Renal Cell Carcinoma 786-O Cells Research Articles

Runx2 silencing sensitized human renal cell carcinoma cells to ABT-737 apoptosis

The prognostic value of Runt-related transcription factor 2 (Runx2) and its involvement in cell growth and motility have been reported in patients diagnosed with renal cell carcinoma (RCC). Since Runx2 may have the potential to be a target for the purpose of antitumor intervention, there is an urgent need to gain insight into its oncogenic properties. Using human 786-O, Caki-1 and ACHN RCC cells as models, the silencing of cellular Runx2 expression brought about a reduction in cyclin D1 and β-catenin expression, cell growth and migration without any significant cell death. Runx2-silenced cells turned into apoptosis vulnerable in the presence of ABT-737, a BH3 mimetic Bcl-2 inhibitor. Data from biochemical and molecular studies have revealed a positive correlation between Runx2 expression and Akt phosphorylation, Mcl-1 expression, and fibronectin expression. Results of genetic silencing studies have indicated the potential involvement of Mcl-1 and fibronectin in the decision of RCC cell ABT-737 resistance and sensitivity. The regulatory roles of the PI3K/Akt axis in the expression of Mcl-1 and fibronectin were suggested by means of the results taken from experiments involving pharmacological study of the PI3K/Akt. Since overexpression and prognostic roles of Runx2, activated Akt, Mcl-1, fibronectin, cyclin D1, and β-catenin have been revealed in RCC, it is important to explore the precise mechanisms underlying Runx2 oncogenic effects. Although the linking details between Runx2 and PI3K/Akt have yet to be identified, our findings suggest that Mcl-1 and fibronectin are downstream effectors of Runx2 via a regulatory axis of the PI3K/Akt and their promotion of cell growth, migration, and ABT-737 resistance in RCC cells.

49 Inhibition of AXL along with c-Met potentially halts resistance development in renal cell carcinoma

Abstract Background c-Met, a receptor tyrosine kinase (RTK), is overexpressed in renal cell carcinoma (RCC) and correlates with a decreased survival rate. Upon binding to its specific ligand, Hepatocyte Growth Factor (HGF), c-Met activates pro-tumorigenic signaling pathways. Cabozantinib (Cabo) inhibits c-Met and a few other RTKs, including AXL, and is approved for the treatment of patients with advanced-stage RCC. AXL and its ligand, GAS6, are overexpressed in RCC and are also markers of poor prognosis. The pro-tumorigenic role of AXL and its potential crosstalk with c-Met in renal cancer need to be thoroughly investigated. Methods We generated a Cabo-resistant (CaboR) cell line from wild-type Cabo-sensitive (CaboS) Caki-1 clear-cell RCC cells, and CRISPR/Cas9-mediated AXL knock-out cells (AXL-KO) from 786-O clear-cell RCC cells. Chromatin immunoprecipitation and sequencing (ChIP-seq) was performed to profile H3K27ac, a histone post-translational modification associated with active promoters and enhancers, in CaboR and CaboS cell lines. We also performed RNA sequencing (RNA-seq) on CaboR and CaboS and evaluated differentially expressed genes and enriched pathways using gene set enrichment analysis (GSEA). Transcriptional profiles of control clones and AXL-KO cells were also compared to identify the effect of AXL-KO on pro-tumorigenic signaling. We utilized an AXL-specific small molecule inhibitor, TP-0903, in combination with Cabo to validate our data. Finally, we studied how AXL silencing or inhibition may affect resistance to Cabo. Results Our findings revealed that AXL forms a complex with c-Met and may have a significant crosstalk which can be involved in therapeutic resistance. We found that prolonged treatment with c-Met inhibitors Cabo, Crizotinib, and PF-4217903, induced c-Met and AXL overexpression in RCC cells. Interestingly, c-Met inhibitor(s)-induced overexpressed c-Met can also be increasingly phosphorylated (at low concentrations) in the presence of HGF, which may cause enhanced downstream tumor-promoting signaling. We found that silencing AXL can prevent this c-Met-inhibitors-mediated c-Met overexpression. Moreover, we found marked activation of the promoters and enhancers of several transcription factors, including ETV1, HOXA9, and FOXK1, in CaboR cells compared to CaboS cells. In the CaboR cells, genes involved in oxidative phosphorylation, progression through the division cycle, DNA replication, the NRF2 pathway, and DNA repair were upregulated, whereas genes associated with glycolysis, angiogenesis, hypoxia, and the Met pathway were downregulated (Figure 1). Finally, silencing AXL (using siRNA) or inhibiting AXL (using TP0903) in CaboR cells, induced significant apoptosis. Conclusions Together, our data suggest that CaboR cells have a distinct epigenomic and transcriptomic profile, and that targeting AXL along with c-Met inhibition can be beneficial in preventing acquired therapeutic resistance in RCC.

Disruption of Autophagic Flux and Treatment with the PDPK1 Inhibitor GSK2334470 Synergistically Inhibit Renal Cell Carcinoma Pathogenesis.

Background: Renal cell carcinoma (RCC) frequently exhibits activating PI3K-Akt-mTOR pathway mutations. 3-Phosphoinositide-dependent kinase 1 (PDPK1 or PDK1) has been established to play a pivotal role in modulating PI3K pathway signaling. mTOR is the main autophagy-initiating factor. However, limited advances have been made in understanding the relationship between PDPK1 and autophagy in RCC. Methods: GSK2334470 (GSK470), a novel and highly specific inhibitor of PDPK1, was selected to investigate the anticancer effects in two RCC cell lines. Cell growth was assessed by CCK-8 test and colony formation. Changes in the protein levels of key Akt/mTOR pathway components and apoptosis markers were assessed by Western blotting. Autophagy was assessed by using LC3B expression, transmission electron microscopy, and a tandem mRFP-EGFP-LC3 construct. The effect of PDPK1 and autophagy inhibitor chloroquine in RCC in vivo was examined in a mouse tumor-bearing model. Results: GSK470 significantly inhibited cell proliferation and induces apoptosis in A498 and 786-O RCC cells. GSK470 downregulates the phosphorylation of PDPK1, thereby inhibiting downstream phosphorylation of Akt1 at Thr308 and Ser473 and mTOR complex 1 (mTORC1) activity. Treatment with insulin-like growth factor-1 (IGF-1) partially restored GSK470-induced behaviors/activities. Interestingly, treatment of A498 and 786-O cells with GSK470 or siPDPK1 induced significant increases in the hallmarks of autophagy, including autophagosome accumulation, autophagic flux, and LC3B expression. Importantly, GSK470 and chloroquine synergistically inhibited the growth of RCC cells in vitro and in xenograft models, supporting the protective role of autophagy activation upon blockade of the PDPK1-Akt-mTOR signaling pathway. Conclusion: Our study provides new insight into PDPK1 inhibition combined with autophagy inhibition as a useful treatment strategy for RCC.

S100 Calcium-Binding Protein A8 Functions as a Tumor-Promoting Factor in Renal Cell Carcinoma via Activating NF-κB Signaling Pathway

Background Renal cell carcinoma (RCC), arising from the renal tubular epithelium, is one of the most common types of genitourinary malignancies. Based on the Gene Expression Omnibus (GEO) database (GSE100666), S100 calcium-binding protein A8 (S100A8) was highly expressed in RCC tissues. S100A8, an inflammatory regulatory factor, has emerged as an important mediator associated with the occurrence and development of cancer. Materials and Methods The Gene Expression Omnibus (GEO) database was used to identify the key genes and investigate the main signaling pathways in RCC. Human RCC samples and corresponding adjacent normal tissues were collected in our hospital. The expression of S100A8 in human RCC samples was detected using western blotting and immunohistochemical analysis. S100A8 overexpression or knockdown was mediated by using Lipofectamine 3000 in human renal cell carcinoma cell line 786-O and ACHN cells. Basic experiments, including MTT and cell apoptosis assays, were utilized for investigating the function of S100A8 in RCC. Furthermore, the levels of inflammation were also evaluated in 786-O and ACHN cells. Results In the current study, we found that downregulation of S100A8 inhibited proliferation and promoted apoptosis in 786-O and ACHN RCC cells. Of note, S100A8 silencing downregulated the phosphorylation of NF-κB p65, thereby decreasing the levels of TNF-α, cleaved caspase1, and MMP9. By contrast, S100A8 upregulation could increase these expressions. Conclusion Overall, S100A8 knockdown restrained RCC malignant biological properties, which was associated with the deactivation of the NF-κB signaling pathway. This present study demonstrates new insights that S100A8 may be a potential therapeutic target in RCC.

ROCK2-RNA interaction map reveals multiple biological mechanisms underlying tumor progression in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of kidney cancer in adults. Despite new therapeutic modalities, the outcomes for RCC patients remain unsatisfactory. Rho-associated coiled-coil forming protein kinase 2 (ROCK2) has previously been shown to be upregulated in RCC, and its expression was negatively correlated with patient survival. However, the precise molecular function of ROCK2 has remained unclear. Herein, using RNA-seq analysis of ROCK2 knockdown and control cells, we identified 464 differentially expressed genes, and 1287 alternative splicing events in 786-O RCC cells. Furthermore, mapping of iRIP-seq reads in 786-O cells showed a biased distribution at 5' UTR, intronic and intergenic regions. By comparing ROCK2-regulated alternative splicing and iRIP-seq data, we found 292 overlapping genes that are enriched in multiple tumorigenic pathways. Taken together, our work defined a complex ROCK2-RNA interaction map on a genomic scale in a human RCC cell line, which deepens our understanding of the molecular function of ROCK2 in cancer development.

Melatonin acts synergistically with pazopanib against renal cell carcinoma cells through p38 mitogen-activated protein kinase-mediated mitochondrial and autophagic apoptosis.

Mounting evidence indicates that melatonin has possible activity against different tumors. Pazopanib is an anticancer drug used to treat renal cell carcinoma (RCC). This study tested the anticancer activity of melatonin combined with pazopanib on RCC cells and explored the underlying mechanistic pathways of its action. The 786-O and A-498 human RCC cell lines were used as cell models. Cell viability and tumorigenesis were detected with the MTT and colony formation assays, respectively. Apoptosis and autophagy were assessed using TUNEL, annexin V/propidium iodide, and acridine orange staining with flow cytometry. The expression of cellular signaling proteins was investigated with western blotting. The in vivo growth of tumors derived from RCC cells was evaluated using a xenograft mouse model. Together, melatonin and pazopanib reduced cell viability and colony formation and promoted the apoptosis of RCC cells. Furthermore, the combination of melatonin and pazopanib triggered more mitochondrial, caspase-mediated, and LC3-II-mediated autophagic apoptosis than melatonin or pazopanib alone. The combination also induced higher activation of the p38 mitogen-activated protein kinase (p38MAPK) in the promotion of autophagy and apoptosis by RCC cells than melatonin or pazopanib alone. Finally, tumor xenograft experiments confirmed that melatonin and pazopanib cooperatively inhibited RCC growth in vivo and predicted a possible interaction between melatonin/pazopanib and LC3-II. The combination of melatonin and pazopanib inhibits the growth of RCC cells by inducing p38MAPK-mediated mitochondrial and autophagic apoptosis. Therefore, melatonin might be a potential adjuvant that could act synergistically with pazopanib for RCC treatment.

Accurate detection and delineation boundary of renal cell carcinoma based on dual-targeted magnetic-fluorescent carbon dots

Accurate tumor localization and tumor boundary delineation are significant to improve the effectiveness of cancer therapeutics. In the present study, a fluorescence/magnetic resonance (MR) nanoprobe AS1411-CLT1-Gd-CDs with dual targeting function was developed for precise detection and delineation boundary of renal cell carcinoma (RCC). The dual-targeted AS1411-CLT1-Gd-CDs nanoprobe was initially established by co-conjugating peptide CLT1 and aptamer AS1411 to magnetic-fluorescent gadolinium(III)-doped carbon dots (Gd-CDs). The proposed AS1411-CLT1-Gd-CDs with wide-range multi-color fluorescence emission could target-specific image 786-O RCC cells through three distinct fluorescence channels and achieved the quantitativedetermination of 786-O cells with a low detection limit of 552 cells mL−1. Besides, the dual-targeted AS1411-CLT1-Gd-CDs nanoprobe could accurately delineate tumor boundary and generate greater and prolonged plateau contrast enhancement than the non-targeted and single-targeted Gd-CDs. Furthermore, the small-sized nanoprobe could be easily excreted from the body by the renal excretory system after imaging without any obvious long-term toxicity. These studies demonstrated that the proposed dual-targeted nanoprobe appeared as a very promising fluorescence/MR imaging agent for accurate identification and localization of renal cancer tumors, which could be beneficial to the early diagnosis and treatment of RCC.

Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway.

Simple SummaryAdvanced renal cell carcinoma (RCC) remains an incurable disease, despite the establishment of new therapeutic options during the last decades. Unfortunately, drug resistance inevitably evolves, making better treatment strategies essential. Shikonin (SHI) from traditional Chinese medicine has shown promising antitumor properties with other tumor entities. Thus, in the current investigation, we evaluated the impact of SHI on therapy-sensitive and therapy-resistant RCC cells. SHI led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. This was accompanied by cell cycle arrest and reduction in cell cycle activating proteins, which results in blockage of cell division. SHI also activated necrosome complex proteins, leading to necroptosis, a programmed cell death. Furthermore, SHI inhibited the AKT/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise in improving the treatment of RCC, even in its advanced form.Therapy resistance remains a major challenge in treating advanced renal cell carcinoma (RCC), making more effective treatment strategies crucial. Shikonin (SHI) from traditional Chinese medicine has exhibited antitumor properties in several tumor entities. We, therefore, currently investigated SHI’s impact on progressive growth and metastatic behavior in therapy-sensitive (parental) and therapy-resistant Caki-1, 786-O, KTCTL-26, and A498 RCC cells. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis), and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI significantly inhibited the growth, proliferation, and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the AKT/mTOR pathway. Adhesion and motility were cell line specifically affected by SHI. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC.

Isothiocyanate From Moringa oleifera Seeds Inhibits the Growth and Migration of Renal Cancer Cells by Regulating the PTP1B-dependent Src/Ras/Raf/ERK Signaling Pathway.

Moringa oleifera Lam. is a tropical and subtropical plant that has been used for centuries as both food and traditional medicine. 4-[(α-L-Rhamnosyloxy) benzyl] isothiocyanate (MIC-1) is an active substance in M. oleifera, with anti-cancer activity. However, whether MIC-1 exerts anti-renal cancer effects is unknown. Therefore, the aim of the present study was to evaluate the effects of MIC-1 on the growth and migration of renal cell carcinoma (RCC) cells and to identify the putative underlying mechanism. We found that, among 30 types of cancer cells, MIC-1 exerted the strongest growth inhibitory effects against 786-O RCC cells. In addition, MIC-1 (10 μM) significantly inhibited the growth of five RCC cell lines, including 786-O, OSRC-2, 769-P, SK-NEP-1, and ACHN cells, but was not toxic to normal renal (HK2) cells. Also, MIC-1 suppressed 786-O and 769-P cell migration and invasion abilities, and reduced the expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, MIC-1 induced apoptosis and cell cycle arrest, increased Bax/Bcl-2 ratio, and decreased cell cycle-related protein expression in 786-O cells and 769-P cells. Molecular docking and small-molecule interaction analyses with PTP1B both showed that MIC-1 inhibited PTP1B activity by binding to its active site through hydrogen bonding and hydrophobic interactions. Additionally, MIC-1 could suppress the growth and migration of 786-O cells by inhibiting PTP1B-mediated activation of the Src/Ras/Raf/ERK signaling pathway. In vivo experiments further showed that MIC-1 markedly inhibited the growth of xenograft tumors in mice, and greatly increased Bax/Bcl-2 ratio in tumor tissues. In addition, MIC-1 had no effect on the PTP1B-dependent Src/Ras/Raf/ERK signaling pathway in HCT-116 cells, Hep-G2 cells, and A431 cells. Overall, our data showed that MIC-1 could be a promising, non-toxic, natural dietary supplement for the prevention and treatment of renal cancer.

Chinese Medicine Leptochloa chinensis Inhibits the Malignant Behaviors of Renal Cell Carcinoma 786-O Cells by Regulating the mTOR Pathway.

Background Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system that seriously threatens human life and health. This study aims to explore the role of the traditional Chinese medicine Leptochloa chinensis in the pathogenesis of RCC. Meanwhile, this study also revealed the molecular biological mechanism of its antitumor activity. Methods Human RCC 786-O cells were cultured in the RPMI-1640 medium, which contains different concentrations of Leptochloa chinensis (1,000, 3,000, and 9,000 μg/ml). MTT and flow cytometry assays were used to detect the viability of 786-O cells. Transwell and wound healing assays were used to detect cell metastasis. The protein expression was observed by western blot analysis. Results Leptochloa can inhibit cell proliferation and induce apoptosis in RCC 786-O cells. In addition, Leptochloa can weaken the migration and invasion of 786-O cells. More importantly, Leptochloa can block the mTOR pathway by inhibiting the protein expression of p-mTOR. Moreover, the high concentration of Leptochloa chinensis has a better inhibitory effect on 786-O cells. Conclusion The traditional Chinese medicine Leptochloa chinensis inhibits the viability and metastasis of 786-O cells by blocking the mTOR pathway.

MP39-11 ESTROGEN RECEPTOR Β PROMOTES RENAL CELL CARCINOMA MALIGNANCY AND TKI RESISTANCE VIA REGULATING LNCRNA AND CIRCRNA ASSOCIATED CERNA NETWORK

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-11 ESTROGEN RECEPTOR Β PROMOTES RENAL CELL CARCINOMA MALIGNANCY AND TKI RESISTANCE VIA REGULATING LNCRNA AND CIRCRNA ASSOCIATED CERNA NETWORK Yixi Hu, Jie Ding, Junfei Gu, Zhenwei Han, Wenbiao Ren, Edward M. Messing, Chawnshang Chang, Jean V. Joseph, and Shuyuan Yeh Yixi HuYixi Hu More articles by this author , Jie DingJie Ding More articles by this author , Junfei GuJunfei Gu More articles by this author , Zhenwei HanZhenwei Han More articles by this author , Wenbiao RenWenbiao Ren More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Jean V. JosephJean V. Joseph More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney cancer accounts for at least 3% of malignant diseases, and the incidence and mortality appear to be rising. Recent studies indicated that the estrogen receptor beta (ERβ), but not the ER alpha, could affect the progression of renal cell carcinoma (RCC), however, its downstream signals remain to be further elucidated. METHODS: Human 786-O, A498, ACHN, and Caki-1 RCC cell lines were used. The RNAs of 10 nM E2 treated and shERβ transfected 786-O cells were isolated for RNA-Seq and non-coding RNA analysis. Invasion assays were performed using the matrigel transwell and 3D invasion assays. The q-PCR analysis and western blot were performed to evaluate the RNA and the protein expressions. HOTAIR promoter was constructed into the pGL3 for luciferase assays. Protein-DNA complexes were detected by Chromatin immunoprecipitation (ChIP) assay. RNA pull-down assays were performed to detect the binding of putative miRNAs. A preclinical xenograft mouse RCC model was used to validate ERβ signal-promoted ceRNA network. RESULTS: RNAseq data of RCC patients were retrieved from the TCGA database and analysis results showed high expressions of ERβ and its down-stream signals were associated with advanced stages and poor prognosis in RCC patients. Mechanism dissection revealed that targeting ERβ with ERβshRNA and stimulating the transactivation of ERβ with 17β-estradiol or environmental endocrine disrupting chemicals, resulted in altering the lncRNA HOTAIR expression. The ERβ-modulated HOTAIR can function via antagonizing several miRNAs (miR-138, -200c, -204, or -217) to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC invasion. In addition, we found ERβ can suppress the expression of the circular RNA, circATP2B1, which then led to a reduced miR-204-3, increased fibronectin 1 (FN1) expression, and enhanced RCC cell invasion. Our results further showed that the expression levels of those ERβ regulated long non-coding RNAs (lncRNAs), circRNAs, and miRNAs could predict the malignancy or TKI resistance of RCC. CONCLUSIONS: We provide evidence that expression levels of ERβ and ERβ-regulated CeRNA network could be used to predict metastasis and overall survival in RCC patients. The identification of the ERβ-lncRNA or circRNA regulated signal pathways may provide physicians with new biomarkers and/or therapeutic targets to better control RCC progression in the future. Source of Funding: This work was partially supported by URMC Urology Department Research Fund and George Whipple H. Professorship Endowment © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e708-e709 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yixi Hu More articles by this author Jie Ding More articles by this author Junfei Gu More articles by this author Zhenwei Han More articles by this author Wenbiao Ren More articles by this author Edward M. Messing More articles by this author Chawnshang Chang More articles by this author Jean V. Joseph More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Loading ...

LncRNA LINC00944 Promotes Tumorigenesis but Suppresses Akt Phosphorylation in Renal Cell Carcinoma.

Long non-coding RNA (lncRNA) is a kind of RNA that possesses longer than 200 nucleotides and lacks protein coding function. It was recognized as a junk sequence for a long time. Recent studies have found that lncRNAs are actively functioning in almost every aspect of cell biology and involved in a variety of biological functions. LncRNAs are closely related to a variety of human diseases, especially tumors. Recently, lncRNAs are being increasingly reported in renal cancer. In our study, we identified the expression of lncRNA LINC00944 is significantly elevated in renal cell carcinoma (RCC) tissues and cell lines and high LINC00944 expression is significantly correlated with the tumor stage and prognosis of RCC. The knockdown of LINC00944 by CRISPR/dCas9-KRAB in higher expressing 786-O and 769-P RCC cells could significantly decrease proliferation and migration and also promote phosphorylation of Akt compared with the control group. Our study is the first to report the function of lncRNA LINC00944 in RCC. And we provide clinicopathological and experimental evidence that lncRNA LINC00944 acts as an oncogene in RCC, suggesting that targeting lncRNA LINC00944 expression might be a promising therapeutic strategy for the treatment of RCC.

Long Non-Coding RNA LOC648987 Promotes Proliferation and Metastasis of Renal Cell Carcinoma by Regulating Epithelial-Mesenchymal Transition.

Renal cell carcinoma (RCC) is a type of urinary tumor with a high incidence and is often associated with tumor metastasis. Long non-coding RNA (lncRNA) regulates tumorigenesis, progression, and metastasis. However, the role and the predictive value of lncRNA in RCC progression and metastasis have not been elucidated. The purpose of this study was to evaluate the effect of a newly discovered lncRNA LOC648987 on RCC proliferation and metastasis. LOC648987 was identified by RT-PCR for high expression in human RCC tissues as well as in metastatic RCC tissues. In the cell experiments, we infected the RCC cell lines ACHN and 786-O cells with LOC648987-shRNA and its negative control (shNC). The results showed that the knockdown of LOC648987 inhibited the proliferation of ACHN and 786-O cells and colony formation. The cell cycle and the apoptosis progression of ACHN and 786-O cells were assessed using flow cytometry. The knockdown of LOC648987 significantly inhibited the progression of ACHN and 786-O cells from G0/G1 to S phase and promoted cell apoptosis. The metastasis promoting effects of LOC648987 on ACHN and 786-O cells were verified by transwell migration assays, which depended on vimentin and MMP-9 to regulate the epithelial–mesenchymal transition. Finally, the promotion of LOC648987 on RCC tumorigenesis was evaluated in BALb/c nude mice. These data confirmed that lncRNA LOC648987 promoted RCC cell proliferation and tumor metastasis and regulated the expression of EMT-related proteins in RCC cells.

BRD4 inhibition sensitizes renal cell carcinoma cells to the PI3K/mTOR dual inhibitor VS-5584.

Activation of the PI3K/AKT/mTOR pathway promotes the progression of renal cell carcinoma (RCC). This study tested the anti-RCC cell activity of the PI3K/mTOR dual inhibitor, VS-5584. We show that VS-5584 inhibited PI3K/AKT/mTORC1/2 activation in established (786-O and A498 lines) and primary RCC cells, thereby suppressing cell survival, proliferation, migration and cell cycle progression. VS-5584 induced significant apoptosis in RCC cells. A daily single oral dose of VS-5584 (20 mg/kg) significantly inhibited 786-O tumor growth in vivo. VS-5584 treatment of 786-O tumor xenografts and RCC cells resulted in feedback upregulation of bromodomain-containing protein 4 (BRD4). Furthermore, BRD4 inhibition (by JQ1 and CPI203), knockdown or complete knockout potentiated VS-5584-induced RCC cell death and apoptosis. Conversely, forced overexpression of BRD4 attenuated the cytotoxicity of VS-5584 in 786-O cells. Collectively, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.

Abstract 6439: CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma

Abstract Objective: The objective of this work was to obtain highly sensitive purification and post-capture analysis of renal cell carcinoma (RCC) circulating tumor cells (CTCs) using CapioCyte™ technology. Background: Circulating tumor cells (CTCs) may be a highly valuable source of actionable information for the practice of precision medicine. However, CTCs are exceptionally rare in peripheral blood. The CapioCyte™ chip purifies live CTCs through a combination of biomimetic cell rolling and dendrimer-mediated multivalent immunorecognition. The CapioCyte™ platform has been previously shown to isolate CTCs with high sensitivity from peripheral blood collected from patients with head and neck cancer using a mixture of antibodies against epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) [Myung et al., Clinical Cancer Research 2018, 24(11):2539-2547]. However, this antibody cocktail is not universally applicable to all types of cancer. In particular, renal cell carcinoma (RCC), having no clinically actionable circulating biomarkers, often do not express HER2. To address this, we have employed a new antibody mixture targeting EpCAM, EGFR, carbonic anhydrase IX (CAIX), and hepatocyte growth factor receptor (c-MET). Our study presents enhanced capture of CTCs from renal cancer, demonstrating the modular nature of our CapioCyte™ technology. Methods: Antibody-coated beads were used to determine the functional recognition of ACHN and 786-O RCC cell lines. Flow channel experiments and atomic force microscopy (AFM) force spectroscopy measurements were used to quantify off-rate kinetics. A clinical pilot study of 7 RCC patients (pre-treatment) and 5 healthy controls was performed to validate the CapioCyte™ surface tuned for RCC capture. In recent work, CTCs recovered from the CapioCyte™ surface were sequenced using 10X Chromium system with v3 chemistry and analyzed using RaceID clustering and Gene Set Enrichment Analysis (GSEA). Results: The optimized mixture of EpCAM, CAIX, EGFR, and c-MET showed 80% enhancement in the capture of RCC cell lines compared to the mixture of EpCAM, EGFR, and HER2. Force spectroscopy measurements were used to show that dendrimers contributed to an order of magnitude enhancement in off-rate kinetics. In patient samples, the RCC capture surface isolated a total of 9.8 +/- 5.1 CTC ml-1 whole blood compared to just 1.8 +/- 2.0 CTC ml-1 with the conventional three-antibody mixture. Finally, preliminary results show the detection of oncogenic signatures within two additional peripheral blood samples. Conclusions: The prototype CapioCyte™ platform is a flexible liquid biopsy that can be tuned for various disease indications. Here, enhanced capture of RCC CTCs was demonstrated in clinical blood samples with high sensitivity. Proof-of-concept RNA sequencing shows the promise of CapioCyte™ in providing further clinically-actionable information from CTCs. Citation Format: Jiyoon Bu, Michael J. Poellmann, Marco Reyes-Martinez, Andrew Armstrong, Daniel George, Tian Zhang, Andrew Z. Wang, seungpyo hong. CapioCyte™ technology for efficient capture and downstream analysis of circulating tumor cells from renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6439.

Bidirect effects from cisplatin combine with rosmarinic acid (RA) or hot water extracts of Glechoma hederacea (HWG) on renal cancer cells

BackgroundCisplatin (CDDP) is a chemotherapeutic drug which also causes adverse side effects. Glechoma hederacea is a traditional Chinese herb belonging to the Labiatae family and has many biological activities. Our previous study indicated that rosmarinic acid (RA) was the most abundant phytochemical in G. hederacea. However, the antioxidant or anti-inflammatory effects of the combined treatment of G. hederacea, RA and CDDP on human renal cell carcinoma (RCC) 786-O cells have not been clearly demonstrated. We aimed to investigate the bioefficacy of hot water extracts of G. hederacea (HWG) and RA in inhibiting RCC 786-O cell activity and its synergism with CDDP against metastatic renal cancer cell.MethodsBioactivities of the combination treatment of HWG, RA, HWG/CDDP and RA/CDDP were assessed using the MTT assay and transwell migration, and the crude extract/compound efficacy was evaluated using wound healing migration assays, flow cytometry and western blotting.ResultsOur study indicates that CDDP inhibits 786-O cell proliferation and migration and HWG and RA protect against these effects. On the other hand, HWG and RA demonstrate a low cytotoxic effect in human renal proximal tubular epithelial cell line -2 (HK-2 cells). Cell cycle analysis found that HWG/CDDP and RA/CDDP combined treatment exerted cytotoxicity by inducing G2/M arrest and apoptosis. RA in combined with CDDP significantly inhibiting the expression of p-FAK (Tyr 925) in RCC 786-O cells in vitro.ConclusionWe propose that the inhibition of RA on RCC 786-O cell invasion and migration may partly occur through the downregulation of FAK phosphorylation. The HWG/CDDP and RA/CDDP combined treatments may be effective strategies for intervention of RCC 786-O cell activity.

An increased level of MiR-222-3p is associated with TMP2 suppression, ERK activation and is associated with metastasis and a poor prognosis in renal clear cell carcinoma.

Renal cell carcinoma (RCC) is the most common malignancy involving the kidneys and a major cause of cancer mortality. The involvement of microRNA (miRNA) expression in the tumorigenesis and progression of RCC still not been previously highlighted. We aimed to explore the potential role of miR-222-3p in renal cell carcinoma (RCC). We first found that miR-222-3p was elevated significantly in the RCC tissues as compared to the non-tumor counterparts. We also found that a higher level of miR-222-3p in different RCC cell lines than the HK-2 cells. In vitro validation experiment using miR-222-3p mimic molecules significantly induced expression of EMT marker vimentin and downregulated E-cadherin in both 769-P and 786-O RCC cells. In contrary, when miR-222-3p was downregulated by its inhibitor, the reverse observations were made. We then demonstrated a reversal association between the expression level of miR-222-3p and TIMP2/ERK where TIMP2 functions as a tumor suppressor. In a small cohort of 45 clinical samples, we found that miR-222-3p expression level was elevated and was associated with a poorer survival of the patients. Patients with higher miR-222-3p expression showed had a statistically shorter overall survival than those patients of lower miR-222-3p level (HR, 5.789; p= 0.02). Collectively, we showed that miR-222-3p functioned as a tumor progression marker and could be a target for future drug development.

Corrigendum] MicroRNA‑429 inhibits cancer cell proliferation and migration by targeting AKT1 in renal cell carcinoma

[This corrects the article DOI: 10.3892/mco.2019.1940.].

Autophagy suppresses resveratrol-induced apoptosis in renal cell carcinoma 786-O cells.

As a polyphenolic compound, resveratrol (Res) is widely distributed in a variety of plants. Previous studies have demonstrated that Res can inhibit various different types of tumor growth. However, its role in renal cell carcinoma (RCC) remains largely unknown. The present study first demonstrated that Res inhibited cell viability and induced apoptosis in RCC 786-O cells. Further experiments revealed that Res damaged the mitochondria and activated caspase 3. In contrast, Z-VAD-FMK, a pan-caspase inhibitor, suppressed Res-induced apoptosis. Reactive oxygen species (ROS) were involved in the process of Res-induced apoptosis, and antioxidant N-acetyl cysteine could significantly attenuate this. Furthermore, Res activated c-Jun N-terminal kinase via ROS to induce autophagy, whereas inhibition of autophagy with chloroquine or Beclin 1 small interfering RNA aggravated Res-induced apoptosis, indicating that autophagy served as a pro-survival mechanism to protect 786-O cells from Res-induced apoptosis. Therefore, a combination of Res and autophagy inhibitors could enhance the inhibitory effect of Res on RCC.

G250 Antigen-Targeting Drug-Loaded Nanobubbles Combined with Ultrasound Targeted Nanobubble Destruction: A Potential Novel Treatment for Renal Cell Carcinoma.

PurposeWe intended to design G250 antigen-targeting temsirolimus-loaded nanobubbles (G250-TNBs) based on the targeted drug delivery system and to combine G250-TNBs with ultrasound targeted nanobubble destruction (UTND) to achieve a synergistic treatment for renal cell carcinoma (RCC).MethodsThe filming-rehydration method was combined with mechanical shock and electrostatic interactions to prepare temsirolimus-loaded nanobubbles (TNBs). G250-TNBs were prepared by attaching anti-G250 nanobodies to the surface of TNBs using the biotin-streptavidin-bridge method. The ability of G250-TNBs to target the G250 antigen of RCC cells and the synergistic efficacy of G250-TNBs and UTND in the treatment of RCC were assessed.ResultsThe average diameter of the prepared G250-TNBs was 368.7 ± 43.4 nm, the encapsulation efficiency was 68.59% ± 5.43%, and the loading efficiency was 5.23% ± 0.91%. In vitro experiments showed that the affinity of G250-TNBs to the human RCC 786-O cells was significantly higher than that of TNBs (P <0.05), and the inhibitory effect on 786-O cell proliferation and the induction of 786-O cell apoptosis was significantly enhanced in the group treated with G250-TNBs and UTND (G250-TNBs+ UTND group) compared with the other groups (P <0.05). In a nude mouse xenograft model, compared with TNBs, G250-TNBs could target the transplanted tumors and thus significantly enhance the ultrasound imaging of the tumors. Compared with all other groups, the G250-TNBs+UTND group exhibited a significantly lower tumor volume, a higher tumor growth inhibition rate, and a higher apoptosis index (P <0.05).ConclusionThe combined G250-TNBs and UTND treatment can deliver anti-tumor drugs to local areas of RCC, increase the local effective drug concentration, and enhance anti-tumor efficacy, thus providing a potential novel method for targeted therapy of RCC.