression in normal renal cell specimens. Pre-miR-21 could upregulate the expression of miR-21 and downregulate the expression of TCF21, and anti-miR-21 showed the opposite effects. siRNA-TCF21 decreased the expression of the TCF21 protein, and the expression of KISS1 was downregulated in Caki-1 cells with TCF21 gene silencing. pcDNA3.1-KISS1 transfection upregulated the expression of the KISS1 protein, and the invasion ability of Caki-1 cells with KISS1 overexpression decreased markedly. Aberrantly expressed miR-21 might regulate the TCF21-KISS1-associated renal cell carcinoma cell invasion pathway, and this miRNA signature could offer a novel potential therapeutic strategy for renal cell carcinoma.

MicroRNA-708 Induces Apoptosis and Suppresses Tumorigenicity in Renal Cancer Cells

Cancer pathogenesis is restricted by stresses that compromise cell division and survival. In this study, we identify miR-708, a little studied member of a set of microRNAs that have been implicated in stress control, as an important tumor suppressor in renal cell carcinoma (RCC). miR-708 expression was attenuated widely in human RCC specimens. Restoration of miR-708 expression in RCC cell lines decreased cell growth, clonability, invasion, and migration and elicited a dramatic increase in apoptosis. Moreover, intratumoral delivery of miR-708 was sufficient to trigger in vivo regression of established tumors in murine xenograft models of human RCC. Investigation of the targets of miR-708 identified the inhibitor of apoptosis protein survivin as important. siRNA-mediated knockdown of survivin partially phenocopied miR-708 overexpression suggesting that the proapoptotic role of miR-708 may be mediated primarily through survivin regulation. Additionally, we identified the E-cadherin regulators ZEB2 and BMI1 as likely miR-708 targets. Taken together, our findings define a major tumor suppressive role for miR-708, which may offer an attractive new target for prognostic and therapeutic intervention in RCC.

Gene microarray analysis of human renal cell carcinoma: The effects of HDAC inhibition and retinoid treatment

Histone deacetylase (HDAC) inhibitor treatments can augment the anti-tumor effects of retinoids in renal cancer cells. We studied the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and 13-cis retinoic acid (cRA) on two human renal cell carcinoma (RCC) lines. Cells were cultured in the presence of each drug for six days to determine the responses to monotherapy and to combination therapy. The proliferation of SKRC06 was inhibited with cRA treatment; SKRC39 was not. However, both RCC lines were sensitive to growth inhibition by dibutyryl cyclic AMP, with or without 13-cis RA. SAHA alone also reduced cell proliferation in both cell lines. To identify the alterations in gene expression that correlate with the responsiveness to treatment, gene microarray analyses were performed. Several retinoid-regulated genes exhibited much higher mRNA levels in SKRC06 than in SKRC39, even in the absence of drugs; these included crabp2, rar, and cyp26A1. Combination treatment of cells with both SAHA and cRA induced several transcripts with known anti-cancer/immunomodulatory effects, including dhrs9, gata3, il1, phlda1, txk and vhl. Immunostaining confirmed the decreased expression of gata3 in human RCC specimens compared to normal kidney. Together, our results show that treatment of RCC with cRA and/or SAHA increases the expression of several genes and gene families that result in reduced cell proliferation.

Receptors for Luteinizing Hormone Releasing Hormone Expressed on Human Renal Cell Carcinomas Can Be Used for Targeted Chemotherapy with Cytotoxic Luteinizing Hormone Releasing Hormone Analogues

To determine the expression of luteinizing hormone releasing hormone (LHRH) receptors in specimens and cell lines of human renal cell carcinoma (RCC) and to evaluate the antitumor efficacy of targeted therapy with a cytotoxic analogue of LHRH, AN-207, in vivo. AN-207, consisting of [D-Lys(6)] LHRH linked to a cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201), binds with high affinity to LHRH receptors and can be targeted to tumors expressing these receptors. The expression of LHRH receptors was investigated in 28 surgically removed specimens of human renal cell carcinoma (RCC) by immunohistochemistry and in three human RCC cell lines A-498, ACHN, and 786-0 by radioreceptor assays, Western immunoblotting, and reverse transcription-PCR analysis. Antitumor efficacy of AN-207 was examined in experimental models of these cell lines. Positive staining for LHRH receptors was found in all (28 of 28) of the examined human RCC specimens. mRNA for LHRH receptor, receptor protein, and LHRH binding sites were detected in all three cell lines. AN-207 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786-0 xenografts in vivo producing a 67.8% to 73.8% decrease in tumor volume and a 62.2% to 77.3% reduction in tumor weight. Nontargeted cytotoxic radical AN-201 had no significant antitumor effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptyl suppressed tumor inhibitory effects of AN-207. Our findings indicate that LHRH receptors are expressed in human RCC specimens and can be used for targeted chemotherapy with cytotoxic LHRH analogues.

Estrogen Receptor–Binding Fragment–Associated Antigen 9 Is a Tumor-Promoting and Prognostic Factor for Renal Cell Carcinoma

The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) has been identified as a primary estrogen-responsive gene in human breast cancer MCF7 cells. A high expression of EBAG9 has been observed in invasive breast cancer and advanced prostate cancer, suggesting a tumor-promoting role of the protein in malignancies. Here we show that intratumoral (i.t.) administration of small interfering RNA against EBAG9 exerted overt regression of tumors following s.c. implantation of murine renal cell carcinoma (RCC) Renca cells. Overexpression of EBAG9 did not promote the proliferation of culture Renca cells; however, the inoculated Renca cells harboring EBAG9 (Renca-EBAG9) in BALB/c mice grew faster and developed larger tumors compared with Renca cells expressing vector alone (Renca-vector). After renal subcapsular implantation, Renca-EBAG9 tumors significantly enlarged compared with Renca-vector tumors in BALB/c mice, whereas both Renca-EBAG9 and Renca-vector tumors were developed with similar volumes in BALB/c nude mice. No apparent difference was observed in specific cytotoxic T-cell responses against Renca-EBAG9 and Renca-vector cells; nonetheless, the number of infiltrating CD8+ T lymphocytes was decreased in Renca-EBAG9 subcapsular tumors. Furthermore, immunohistochemical study of EBAG9 in 78 human RCC specimens showed that intense and diffuse cytoplasmic immunostaining was observed in 87% of the cases and positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients. Multivariate analysis revealed that high EBAG9 expression was an independent prognostic predictor for disease-specific survival (P = 0.0485). Our results suggest that EBAG9 is a crucial regulator of tumor progression and a potential prognostic marker for RCC.

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.