Human Renal Cell Carcinoma Models Research Articles

Development of siRNA Delivery Targeting the Tumor Microenvironment with a New Functional Device

The tumor microenvironment plays a key role in cancer progression, drug resistance, metastasis, etc. To establish a new therapeutic strategy based on control of the tumor microenvironment, I have developed a lipid nanoparticle (LNP)-based in vivo small interfering RNA (siRNA) delivery system equipped with a targeting ligand. First, I established an LNP that induces membrane fusion in response to acidification after internalization by cells using the original pH-sensitive cationic lipid YSK05. A modification of polyethylene glycol to YSK05-containing LNPs allowed significant gene silencing in the human renal cell carcinoma model. Then, I attempted to establish a tumor vasculature-targeting LNP because the vasculature is responsible for the tumor microenvironment. Cyclic RGD peptide is known to be a ligand against integrin αVβ3, which is highly expressed on tumor endothelial cells (TECs). Optimized cyclic RGD peptide-modified LNP (RGD-LNP) suppressed gene expression in TECs to 50%. The inhibition of vascular endothelial cell growth factor receptor 2 (VEGFR2), which is a dominant factor in angiogenesis, by the injection of RGD-LNP significantly delayed tumor growth. Finally, I examined the effect of RGD-LNP on the tumor microenvironment. The suppression of VEGFR2 increased pericyte coverage and endothelial junctions, which indicate maturation of the vasculature. In RGD-LNP-treated mice, systemically administered nanoparticles encapsulating doxorubicin were distributed in a larger area than in untreated mice. Moreover, the therapeutic effect of doxorubicin-loaded liposomes was significantly enhanced by RGD-LNP. In conclusion, I succeeded in developing a new therapy based on regulation of the tumor microenvironment.

A comparative study of four methods of establishing orthotopic human renal cell carcinoma models in nude mice

A comparative study of four methods of establishing orthotopic human renal cell carcinoma models in nude mice

Effect of Targeting Clusterin Using OGX-011 on Antitumor Activity of Temsirolimus in a Human Renal Cell Carcinoma Model.

It has not been well documented that the modulation of stress response mediates the efficacy of the mammalian target of rapamycin (mTOR) inhibitor in renal cell carcinoma (RCC). The objective of this study was to investigate whether the activity of the mTOR inhibitor temsirolimus against RCC could be enhanced by OGX-011, an antisense oligodeoxynucleotide (ODN) targeting the stress-activated chaperone clusterin. We investigated the efficacy of combined treatment with temsirolimus plus OGX-011 in a human RCC Caki-1 model focusing on the effects on apoptotic and autophagic pathways. Although clusterin expression was increased by temsirolims, additional treatment of Caki-1 with OGX-011 significantly inhibited clusterin upregulation (p < 0.05). Combined treatment of temsirolimus and OGX-011 synergistically enhanced the sensitivity of Caki-1 to temsirolimus (p < 0.01), reducing the IC50 by approximately 50%. Apoptotic changes were marked in Caki-1 following combined treatment with a sublethal dose of temsirolimus and OGX-011, accompanying the significant downregulation of Mcl-1 (p < 0.05), but not with either agent alone. Furthermore, this combined treatment markedly blocked the temsirolimus-induced activation of autophagy in Caki-1 (p < 0.01). In-vivo systemic administration of temsirolimus plus OGX-011 significantly inhibited the growth of Caki-1 tumors compared with that of temsirolimus plus control ODN (p < 0.05). Silencing of clusterin using OGX-011 resulted in the further enhancement of proapoptotic activity as well as the marked attenuation of the autophagic pathway induced by temsirolimus in a human RCC model. Thus, the combined use of OGX-011 could be a promising strategy through the enhanced cytotoxic activity of temsirolimus against RCC.

MP92-08 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOUR ACTIVITY OF AXITINIB IN A HUMAN RENAL CELL CARCINOMA MODEL

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-08 CLUSTERIN INHIBITION USING OGX-011 SYNERGISTICALLY ENHANCES ANTITUMOUR ACTIVITY OF AXITINIB IN A HUMAN RENAL CELL CARCINOMA MODEL Satoshi Imai, Hideaki Miyake, Martin Gleave, and Masato Fujisawa Satoshi ImaiSatoshi Imai More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , Martin GleaveMartin Gleave More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2641AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clusterin is expressed in various malignant tumors, including renal cell carcinoma (RCC), and associated with resistance to a wide variety of therapeutic stimuli. OGX-011 is a second-generation antisense oligodeoxynucleotide (ODN) currently in late-stage clinical development that potently inhibits clusterin expression and enhances the efficacy of anticancer therapies in several types of human cancer. The objective of this study was to investigate whether inactivation of clusterin with OGX-011 enhances the effect of axitinib, a potential vascular endothelial growth (VEGF)-targeted agent, in a human RCC ACHN model. METHODS Effects of a combined treatment with OGX-011 and axitinib on ACHN cells were examined by MTT assay, DNA fragmentation assay and Western bolt analyses of proteins involved in apoptosis, signal transduction and autophagy. In vivo efficacy of this combined therapy was assessed in nude mice given subcutaneous injection of ACHN cells. RESULTS Although clusterin expression was increased by axitinib, additional treatment of ACHN with OGX-011 significantly blocked the upregulation of clusterin induced by axitinib. Despite the lack of a significant effect on the growth of ACHN, OGX-011 synergistically enhanced the sensitivity to axitinib, reducing the IC50 by more than 50%. Apoptotic changes were intensively detected in ACHN cells after combined treatment with OGX-011 and a sublethal dose of axitinib, but not either agent alone. Furthermore, this combined treatment resulted in the marked downregulation of phosphorylated Akt and p44/42 mitogen-activated protein kinase in ACHN cells compared with treatment with either agent alone, while the expression levels of Bcl-2, Bcl-xL and p53 after the combined treatment were significantly upregulated compared with those after treatment with either agent alone. In vivo systemic administration of OGX-011 plus axitinib significantly decreased the ACHN tumour volume compared with control ODN plus axitinib. Although no significant differences in the proportions of cells positive for Ki-67 immunostaining or TUNEL assay between the group treated with OGX-011 and that with control ODN, the inhibition of cell proliferation and induction of apoptosis in ACHN tumors treated with OGX-011 and axitinib were markedly remarkable than those in ACHN tumors treated with control ODN and axitinib. CONCLUSIONS Combined use with OGX-011 may be useful in enhancing the cytotoxic effect of axitinib on RCC by inducing apoptosis and inactivating major signal transduction pathways. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1164-e1165 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Satoshi Imai More articles by this author Hideaki Miyake More articles by this author Martin Gleave More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Combination strategy targeting VEGF and HGF/c-met in human renal cell carcinoma models.

Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting.

Inhibition of Hedgehog signalling by NVP-LDE225 (Erismodegib) interferes with growth and invasion of human renal cell carcinoma cells.

Background:Multiple lines of evidence support that the Hedgehog (Hh) signalling has a role in the maintenance and progression of different human cancers. Therefore, inhibition of the Hh pathway represents a valid anticancer therapeutic approach for renal cell carcinoma (RCC) patients. NVP-LDE225 is a Smoothened (Smo) antagonist that induces dose-related inhibition of Hh and Smo-dependent tumour growth.Methods:We assayed the effects of NVP-LDE225 alone or in combination with everolimus or sunitinib on the growth and invasion of human RCC models both in vitro and in vivo. To this aim, we used a panel of human RCC models, comprising cells with acquired resistance to sunitinib – a multiple tyrosine kinase inhibitor approved as a first-line treatment for RCC.Results:NVP-LDE225 cooperated with either everolimus or sunitinib to inhibit proliferation, migration, and invasion of RCC cells even in sunitinib-resistant (SuR) cells. Some major transducers involved in tumour cell motility, including paxillin, were also efficiently inhibited by the combination therapy, as demonstrated by western blot and confocal microscopy assays. Moreover, these combined treatments inhibited tumour growth and increased animal survival in nude mice xenografted with SuR RCC cells. Finally, lung micrometastasis formation was reduced when mice were treated with NVP-LDE225 plus everolimus or sunitinib, as evidenced by artificial metastatic assays.Conclusions:Hedgehog inhibition by NVP-LDE225 plus sunitinib or everolimus bolsters antitumour activity by interfering with tumour growth and metastatic spread, even in SuR cells. Thus, this new evidence puts forward a new promising therapeutic approach for RCC patients.

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma

Angiogenesis, the growth of new vessels from pre-existing ones, is an important feature of tumor growth that has been exploited as a therapeutic target in oncology. Given its key role in facilitating blood vessel sprouting, VEGF has been a major focus of anti-angiogenic strategies, but the observation of resistance in some clinical trials utilizing such agents has led to a search for new or complementary targets in angiogenesis process. The Angiopoietin/Tie2 pathway and in particular the Angiopoietin-2 (Ang-2) ligand which is critically involved in the destabilization of normal vasculature, has been identified as one such target. The current study investigated the potential benefits of combining an Ang-2 targeted therapy with small molecule VEGF targeted agents (Sunitinib, Cediranib) in a human renal cell carcinoma model. The results showed that while both Ang-2 and VEGF interference on their own impaired tumor growth and new blood vessel formation, the combination of agents that targeted both pathways resulted in significantly superior anti-tumor and anti-angiogenic effects.

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma

Angiogenesis, the growth of new vessels from pre-existing ones, is an important feature of tumor growth that has been exploited as a therapeutic target in oncology. Given its key role in facilitating blood vessel sprouting, VEGF has been a major focus of anti-angiogenic strategies, but the observation of resistance in some clinical trials utilizing such agents has led to a search for new or complementary targets in angiogenesis process. The Angiopoietin/Tie2 pathway and in particular the Angiopoietin-2 (Ang-2) ligand which is critically involved in the destabilization of normal vasculature, has been identified as one such target. The current study investigated the potential benefits of combining an Ang-2 targeted therapy with small molecule VEGF targeted agents (Sunitinib, Cediranib) in a human renal cell carcinoma model. The results showed that while both Ang-2 and VEGF interference on their own impaired tumor growth and new blood vessel formation, the combination of agents that targeted both pathways resulted in significantly superior anti-tumor and anti-angiogenic effects.

Clusterin inhibition using OGX-011 synergistically enhances antitumour activity of sorafenib in a human renal cell carcinoma model

Background:The objective of this study was to investigate whether the therapeutic activity of sorafenib could be enhanced by combining with OGX-011, an antisense oligodeoxynucleotide (ODN) targeting clusterin, in renal cell carcinoma (RCC).Methods:We investigated the effects of combined treatment with OGX-011 and sorafenib on a human RCC ACHN model both in vitro and in vivo.Results:Although clusterin expression was increased by sorafenib, additional treatment of ACHN with OGX-011 significantly blocked the upregulation of clusterin induced by sorafenib. Despite the lack of a significant effect on the growth of ACHN, OGX-011 synergistically enhanced the sensitivity to sorafenib, reducing the IC50 by >50%. Apoptotic changes were intensively detected in ACHN after combined treatment with OGX-011 and a sublethal dose of sorafenib, but not either agent alone. Furthermore, this combined treatment resulted in the marked downregulation of phosphorylated Akt and p44/42 mitogen-activated protein kinase in ACHN compared with treatment with either agent alone. In vivo systemic administration of OGX-011 plus sorafenib significantly decreased the ACHN tumour volume compared with control ODN plus sorafenib.Conclusion:Combined use with OGX-011 may be useful in enhancing the cytotoxic effect of sorafenib on RCC by inducing apoptosis and inactivating major signal transduction pathways.

Abstract 2329: Anti-tumor efficacy of Dll4 blockade in combination with sunitinib or aflibercept in primary human renal cell carcinoma models

Abstract Background: The Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis, particularly angiogenic sprouting. Blockade of the Dll4-Notch pathway leads to an excessive production of aberrant non-functional tumor vascular structures and is associated with reduced tumor growth. REGN1035, an antibody that selectively binds and blocks murine Dll4, has demonstrated potent inhibition of tumor growth in a broad spectrum of xenograft models. The aim of this study was to assess the effect of REGN1035 alone and in combination with anti-VEGF therapy in two preclinical models of primary RCC. We hypothesized that combining REGN1035 with sunitinib or the VEGF blocker aflibercept would provide greater anti-tumor and anti-angiogenesis efficacy compared to single agent treatments. Methods: Two human RCC models isolated from skin metastases from a patient with sporadic ccRCC (RP-01) and a patient with hereditary ccRCC (RP-02), respectively, were bilaterally implanted subcutaneously into the flank area of three sets of 32 SCID mice (8 mice/group). When tumor volume reached ∼ 25 mm2, mice were treated with vehicle (human Fc control protein, 4.89 mg/kg, 1x/wk, s.q.), REGN1035 (5 mg/kg, 1x/wk, s.q.), sunitinib (40 mg/kg, 5x/wk, oral gavage), aflibercept (10 mg/kg, 2x/wk, s.q.) or REGN1035 plus sunitinib or aflibercept. Tumor growth was assessed with serial caliper measurements. Once tumor volume of vehicle mice reached 200 mm2, mice were sacrificed and tumor tissue was excised and analyzed. Results: In the RP-01 and RP-02 models, sunitinib and REGN1035 induced 60-69% and 71-81% inhibition (p &amp;lt; .01 vs. control), respectively, while the combination group showed 89-114% inhibition (p &amp;lt; .001 vs. single agents), respectively. No overt signs of toxicity (i.e., weight loss, diarrhea) were observed in any of the experimental groups. Similar results were observed by combining the VEGF blocker aflibercept with REGN1035. HIF-1α and HIF-2α protein levels in the tumor were both found to be downregulated in the combination treated mice, as compared to the single agent treated mice as monitored by Western blotting analysis. A reduction in microvessel density, as measured by CD31 and VEGF immunohistochemistry, was observed in the sunitinib treated group, whereas an increase in abnormal vascular structures was seen in the REGN1035 group. Summary: Overall, the data demonstrate that combination therapy with REGN1035 and sunitinib or aflibercept resulted in significant anti-tumor efficacy including tumor regression in primary human RCC xenografts and highlights the potential therapeutic application of combined VEGF and Dll4 blockade for the treatment of RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2329. doi:1538-7445.AM2012-2329

Epigenetic Modulation of Retinoic Acid Receptor β2 by the Histone Deacetylase Inhibitor MS-275 in Human Renal Cell Carcinoma

Histone deacetylase (HDAC) inhibitors have been shown to reverse epigenetic repression of certain genes, including retinoic acid receptor beta2 (RARbeta2). In this study, we examined whether RARbeta2 expression is repressed in human renal cell carcinoma (RCC) and whether the HDAC inhibitor MS-275 may revert its epigenetic repression. Six human tumor RCC cell lines were analyzed for RARbeta2 gene expression and for methylation and acetylation status at the promoter level. Modulation of RARbeta2 expression and correlation with antitumor activity by combination of MS-275 with 13-cis-retinoic acid (CRA) was assessed in a RARbeta2-negative RCC cell line. RARbeta2 expression was either strongly present, weakly expressed, or absent in the RCC cell lines analyzed. Methylation-specific PCR indicated that the RARbeta2 promoter was partially methylated in three of the cell lines. CRA treatment did not inhibit clonogenic growth in the RARbeta2-negative cell line RCC1.18, whereas MS-275 induced a dose-dependent inhibitory effect. A greater inhibitory effect was observed with combination treatment (MS-275 + CRA). Treatment with MS-275 was associated with histone acetylation at the promoter level and synergistic gene reexpression of RARbeta2 in combination with CRA. RARbeta2 reexpression was associated with synergistic induction of the retinoid-responsive gene HOXA5. In vivo, single-agent CRA treatment showed no significant effect, whereas MS-275 and the combination induced a regression of RCC1.18 tumor xenografts. Discontinuation of treatment produced tumor recurrence in MS-275-treated mice, whereas animals treated with the combination remained tumor free. The HDAC inhibitor MS-275 seems to revert retinoid resistance due to epigenetic silencing of RARbeta2 in a human RCC model and has greater antitumor activity in combination with CRA compared with single agents. Thus, the combination of HDAC inhibitors and retinoids may represent a novel therapeutic approach in patients with RCC.