RCC Lines Research Articles

The key role of myostatin b in somatic growth in fishes derived from distant hybridization.

The basic mechanism of heterosis has not been systematically and completely characterized. In previous studies, we obtained three economically important fishes that exhibit rapid growth, WR (WCC ♀ × RCC ♂), WR-II (WR ♀ × WCC ♂), and WR-III (WR-II ♀ × 4nAU ♂), through distant hybridization. However, the mechanism underlying this rapid growth remains unclear. In this study, we found that WR, WR-II, and WR-III showed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species (RCC and WCC). Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes. Notably, the mRNA level of mstnb (myostatin b), which is a negative regulator of myogenesis, was significantly reduced in WR, WR-II, and WR-III compared with the parent species. To verify the function of mstnb, a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique. The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57% compared with that in wild-type siblings. Moreover, the area and number of muscle fibers were significantly increased in mstnb-deficient RCC, indicating hypertrophy and hyperplasia. Furthermore, the muscle protein and fat contents were significantly increased in mstnb-deficient RCC. The molecular regulatory mechanism of mstnb was then revealed by transcription profiling, which showed that genes related to myogenesis (myod, myog, and myf5), protein synthesis (PI3K-AKT-mTOR), and lipogenesis (pparγ and fabp3) were highly activated in hybrid fishes and mstnb-deficient RCC. This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis, protein synthesis, and lipogenesis in hybrid fishes and mstnb-deficient RCC, which provides evidence for the molecular mechanism of heterosis via distant hybridization.

Abstract 2334: Intratumoral classical complement pathway promotes tumor growth in renal cancer

Abstract Objective: The complement system has been considered for long as anti-tumoral effector in the context of monoclonal antibody therapy. Nevertheless, recent studies highlighted a pro-tumoral effect of complement, acting on angiogenesis, tumor cells proliferation and immunosuppression. The kidney is capable to produce a whole spectrum of complement components. Nevertheless, the role of complement activation for the progression of renal cancer remains poorly understood. In the present work we address the presence of complement components and their impact on the clinical outcome of patients with clear cell renal carcinoma (ccRCC). In addition, we evaluate the tumor growth of complement proteins-expressing tumor cell lines in complement-deficient mice. Methods: The production of complement proteins by RCC lines was measured by RT-qPCR and by western blot (WB). Their expression in the tumor tissue was evaluated by immunohistochemistry and multicolor staining by immunofluorescence (IF) on sections from paraffin embedded tumors. The capacity of RCC cells-produced proteins to activate complement was evaluated by ELISA and western blot. The presence of complement was evaluated by immunohistochemistry on tumor sections from 3 retrospective cohorts composed by primary ccRCC patients (over 300 cases total). Complement producing tumor cell lines were grafted in WT and classical complement pathway deficient mice and the tumor growth was followed. Results: We found that C1q is expressed in the ccRCC tumors by the infiltrating macrophages, having a particular phenotype. The remaining complement proteins are produced by the tumor cells. A dynamic interaction between the complement proteins produced by the tumor cells and the immune microenvironnement led to a local activation of complement revealed in vitro and in situ. The high level of C1q and complement activation correlated with a poor prognosis for patients. Moreover, the tumor growth was reduced in C1q-/- and C4-/- mice. Conclusion: Early steps of the classical complement pathway can be activated locally and are associated with a pro-tumoral effect in mice models and in patients with renal cancer. Keywords: renal cancer; C1q; classical pathway Citation Format: Marie Daugan, Lubka Roumenina, Rémi Noé, Florent Petitprez, Catherine Sautès-Fridman, Wolf H. Fridman. Intratumoral classical complement pathway promotes tumor growth in renal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2334.

MP71-19 ESTABLISHING AN EXPERIMENTAL MODEL FOR TARGETING RENAL CELL CARCINOMA USING MESENCHYMAL STEM CELLS IN THE AVIAN CHORIOALLANTOIC MEMBRANE

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2016MP71-19 ESTABLISHING AN EXPERIMENTAL MODEL FOR TARGETING RENAL CELL CARCINOMA USING MESENCHYMAL STEM CELLS IN THE AVIAN CHORIOALLANTOIC MEMBRANE Zhihao (James) Xu, Hua Chen, Desmond Pink, Katia Carmine-Simmen, John Lewis, and Ronald Moore Zhihao (James) XuZhihao (James) Xu More articles by this author , Hua ChenHua Chen More articles by this author , Desmond PinkDesmond Pink More articles by this author , Katia Carmine-SimmenKatia Carmine-Simmen More articles by this author , John LewisJohn Lewis More articles by this author , and Ronald MooreRonald Moore More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1465AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastatic renal cell carcinoma (mRCC) is a lethal urologic disease with 10 year survival less than 5 percent. Recent developments in cell signalling and small molecule therapy for mRCC have impacted progression free survival (PFS) only. High-dose IL-2 immunotherapy can produce durable complete responses (CR) but the response rate is low (16%), highlighting the need for novel therapy. Mesenchymal stem cells (MSCs) have the capacity to mobilize to sites of inflammation. Recently, MSCs were reported to traffic to murine RCC lung metastases via inflammatory signals, opening the potential of delivering tumor site-specific treatment. We hypothesize that human MSCs could target human RCC engrafted in the chorioallantoic membrane (CAM) of the avian embryo. In this project, we investigated potential use of the avian CAM model to study MSC RCC homing. This immunodeficient model is ideal for imaging tumor growth, and cell trafficking. METHODS We first transduced 786-O RCC lines to express green fluorescent protein (GFP), blue fluorescent protein (BFP), or mApple respectively using lentiviral-based vectors. Successful transduction was selected using blasticidin antibiotic treatment. Concurrently, human adipose derived MSCs were isolated from patient perirenal fat, cultured, and characterized using flow cytometry. For the avian CAM model, fluorescent 786-O cells were initially injected into the CAM to establish a viable tumor. The chicken egg vascular endothelium was fluorescently labelled with lectin. MSCs, labelled with a fluorescent plasma membrane stain, were injected IV and observed using intravital imaging via confocal microscopy. RESULTS At 6 weeks post MSC isolation, flow cytometry results indicated that the patient adipose derived primary culture was CD73, CD90, CD105, CD44, CD166 positive and CD45, CD19, CD106, CD146 negative, consistent with MSC expression. All three fluorescent 786-O cell lines were observed by fluorescent microscopy to establish viable and vascularized tumors in the CAM, 7 days post injection. The injected commercial and patient-derived human MSCs were observed to be in proximity to the RCC tumor consistently over periods of 18 hrs and 3 hrs respectively. However, MSCs were not observed to specifically traffic to the RCC tumor. CONCLUSIONS Our results demonstrated that both viable human RCC tumors and human MSCs were established and imaged in the avian CAM model. The stage is set for future studies exploring MSC tumor homing. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e923 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Zhihao (James) Xu More articles by this author Hua Chen More articles by this author Desmond Pink More articles by this author Katia Carmine-Simmen More articles by this author John Lewis More articles by this author Ronald Moore More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MicroRNA-182 suppresses clear cell renal cell carcinoma migration and invasion by targeting IGF1R.

The purpose of our study was aimed to determine the functional role of microRNA (miR)-182 in clear cell renal cell carcinoma (ccRCC) and try to clarify its underlying molecular mechanism. Expression of miR-182 in both cancer and peripheral blood samples was analyzed by quantitative real-time PCR (qRT-PCR). Human RCC line Caki-1 cells were transfected with miR-182 mimic, miR-182 inhibitor, or negative controls, and then the cell viability, colony-formation ability, migration, and invasion assay were determined. Luciferase reporter assay, qRT-PCR and Western blotting were used to determine whether insulin-like growth factor 1 receptor (IGF1R) was a target of miR-182. Further, small interfering RNA (siRNA) against IGF1R was co-transfected with miR-182 inhibitor into cells, and then the effects on migration and invasion were assessed. MiR-182 was down-regulated in both cancer and blood samples compared to the matched non-tumor adjacent tissues and healthy volunteers, respectively (both P<0.05). Compared to the control group, cell viability, colony-forming ability, and numbers of migrated and invaded cells were significantly decreased by transfection with miR-182 mimic but were markedly increased by miR-182 inhibitor (all P < 0.05). Luciferase reporter assay confirmed that IGF1R was a target gene of miR-182, and IGF1R was negatively regulated by miR-182. Co-transfection of miR-182 inhibitor with si-IGF1R reversed the effect of miR-182 inhibitor on the migration and invasion of the cells. MiR-182 functions as an anti-oncogene in ccRCC, and miR-182-mediated inhibition of cell migration and invasion might be through directly targeting IGF1R.

A Kinome-Wide siRNA Screen Identifies Multiple Roles for Protein Kinases in Hypoxic Stress Adaptation, Including Roles for IRAK4 and GAK in Protection against Apoptosis in VHL–/– Renal Carcinoma Cells, Despite Activation of the NF-κB Pathway

Hypoxia induces changes to cancer cells that make them more resistant to treatment. We have looked at signaling pathways that facilitate these changes by screening the human kinome for effects on hypoxic responses in SW480 colon cancer cells. Hits identified in the screen were examined for effects on multiple molecular responses to hypoxia, including the endoplasmic reticulum stress and DNA damage responses in colon, melanoma, and renal cancer lines. To validate the hits from the small interfering RNA studies, we developed cell lines expressing stable short hairpin RNAs (shRNAs) in the A498 renal carcinoma cell line. Several lines, including those expressing shRNAs against DYRK1B, GAK, IHPK2, IRAK4, and MATK, showed an inability to form spheroid cultures. In addition, shRNAs targeting IRAK4 and GAK were incapable of 2D growth under anoxia. In the GAK shRNA-expressing line, nuclear factor–κB (NF-κB) was localized to the nucleus, but in the IRAK4 shRNA line, NF-κB levels were increased but the extent of nuclear localization was unchanged. Dominant negative mutants of IRAK4 and GAK also showed strong apoptotic effects in A498 cells under anoxia, supporting a direct link between these kinases and survival of the VHL–/– RCC line, which is typically highly resistant to hypoxic stress as a result of high and constitutive levels of Hif-1α.

Expression of Human Organic Cation Transporter 3 in Kidney Carcinoma Cell Lines Increases Chemosensitivity to Melphalan, Irinotecan, and Vincristine

Renal cell carcinoma (RCC) is usually chemoresistant. This chemoresistance could be overcome if specific cytostatics are applied for which the RCC expresses an uptake transporter. In the present study, we investigated the expression of solute carrier (SLC) transporters in different RCC lines and their ability to interact with chemotherapeutics. We tested five RCC lines for the expression of different SLCs by reverse transcription-PCR and TaqMan real-time PCR. In two of five RCC lines, A498 and 7860, we observed a highly significant expression of SLC22A3 (hOCT3). Uptake of the organic cation [(3)H]MPP (4-methyl-pyridinium iodide) into these cells and also into hOCT3 stably transfected Chinese hamster ovary (CHO) cells was inhibited by irinotecan, vincristine, and melphalan. The K(i) values [determined from Dixon plots] for irinotecan, vincristine, and melphalan were 1.72 +/- 0.45 micromol/L, 17 +/- 4.81 micromol/L, and 366 +/- 51 micromol/L, respectively. Cytotoxic activities of the selected drugs were tested by [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on CHO-hOCT3, A498 (high expression of hOCT3), and ACHN cell lines (low expression of hOCT3). The growth of CHO-hOCT3 was inhibited by 20% more with irinotecan and by 50% more with vincristine compared with nontransfected CHO cells. Melphalan produced 20% to 30% more inhibition in hOCT3-expressing cells compared with nonexpressing control cells. Similar results were obtained for A498 and ACHN cells. Thus, our data support the hypothesis that the sensitivity of tumor cells to chemotherapeutic treatment depends on the expression of transporter proteins mediating specific drug accumulation into target cells.

Indirubin-3‘-Monoxime, an Inhibitor of Cyclin Dependent Kinases, Induces Growth Inhibition and Apoptosis in Renal Cell Cancer in vitro

The compound indirubin is the active constituent of a traditionalChinese phytomedicine (Danggui Longhui Wan).This preparation has been used for years in the treatmentof chronic myelocytic leukemia. A derivate of the originalindirubin compound is indirubin-3'-monoxime which is aselective and potent inhibitor of cyclin-dependent kinases.In this study we investigated if indirubin-3'-monoximecan induce apoptosis and tumor cell death in 3 human(A498, CAKI-1, CAKI-2) and 1 murine (RENCA) renal cellcancer cell lines. The growth inhibitory and apoptosis inductionproperties were evaluated by EZ4U, a cytotoxicassay and by flow cytometry of annexin-V/PI stainingduring treatment with doses ranging from 5.0 to 15.0 µmindirubin-3'-monoxime over 72 h. To further evaluate theunderlying molecular actions of indirubin-3'-monoxime,survivin, a major antiaptotic protein, was additionallydetermined by intracellular flow cytometry. Our resultsshow that indirubin-3'-monoxime induces growth arrestand apoptosis in all RCC cell lines. All RCC lines expressedsurvivin. However, no clear correlation between apoptosisinduction and expression of survivin was found. As treatmentof metastatic RCC remains a challenge, the need for continuing assessment of novel agents in the treatment ofthis disease is mandatory. Indirubin-3'-monoxime seems acandidate for further evaluation.

Stromal Cell–Derived Factor-1α and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor

The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1alpha (HIF-1alpha). In hemangioblastomas and CC-RCCs, HIF-1alpha is constitutively overexpressed leading to increased transcription of HIF-1-regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1alpha, CXCR4, and its ligand stromal cell-derived factor-1alpha (SDF-1alpha) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1alpha within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1alpha was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1alpha protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1alpha mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1alpha mRNA was significantly decreased in HIF-1alpha-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1alpha and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.

Therapeutic efficacy of RAGE-1 and MAGE-9 peptide specific cytotoxic T cell clones in renal cell carcinoma

RAGE-1 and MAGE-9 are expressed on 61% and 40% of RCC. Six MAGE-9 and 14 RAGE-1 derived peptides were found to be immunogenic in the context of the HLA-A2.1 MHC complex. CTL were generated by co-culture with peptide pulsed dendritic cells (DC) or peptide pulsed CD40-activated B cells. The latter are as efficient in antigen presentation as DC, but have the advantage of being easier to generate in great quantitities and to display functional activity for a prolonged period of time. Therefore, they are well suited for the generation of CTL clones to be used in adoptive transfer. Three MAGE-9 and RAGE-1 specific CTL clones were generated. The clones were strictly peptide-specific and displayed high cytotoxic activity not only against peptide-loaded T2 cells, but also against HLA-A2.1-positive RCC lines that naturally expressed MAGE-9, RAGE-1 or both. The in vivo efficacy of these MAGE-9 and RAGE-1 peptide specific CTL clones is currently being evaluated in human RCC-bearing SCID, which received after intraperitoneal RCC injection repeated applications of the CTL clones at weekly intervals. So far, the majority of control mice became moribund, but only one mouse receiving a MAGE-9 and a RAGE-1 specific CTL developed a tumor, that had retained MHC class I as well as MAGE-9 and RAGE-1 expression. CTL recovered from these mice at 2 and 3 weeks after the last T cell transfer revealed that CTL had retained their specificity and cytotoxic activity was only slightly reduced.

Tumor associated antigens in human renal cell carcinoma: MHC restricted recognition by cytotoxic T lymphocytes.

Based on previous studies in human melanoma leading to the molecular cloning of genes encoding peptide antigens recognized by MHC-restricted cytotoxic T lymphocytes (CTL) we extended our efforts to renal cancer systems established in tissue culture. In two patients we obtained stable CD8+ CTL clones with high cytolytic activity for the corresponding autologous tumor cell line in vitro. Neither autologous EBV-transformed B lymphocytes or PHA-activated PBL nor natural killer targets K562 were lysed by these CTL clones. MZ1257-RCC CTL5-30 lysed autologous tumor cells as well as normal kidney cell cultures in an HLA-A2 restricted fashion. Further specificity analysis showed cross reactivity with HLA-A2 matched renal cancer cell lines, melanoma cell lines and tumor cell lines of other origins. HLA-A2 negative target cells were not lysed. The restriction element for T cell recognition in another renal cancer system, MZ1973-RCC, appeared to be HLA-B8 as tested with CTL 5-10. Crossreactivity of CTL 5-10 was documented with one HLA-B8 positive RCC line. Tumor cell lines of other origin were not lysed by CTL 5-10. For further study of CTL-defined epitopes, peptides were extracted from tumor cell lines. Peptides from cultured cell lines were acid-eluted and fractionated by reversed phase HPLC. The peptide fractions were tested in cytotoxicity assays for their ability to reconstitute RCC epitopes by addition to the HLA-A2.1 positive antigen processing mutant cell line 721.124xCEM.T2. One HPLC peak was identified containing an epitope for the HLA-A2 restricted MZ1257-RCC CTL 5-30. These peptide epitopes were present in the autologous renal cancer cell line MZ1257-RCC as well as in an allogeneic HLA-A2 positive RCC cell line MZ1973 and melanoma cell line SK29-MEL, but were absent in peptides eluted from autologous EBV-B cells. We conclude that renal cell carcinoma may induce autologous CTL. Epitopes recognized by MHC-restricted CTL on RCC may be shared by HLA-matched allogeneic renal cancer or even tumors of other origin, such as melanoma. The current challenge is the determination of the amino acid sequences of these CTL-defined peptides as a step to further characterize the nature of these tumor associated antigens.

Induction of autologous tumor-specific cytotoxic T-lymphocyte activity against a human renal carcinoma cell line by B7-1 (CD8O) costimulation.

Recently mouse models have shown that expression of costimulatory molecules such as B7-1 on tumor cells can induce tumor-specific immunity, suggesting that tumor cells modified to express costimulatory molecules can be a potential tumor vaccine. To investigate the importance of B7-1 co-stimulation in induction of autologous tumor immunity in humans, we established a renal carcinoma cell line, RCC-1, from a tumor resection and studied the patient's antitumor immune responses in vitro. The RCC-1 cell line constitutively expressed major histocompatibility complex (MHC) class I, intercellular adhesion molecule (ICAM)-1, and leukocyte function-associated antigen (LFA)-3 molecules, and MHC class II molecules were induced by interferon-gamma (IFN-gamma) treatment in vitro. However, neither RCC-1- nor IFN-gamma-treated RCC-1 cells expressed B7-1, and both failed to induce T-cell proliferative responses in mixed lymphocyte and tumor cell reaction (MLTR) assays, suggesting that the costimulatory signals provided by cell adhesion molecules such as ICAM-1 and LFA-3 were not sufficient to elicit an antitumor immune response. However, on transfection of the human B7-1 into RCC-1, these cells were able to induce a significant T-cell proliferation in MLTR assays. This T-cell response could be blocked by anti-B7 mAb treatment of the tumor cells. RCC-1B7 cells also induced the generation of tumor-specific cytolytic T lymphocytes to the parent RCC-1 cells in vitro, with little nonspecific cytolysis of an unrelated RCC line, A498, or autologous phytohemagglutinin (PHA) blasts. This specific cytotoxicity could be abrogated by anti-CD8 mAb and complement treatment. In summary, our study indicates that B7-1-CD28 interaction plays a critical role in induction of autologous tumor-specific cytotoxic T lymphocytes (CTLs) in humans, suggesting that the costimulatory molecule transfected tumor cells could be useful in expanding tumor-specific autologous CTL in vitro for adoptive tumor immunotherapy.

Synergistic Effects of Vinblastine and Recombinant Interferon-β on Renal Tumor Cell Lines

Potential synergistic interactions between vinblastine (VBL) and recombinant interferon-beta (rIFN-beta) were assessed using median effect analysis. Calculation of the combination index demonstrated values less than 1 (indicating synergy) over a wide range of drug-induced growth inhibition for each of four different renal carcinoma cell lines (RCC). The degree of synergy observed could not be predicted from the morphology, doubling time, or relative sensitivity of the RCC lines to VBL and rIFN-beta. The optimal ratio of VBL to rIFN-beta in the combination appeared to be close to the ratio of the concentrations of each agent which yielded a 50% inhibition of growth. Although simultaneous presence of VBL and rIFN-beta in the culture medium was not required to demonstrate a synergistic effect, the minimum exposure time for rIFN-beta was determined to be 7 days. The uptake but not the egress of tritiated VBL into RCC cells was enhanced after growth for 4 days in 2.25 ng/ml of rIFN-beta. Median effect analysis can be shown to be independent of the mechanism of action of VBL and rIFN-beta and gives an indication of potential synergistic interactions over a wide range of drug effects. This method may prove useful in the selection of combinations of IFNs and antitumor drugs for clinical study.