ccRCC Cell Line 786-O Research Articles

Exploring the prognostic implications of cuproptosis-associated alterations in clear cell renal cell carcinoma via in vitro experiments

This study investigated the impact of novel copper ionophores on the prognosis of clear cell renal cell carcinoma (ccRCC) and the tumor microenvironment (TME). The differential expression of 10 cuproptosis and 40 TME-pathway-related genes were measured in 531 tumor samples and 71 adjacent kidney samples in The Cancer Genome Atlas database. A risk score model was constructed with LASSO cox to predict the prognosis of ccRCC patients. Forest plot and function enrichment were used to study the biological function of the key genes in depth. The study found that the risk score model accurately predicted the prognosis of ccRCC patients. Patients with high scores had higher immune responses with a higher proportion of anti-tumor lymphocytes and a lower proportion of immunosuppressive M2-like macrophages. However, the high-score group also exhibited a higher proportion of T follicular helper cells and regulatory T cells. These results suggest that cuproptosis-based therapy may be worth further investigation for the treatment of ccRCC and TME. Subsequently, by using RNAi, we established the stable depletion models of FDX1 and PDHB in ccRCC cell lines 786-O and ACHN. Through CCK8, colony formation, and Transwell assays, we observed that the knockdown of FDX1 and PDHB could significantly reduce the capabilities of proliferation and migration in ccRCC cells. In conclusion, this study illuminates the potential effectiveness of copper ionophores in the treatment of ccRCC, with higher risk scores correlating with better TME immune responses. It sets the stage for future cuproptosis-based therapy research in ccRCC and other cancers, focusing on copper’s role in TME.

Deciphering glutamine metabolism patterns for malignancy and tumor microenvironment in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer characterized by metabolic reprogramming. Glutamine metabolism is pivotal in metabolic reprogramming, contributing to the significant heterogeneity observed in ccRCC. Consequently, developing prognostic markers associated with glutamine metabolism could enhance personalized treatment strategies for ccRCC patients. This study obtained RNA sequencing and clinical data from 763 ccRCC cases sourced from multiple databases. Consensus clustering of 74 glutamine metabolism related genes (GMRGs)- profiles stratified the patients into three clusters, each of which exhibited distinct prognosis, tumor microenvironment, and biological characteristics. Then, six genes (SMTNL2, MIOX, TMEM27, SLC16A12, HRH2, and SAA1) were identified by machine-learning algorithms to develop a predictive signature related to glutamine metabolism, termed as GMRScore. The GMRScore showed significant differences in clinical prognosis, expression profile of immune checkpoints, abundance of immune cells, and immunotherapy response of ccRCC patients. Besides, the nomogram incorporating the GMRScore and clinical features showed strong predictive performance in prognosis of ccRCC patients. ALDH18A1, one of the GRMGs, exhibited elevated expression level in ccRCC and was related to markedly poorer prognosis in the integrated cohort, validated by proteomic profiling of 232 ccRCC samples from Fudan University Shanghai Cancer Center (FUSCC). Conducting western blotting, CCK-8, transwell, and flow cytometry assays, we found the knockdown of ALDH18A1 in ccRCC significantly promoted apoptosis and inhibited proliferation, invasion, and epithelial-mesenchymal transition (EMT) in two human ccRCC cell lines (786-O and 769-P). In conclusion, we developed a glutamine metabolism-related prognostic signature in ccRCC, which is tightly linked to the tumor immune microenvironment and immunotherapy response, potentially facilitating precision therapy for ccRCC patients. Additionally, this study revealed the key role of ALDH18A1 in promoting ccRCC progression for the first time.

AUY922 improves sensitivity to sunitinib in clear cell renal cell carcinoma based on network pharmacology and in vitro experiments

Clear Cell Renal Cell Carcinoma (ccRCC), the most prevalent form of renal cell carcinoma (RCC), poses a significant threat to human health due to its rising morbidity and mortality rates. Sunitinib, a pivotal targeted drug for the treatment of ccRCC, presents a significant challenge due to the high susceptibility of ccRCC to resistance. HSP90 inhibitor AUY922 has demonstrated anti-tumor activity in a range of cancer types. However, its efficacy in combination with sunitinib for ccRCC treatment has not been evaluated. In this study, we employed bioinformatics, network pharmacology, and in vitro assays to verify that AUY922 inhibits cell viability, proliferation, and migration of ccRCC cell lines 786-O and ACHN, with IC50s of 91.86 μM for 786-O and 115.5 μM for ACHN. The effect of AUY922 enhancing the inhibitory effect of sunitinib on ccRCC was further confirmed. The CCK-8 assay demonstrated that the IC50 of sunitinib was reduced from 15.10 μM to 11.91 μM for 786-O and from 17.65 μM to 13.66 μM for ACHN, after the combined application of AUY922. The EdU assay and wound healing assay indicated that AUY922 augmented the inhibitory impact of sunitinib on the proliferation and migration of ccRCC cells. Western blot and RT-PCR analyses demonstrated that AUY922 increased the sensitivity of ccRCC cells to sunitinib by targeting the HIF-1α/VEGFA/VEGFR pathway. Our study represents the first investigation into the role and mechanism of AUY922 in enhancing the sensitivity of ccRCC to sunitinib. In conclusion, the findings indicate the potential for AUY922 to enhance the therapeutic efficacy of sunitinib and overcome sunitinib resistance in ccRCC.

MicroRNA-155-5p Targets JADE-1, Promoting Proliferation, Migration, and Invasion in Clear Cell Renal Cell Carcinoma Cells.

Clear cell renal cell carcinoma (ccRCC) incidence has been rising in recent years, with strong association between differential microRNA (miRNA) expression and neoplastic progression. Specifically, overexpression of miR-155-5p has been associated with promoting aggressive cancer in ccRCC and other cancers. In this study, we further investigate the role of this miRNA and one of its protein targets, Jade-1, to better understand the mechanism behind aggressive forms of ccRCC. Jade-1, a tumor suppressor, is stabilized by Von-Hippel Lindau (VHL), which is frequently mutated in ccRCC. Experiments featuring downregulation of miR-155-5p in two ccRCC cell lines (786-O and Caki-1) attenuated their oncogenic potential and led to increased levels of Jade-1. Conversely, knockdown experiments with an anti-Jade-1 shRNA in 786-O and Caki-1 cells showed increased metastatic potential through elevated proliferation, migration, and invasion rates. In a mouse xenograft model, downregulation of miR-155 decreased the rate of tumor implantation and proliferation. Direct interaction between miR-155-5p and Jade-1 was confirmed through a 3'UTR luciferase reporter assay. These findings further elucidate the mechanism of action of miR-155-5p in driving an aggressive phenotype in ccRCC through its role in regulating Jade-1.

MP04-12 PHARMACOLOGICAL INHIBITION OF THE MOLECULAR CHAPERONE HSP70 OVERCOMES BELZUTIFAN RESISTANCE IN CCRCC

You have accessJournal of UrologyCME1 Apr 2023MP04-12 PHARMACOLOGICAL INHIBITION OF THE MOLECULAR CHAPERONE HSP70 OVERCOMES BELZUTIFAN RESISTANCE IN CCRCC Michael Basin, Sarah Backe, Michael Bratslavsky, Mark Woodford, Dimitra Bourboulia, Mehdi Mollapour, and Gennady Bratslavsky Michael BasinMichael Basin More articles by this author , Sarah BackeSarah Backe More articles by this author , Michael BratslavskyMichael Bratslavsky More articles by this author , Mark WoodfordMark Woodford More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , Mehdi MollapourMehdi Mollapour More articles by this author , and Gennady BratslavskyGennady Bratslavsky More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with von-Hippel Lindau (VHL) mutations are predisposed to developing clear cell renal cell carcinoma (ccRCC) through constitutive activation of transcription factor hypoxia-inducible factor 2a (HIF2a). The HIF2a antagonist Belzutifan has recently been FDA approved for treatment VHL disease associated tumors. Results of the recent clinical trial (MK-4682) revealed that a certain proportion of VHL patients progress on therapy. Our objective here was to decipher the molecular mechanisms of Belzutifan mediated downregulation of HIF2a and therefore provide better understanding of drug resistance. The molecular chaperone Hsp70 promotes the stability of hundreds of client proteins, thereby protecting them from degradation. Since HIF2a interacts with Hsp70, we hypothesized that Hsp70 maintains HIF2a stability in ccRCC and is a potential alternative therapeutic target in cases of Belzutifan resistance. METHODS: Cell line models of VHL-null ccRCC (786-0, A498, 769-P) were treated with 2 mM Belzutifan. HIF2a expression levels were evaluated by immunoblotting. Wild-type (WT) or Belzutifan resistant (S304M, G323E) HIF2a were transiently expressed in 786-O cells prior to treatment with Belzutifan or an Hsp70 inhibitor (JG-98). HIF2a degradation was examined by immunoblot. RESULTS: Here, we show that HIF2a is an Hsp70 client in VHL null ccRCC cell line 786-O. Further, we demonstrated a time dependent decrease in HIF2a expression with Belzutifan (2 mM) as early as 2 hours after treatment in three VHL null ccRCC cell lines 786-0, A498, and 769-P cells. Pretreatment of ccRCC cells with the proteasome inhibitor bortezomib blocked Belzutifan mediated downregulation of HIF2a, indicating Belzutifan leads to proteasomal degradation of HIF2a through a VHL independent pathway. Utilizing previously characterized Belzutifan resistant HIF2a-S304M and -G323E mutants we demonstrated that treating ccRCC cells with Hsp70 inhibitor JG-98 leads to mutant-HIF2a degradation, therefore overcoming Belzutifan resistance. CONCLUSIONS: The FDA-approved HIF2a inhibitor Belzutifan causes proteasomal degradation of WT-HIF2a, but not of resistant mutants. Our data indicates that HIF2a stability is reliant on the molecular chaperone Hsp70. Further, treatment with an Hsp70 inhibitor could potentially be used in the clinic to overcome resistance to Belzutifan in patients with VHL disease and ccRCC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e37 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Basin More articles by this author Sarah Backe More articles by this author Michael Bratslavsky More articles by this author Mark Woodford More articles by this author Dimitra Bourboulia More articles by this author Mehdi Mollapour More articles by this author Gennady Bratslavsky More articles by this author Expand All Advertisement PDF downloadLoading ...

MT1X is an oncogene and indicates prognosis in ccRCC

The metallothionein 1 (MT1) family was previously shown to be involved in metal ion homeostasis, DNA damage, oxidative stress, and carcinogenesis. Our team’s previous study showed that MT1X is most closely associated with ccRCC. However, its role in clear cell RCC (ccRCC) remains unclear. The present study aimed to demonstrate MT1X’s prognostic value, potential biologic function, impact on the immune system, and influence on cell growth, the cell cycle, apoptosis, and migration in the setting of ccRCC. The relationship between clinical pathologic features and MT1X was analyzed using bioinformatics. We knocked down MT1X in the ccRCC cell line 786O with si-MT1X to verify the results of the bioinformatic analysis at the cytological level. Apoptosis assay, cell cycle assay, wound-healing assay, colony formation assay, and RT-qPCR were performed. MT1X is correlated with the stage (T and M) and grade and is able to be an independent prognostic factor for ccRCC. The TISIDB database analysis showed a significant correlation between MT1X and tumor-infiltrating lymphocytes such as central memory CD8+ T cells and γΔT cells. MT1X was also positively related to immunomodulators such as TGFB1 and CXCR4. We also found that MT1X knockdown inhibits cell growth, induces apoptosis, arrests cells in the S cell cycle, and inhibits the wound healing proportion in ccRCC. Gene set enrichment analysis and quantitative PCR (q-PCR) analysis found that down-regulation of MT1X reduced the accumulation of hypoxia-associated factors. Bioinformatic analysis associated increased MT1X expression with a worse prognosis. Laboratory experiments confirmed bioinformatic findings. MT1X was also found to be an independent prognostic biomarker for ccRCC and is involved in immune system regulation.

MicroRNA Associated with the Invasive Phenotype in Clear Cell Renal Cell Carcinoma: Let-7c-5p Inhibits Proliferation, Migration, and Invasion by Targeting Insulin-like Growth Factor 1 Receptor.

Differential microRNA (miRNA) expression can portend clear cell renal cell carcinoma (ccRCC) progression. In a previous study, we identified a subset of dysregulated miRNA in small renal masses, pT1 ccRCC (≤5 cm) that are associated with an aggressive phenotype. The present study investigated miRNA expression in clinical stage I (cT1) tumors (≤5 cm), comparing pathologic stage I (pT1) tumors to those upstaged to pathologic stage 3 (pT3) after surgery following identification of renal vein invasion or invasion into adjacent fat tissue within Gerota’s fascia. Twenty cT1 tumors were examined in an miRNA screening, 10 pT1 and 10 pT3 tumors. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of let-7c-5p and its protein target insulin-like growth factor 1 receptor (IGF1R). Cells were transfected with pre-let-7c-5p and assessed through cell proliferation, migration, and invasion assays. IGF1R expression was evaluated through Simple Western, and interaction between let-7c-5p and IGF1R was confirmed via luciferase reporter assay. Screening identified 20 miRNA, including let-7c-5p, that were dysregulated between pT1 and pT3 upstaged tumors. This miRNA was also downregulated in our previous study of pT1 tumors that progressed to metastatic disease. Transfection of ccRCC cells with pre-let-7c-5p significantly inhibited proliferation, migration, invasion, and IGF1R expression. These findings suggest that miRNA dysregulation is involved in ccRCC progression, specifically through invasion, and that let-7c-5p downregulation contributes to the aggressiveness of small ccRCC tumors, in part, through its regulation of IGF1R.

MiR-744-5p inhibits the proliferation, invasion, and migration of clear-cell renal cell carcinoma cells by targeting CCND1

To explore the role of miR-744-5p/CCND1 axis in clear-cell renal cell carcinoma (ccRCC). We examined the expression levels of miR-744-5p in 65 pairs of ccRCC and adjacent tissue specimens and in 5 ccRCC cell lines and human renal tubular epithelial (HK2) cells using qRT-PCR. The ccRCC cell lines 786-O and OSRC2 were transfected with miR-744-5p mimic, CCND1 mimic, or their negative control mimics, and the changes in cell proliferation, migration, and invasion were evaluated with CCK-8, wound healing, and Transwell assays. The downstream target molecules of miR-744-5p were predicted by bioinformatics analysis, and the expression level of CCND1 in ccRCC cells was verified by qRT-PCR and Western blotting. The relationship between miR-744-5p and CCND1 was further validated by dual luciferase reporter assay, and the role of the miR-744-5p/CCND1 axis in ccRCC was explored by rescue experiments. MiR-744-5p was significantly downregulated in ccRCC tissues and cell lines (all P < 0.05), and its overexpression inhibited the proliferation, migration, and invasion of ccRCC cells (all P < 0.05). Bioinformatics analysis and dual luciferase reporter assay showed that CCND1 was a downstream target of miR-744-5p. The results of rescue experiments showed that upregulation of CCND1 could partially reverse the inhibitory effect of miR-744-5p overexpression on ccRCC cell proliferation, migration, and invasion (all P < 0.05). MiR-744-5p inhibits the malignant phenotype of ccRCC cells by targeting CCND1, and the miR-744-5p/CCND1 axis may be a novel target for diagnosis and treatment of ccRCC.

Assembly and localization of extracellular matrix protein fibronectin modulates Natural killer cell infiltration in tumor spheroids.

Abstract Natural Killer cells (NK), the major innate effector cells, with their broad cytotoxicity against tumors are ideal candidates for immunotherapy. Once having entered the malignant site, NK cells encounter a complex environment composed of tumor cells, non-tumor cells along with the extra cellular matrix (ECM). As one of the integral components of the tumor microenvironment (TME), the normal regulation of the extra cellular matrix (ECM) is necessary to prevent tumor progression and metastasis. Nevertheless, the impact of the matrix architecture found in solid tumors on immune cells and especially NK cells is not well characterized. Here we investigated the role of Fibronectin, a fibrous ECM protein, and its assembly and localization on NK cell infiltration into clear cell renal cell carcinoma (ccRCC) spheroids. 3D tumor spheroids established from the ccRCC cell line 786-O (RCC-) that lacks the Von Hippel-Lindau (VHL) gene are incapable of fibronectin assembly in their ECM. However, addition of the VHL gene into the 786-O cells (RCC+) reversed this phenotype facilitating fibronectin assembly. Fibronectin in RCC-spheroids exhibited prominent peripheral localization in contrast to RCC+ spheroids where fibronectin was detected towards the center of the spheroid. Coincident with fibronectin localization, PKH26 labeled Natural killer cell line, NKL, showed significantly increased infiltration into RCC+ spheroids compared to RCC-spheroids that lacked the tumor suppressor VHL. Thus, assembly and localization of fibronectin modulates NK cell infiltration in RCC tumors. Supported by grants from NIH U54MD012388 (NR and AV) and NIH U54CA143925 (NACP-NAU) to NR.

MiRNA-424-5p Suppresses Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma and Attenuates Expression of O-GlcNAc-Transferase.

Simple SummaryThe identification of biomarkers that predict the metastatic potential of tumors is a current area of interest in cancer research. A previous study from our laboratory identified numerous microRNA (miRNA) biomarkers that are differentially expressed in pathologic stage I clear cell renal cell carcinoma (ccRCC) tumors that progress to metastatic disease. This study investigated the role of aberrant expression of one of these miRNA, miR-424-5p, and one of its associated protein targets, O-GlcNAc-transferase (OGT). We examined the influence of miR-424-5p and OGT expression on the proliferation, migration, and invasion of ccRCC cells, and confirmed the direct interaction between miR-424-5p and OGT. These findings suggest that the decrease in miR-424-5p expression observed in these small renal masses leads to an increase in OGT, which facilitates metastasis.MicroRNAs (miRNAs) are non-coding post-transcriptional regulators of gene expression that are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in tumor progression. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors, including miR-424-5p, that are associated with an aggressive phenotype. We investigate the impact of this dysregulated miRNA and its protein target O-GlcNAc-transferase (OGT) to better understand the mechanisms behind aggressive stage I ccRCC. The ccRCC cell lines 786-O and Caki-1 were used to assess the impact of miR-424-5p and OGT. Cells were transfected with pre-miR-424-5p, a lentiviral anti-OGT shRNA, or were treated with the demethylating agent 5-Aza-2′-deoxycytidine. Cell proliferation was measured via MT cell viability assay. Cell migration and invasion were analyzed using Transwell assays. The expression of miR-424-5p was determined through qRT-PCR, while OGT protein expression was evaluated through Western blotting. The interaction between miR-424-5p and OGT was confirmed via luciferase reporter assay. The transfection of ccRCC cells with pre-miR-424-5p or anti-OGT shRNA significantly inhibited cell proliferation, migration, and OGT expression, while miR-424-5p also attenuated cell invasion. Addition of the demethylating agent significantly reduced cell proliferation, migration, invasion, and OGT expression, while significantly increasing the expression of miR-424-5p. Altogether, these findings suggest that epigenetic downregulation of miR-424-5p, which in turn augments OGT expression, contributes to the creation of aggressive forms of stage I ccRCC.

MicroRNAs MiR-15a and MiR-26a cooperatively regulate O-GlcNAc-transferase to control proliferation in clear cell renal cell carcinoma.

MicroRNAs (miRNAs), a group of non-coding post-transcriptional regulators of gene expression, are dysregulated in clear cell renal cell carcinoma (ccRCC) and play an important role in carcinogenesis. Our prior work identified a subset of miRNAs in pT1 ccRCC tumors associated with progression to metastatic disease. To investigate the impact of two of these dysregulated miRNA, miR-15a-5p and -26a-5p, in an effort to elucidate the mechanisms underpinning aggressive forms of stage I ccRCC. The ccRCC cell line 786-O was transfected with pre-miRs-15a-5p and -26a-5p to rescue expression. Cell proliferation was measured via MT Cell Viability Assay. O-GlcNAc-transferase (OGT), a known protein in ccRCC proliferation, was identified by bioinformatics analysis as a target of both miRNA and validated via luciferase reporter assay to confirm binding of each miR to the 3' untranslated region (UTR). OGT protein expression was evaluated via western blotting. Luciferase assay confirmed specificity of miR-15a-5p and -26a-5p for the OGT UTR. Western blot analysis for OGT showed reduced expression following co-transfection of both miRNAs compared to negative control or individual transfection. Co-transfection of these miRNAs greatly reduced proliferation when compared to negative control or the individual transfections. Our results indicate that the dysregulation of miR-15a-5p and -26a-5p contribute cooperatively to the proliferation of ccRCC through their regulation of OGT. These results give insight into the pathogenesis of aggressive early stage ccRCC and suggest potential therapeutic targets for future research.

Celastrol induces lipophagy via the LXRα/ABCA1 pathway in clear cell renal cell carcinoma.

Celastrol is a triterpene derived from the traditional Chinese medicine Tripterygium wilfordii Hook f, which displays potential anticancer activity. In the present study, we investigated the anticancer effects of celastrol against clear cell renal cell carcinoma (ccRCC) and the underlying mechanisms. Using Cancer Genome Atlas (TCGA) database and genotype-tissue expression (GTEx) database we conducted a bioinformatics analysis, which showed that the mRNA levels of liver-X receptors α (LXRα) and ATP-binding cassette transporter A1 (ABCA1) in ccRCC tissues were significantly lower than those in adjacent normal tissues. This result was confirmed by immunoblotting analysis of 4 ccRCC clinical specimens, which showed that the protein expression of LXRα and ABCA1 was downregulated. Similar results were obtained in a panel of ccRCC cell lines (786-O, A498, SN12C, and OS-RC-2). In 786-O and SN12C cells, treatment with celastrol (0.25-2.0 μM) concentration-dependently inhibited the cell proliferation, migration, and invasion as well as the epithelial-mesenchymal transition (EMT) process. Furthermore, we demonstrated that celastrol inhibited the invasion of 786-O cells through reducing lipid accumulation; celastrol concentration-dependently promoted autophagy to reduce lipid storage. Moreover, we revealed that celastrol dramatically activated LXRα signaling, and degraded lipid droplets by inducing lipophagy in 786-O cells. Finally, celastrol promoted cholesterol efflux from 786-O cells via ABCA1. In high-fat diet-promoted ccRCC cell line 786-O xenograft model, administration of celastrol (0.25, 0.5, 1.0 mg·kg-1·d-1, for 4 weeks, i.p.) dose-dependently inhibited the tumor growth with upregulated LXRα and ABCA1 protein in tumor tissue. In conclusion, this study reveals that celastrol triggers lipophagy in ccRCC by activating LXRα, promotes ABCA1-mediated cholesterol efflux, suppresses EMT progress, and ultimately inhibits cell proliferation, migration, and invasion as well as tumor growth. Thus, our study provides evidence that celastrol can be used as a lipid metabolism-based anticancer therapeutic approach.

Mass Spectrometry-Based Metabolic Fingerprinting Contributes to Unveil the Role of RSUME in Renal Cell Carcinoma Cell Metabolism.

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with 50-80% patients exhibiting mutations in the von Hippel-Lindau (VHL) gene. RSUME (RWD domain (termed after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases)-containing protein small ubiquitin-related modifier (SUMO) enhancer) acts as a negative regulator of VHL function in normoxia. A discovery-based metabolomics approach was developed by means of ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (MS) for fingerprinting the endometabolome of a human ccRCC cell line 786-O and three other transformed cell systems (n = 102) with different expressions of RSUME and VHL. Cross-validated orthogonal projection to latent structures discriminant analysis models were built on positive, negative, and a combination of positive- and negative-ion mode MS data sets. Discriminant feature panels selected by an iterative multivariate classification allowed differentiating cells with different expressions of RSUME and VHL. Fifteen identified discriminant metabolites with level 1, including glutathione, butyrylcarnitine, and acetylcarnitine, contributed to understand the role of RSUME in ccRCC. Altered pathways associated with the RSUME expression were validated by biological and bioinformatics analyses. Combined results showed that in the absence of VHL, RSUME is involved in the downregulation of the antioxidant defense system, whereas in the presence of VHL, it acts in rerouting energy-related pathways, negatively modulating the lipid utilization, and positively modulating the fatty acid synthesis, which may promote deposition in droplets.

MicroRNA-133a-5p inhibiting metastatic capacity of renal clear cell carcinoma through regulating MON2.

We aimed at analyzing the correlation between microRNA-133a-5p expression and clinical pathological parameters in patients with clear cell renal cell carcinoma (ccRCC) and exploring the mechanism by which microRNA-133a-5p affects the biological behavior of ccRCC cells. MicroRNA-133a-5p expression in ccRCC tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the relationship between ATG14 expression and clinicopathological parameters of ccRCC patients was analyzed. A control group (NC mimic) and a microRNA-133a-5p overexpression group (microRNA-133a-5p mimic) were set in the ccRCC cell lines ACHN and 786-O, respectively. The impacts of microRNA-133a-5p on the proliferation and invasion of ccRCC cells were evaluated through performing Cell Counting Kit-8 (CCK-8) and transwell tests, respectively. We further explored the interaction between microRNA-133a-5p and its downstream target gene WNK2 by bioinformatics analysis and Luciferase assay. Both in ccRCC tissues and cell lines, microRNA-133a-5p showed a significantly reduced expression, which could be used to predict poor prognosis of ccRCC patients. Upregulation of microRNA-133a-5p markedly blunted the proliferation and migratory capacities of HCC cells. Bioinformatics analysis suggested that microRNA-133a-5p can target MON2. In addition, qPCR assay indicated an increased expression of MON2 in ccRCC cell lines and tissues, which was negatively correlated with microRNA-133a-5p. Finally, in vitro cell reverse experiments suggested that overexpression of MON2 counteracted the inhibitory effects of overexpression of microRNA-133a-5p on the proliferation and metastatic capacity of ccRCC. This study suggests that the reduced expression of microRNA-133a-5p in ccRCC tissue specimens can predict poor prognosis of ccRCC patients. At the same time, microRNA-133a-5p may suppress the proliferation capacity and metastasis of ccRCC cells by acting on MON2.

Esculetin inhibits proliferation, migration, and invasion of clear cell renal cell carcinoma cells

BackgroundEsculetin, the main active ingredient in the Chinese herbal medicineCortex Fraxini, has been shown to possess antitumor activity. However, the effect of esculetin on clear cell renal cell carcinoma (ccRCC) has not been investigated. MethodsMTS assays and colony formation assays were used to study the cytotoxicity of esculetin. The effects of esculetin on cell cycle and apoptosis were analyzed by flow cytometry. Western blot was used to detect cell cycle-, apoptosis-, and EMT-related proteins. Wound-healing assays and transwell assays were performed to study the effect of esculetin on cell migration and invasion in the ccRCC cell lines 786-O and SN12-PM6. ResultsEsculetin exerted cytotoxic activities in 786-O and SN12-PM6 cells in a dose- and time-dependent manner. The compound arrested the cell cycle in G0/G1 and G2/M phase with down-regulation of Cyclin D1, CDK4, CDK6, and c-Myc expression. Esculetin also induced apoptosis and the expression of cleaved caspase 3 increased. Additionally, esculetin significantly inhibited 786-O and SN12-PM6 cell migration and invasion, the expression of E-Cadherin increased, and the expression of N-cadherin and vimentin decreased. ConclusionTaken together, these results suggest that esculetin inhibits proliferation, migration, and invasion of ccRCC and is a potential novel therapeutic agent for the treatment of ccRCC.

Blocking the autocrine regulatory loop of Gankyrin/STAT3/CCL24/CCR3 impairs the progression and pazopanib resistance of clear cell renal cell carcinoma

The poor prognosis of clear-cell renal cell carcinoma (ccRCC) patients is due to progression and targeted drug resistance, but the underlying molecular mechanisms need further elucidation. This study examined the biological function and related mechanisms of gankyrin in ccRCC based on the results of our previous study. To this end, in vitro functional experiments; in vivo models of subcutaneous tumor formation, lung metastasis, and orthotopic ccRCC; and antibody chip detection, co-IP, ChIP assays were performed to examine the biological role and molecular mechanisms of gankyrin in ccRCC. Two hundred fifty-six ccRCC patients were randomly divided into training and validation cohorts to examine the prognostic value of gankyrin and other markers through IHC and statistical analyses. We observed that the gankyrin-overexpressing ccRCC cell lines 786-O and 769-P exhibited increased proliferation, invasion, migration, tumorigenicity, and pazopanib resistance and decreased apoptosis, while gankyrin knockdown achieved the opposite results. Mechanistically, gankyrin recruited STAT3 via direct binding, and STAT3 binding to the CCL24 promoter promoted its expression. Reciprocally, an increase in autocrine CCL24 enhanced the expression of gankyrin and STAT3 activation via CCR3 in ccRCC, forming a positive autocrine-regulatory loop. Furthermore, in vivo experimental results revealed that blocking the positive loop through gankyrin knockdown or treatment with the CCR3 inhibitor SB328437 reversed the resistance to pazopanib and inhibited lung metastasis in ccRCC. Moreover, a positive correlation between gankyrin and STAT3 or CCL24 expression in ccRCC specimens was observed, and improved accuracy for ccRCC patient prognosis was achieved by combining gankyrin and STAT3 or CCL24 expression with existing clinical prognostic indicators, including the TNM stage and SSIGN score. In summary, targeting the gankyrin/STAT3/CCL24/CCR3 autocrine-regulatory loop may serve as a remedy for patients with advanced ccRCC, and combining gankyrin and STAT3 or CCL24 expression with the current clinical indicators better predicts ccRCC patient prognosis.

Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 expression and its contribution to the regulation of biological function of human clear cell renal cell carcinoma cell line

Objective To investigate the clinical significance and biological function of human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) in human clear cell renal cell carcinoma (ccRCC). Methods Down-regulation of HHLA2 was performed by using RNA interference (RNAi) method to investigate the role of HHLA2 in regulation of biological behaviors in human clear cell renal cell carcinoma cell lines 786-O and ACHN. The cell counting kit-8 (CCK-8) assay, wound healing assay, and transwell invasion assay were used to examine the cellular function after HHLA2 knockdown of these cells. We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis. Results To investigate the functions of HHLA2 in human ccRCC, we successfully constructed HHLA2 knockdown expression in human ccRCC cell lines by using the RNAi method. CCK-8 assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell proliferation. The wound healing assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell migration (LV-HHLA2-sh1: 786-O: F=99.340, P<0.01, ACHN: F=113.500, P<0.01; LV-HHLA2-sh2: 786-O: F=35.320, P<0.01, ACHN: F=26.470, P<0.01). Transwell invasion assay showed that decreased HHLA2 expression in ccRCC cell lines 786-O and ACHN significantly attenuated cell invasion. The cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, according to the microarray data, the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly changed after knockdown of HHLA2 expression. After knockdown of HHLA2 in 786-O and ACHN, the expression of E-cadherin was significantly increased, meanwhile the expression of N-cadherin and Vimentin were significantly decreased. Conclusion Our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy. Key words: Human endogenous retrovirus subfamily H long terminal repeat associating protein 2; Human clear cell renal cell carcinoma; RNA interference

Abstract B087: PARK2 expression reduces migration and invasion and deregulates metabolic pathways in ccRCC

Abstract PARK2 is an E3-ubiquitin-ligase, which plays an important role in mitophagy, a macroautophagy process that selectively degrades defective mitochondria. The significant downregulation of mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) were shown to be associated with aggressive disease and poor clinical outcome (Toma et al., 2013). The aim of our study was to investigate the role of of PARK2 in ccRCC. First, we transduced the ccRCC cell line 786-O with a PLVX-EF1a-PARK2-IRES-mcherry construct to overexpress PARK2. Subsequently, we performed functional assays, analyzed the mitochondria and mitochondrial respiration, and RNA-Sequencing. While apoptosis, cell cycle and cell proliferation remained unaffected, we found a marked reduction of migration and invasion in PARK2 overexpressing cells compared to wt controls. PARK2 is involved in the degradation of damaged mitochondria during mitophagy, therefore, we investigated the amount of active and total mitochondria and found more active mitochondria in the PARK2 clones. The overall amount of mitochondria was unchanged. To verify the results and get a further insight into the functionality of the mitochondria mitochondrial respiration was analyzed. As expected, the mitochondrial respiration of the PARK2 clones was upregulated. In addition, we observed a general shift towards metabolic upregulation in 786-O cells expressing PARK2. To find specific pathways affected by the expression of PARK2, we performed RNA Sequencing. Several metabolic pathways were deregulated in the cells expressing PARK2 compared to wt controls. We were able to identify several genes being downregulated in transgenic wild type cells that play a role in the epithelial-to-mesenchymal transition underlining our phenotypic findings of invasion and migration. In conclusion, PARK2 has an impact on the aggressiveness of the disease, the health of the mitochondria and effects a deregulation of metabolic pathways. Lately, several groups reported on the connection between migration/invasion and metabolic changes (Kenny et al., 2019; Yizhak et al., 2014; Gaude and Frezza; 2016, Yao et al., 2018) suggesting a potential link between these two events. Our study adds additional proof towards that link, nevertheless, further research needs to be conducted to identify if these two events are indeed interconnected and dependent on each other. Literature Gaude E and Frezza C. Tissue-specific and convergent metabolic transformation of cancer correlates with metastatic potential and patient survival. Nat Commun. 7:13041. 2016. Kenny TC et al. Mitohormesis Primes Tumor Invasion and Metastasis. Cell Rep. 27(8):2292-2303. 2019. Toma MI et al. PARK2 and PACRG are commonly downregulated in clear-cell renal cell carcinoma and are associated with aggressive disease and poor clinical outcome. Genes Chromosomes Cancer. 52(3):265-73. 2013. Yao L, Guo X, Gui Y. Acetyl-CoA Synthetase 2 Promotes Cell Migration and Invasion of Renal Cell Carcinoma by Upregulating Lysosomal Associated Membrane Protein 1 Expression. Cell Physiol Biochem. 45(3):984-992. 2018. Yizhak K et al. A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration. Mol Syst Biol. 10:744. 2014. Citation Format: Laura K Eßer, Lena Arévalo, Glen Kristiansen, Hubert Schorle, Marieta I Toma. PARK2 expression reduces migration and invasion and deregulates metabolic pathways in ccRCC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B087. doi:10.1158/1535-7163.TARG-19-B087

Effect of EH domain containing protein 2 on the biological behavior of clear cell renal cell carcinoma.

To investigate the effects of EH domain containing protein 2 (EHD2) on clear cell renal cell carcinoma (ccRCC) and provide new insights for the clinical treatment of rental cancer. Forty patients (26 males and 14 females, 62.4 ± 5.7 years old) with ccRCC were selected from January 2015 to December 2016 to serve as research subjects in this study. The EHD2 protein expression in the tumor tissues and adjacent healthy tissues of ccRCC patients were detected by Western Blot assay. The cells of ccRCC cell lines RLC-310 and 786-O were divided into normal control group (control), no-load control group (pLV), EHD2 overexpression group (pLV-EHD2), and EHD2 interference group (pLV-siEHD2). The expression levels of EHD2 protein in each group of cells were detected by western blot. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Wound healing assay was performed to check the cell migration ability. Transwell invasion assay was used to detect the cell invasion ability. Cell apoptosis was detected by flow cytometry. The expression level of EHD2 was significantly increased in pLV-EHD2 group and decreased in pLV-siEHD2 group compared with control group and pLV-siEHD2 group, indicating the successfully established EHD2 overexpression cell line and EHD2 RNA interference cell line. EHD2 overexpression enhanced the proliferation, invasion, and migration but inhibited the apoptosis of ccRCC cells, while EHD2 interference showed opposite functions. EHD2 interference can inhibit the development of ccRCC by inhibiting the proliferation, invasion, and migration, and EHD2 can potentially serve as a molecular target for the clinical treatment of ccRCC.

Overexpression of HHLA2 in human clear cell renal cell carcinoma is significantly associated with poor survival of the patients

BackgroundIt is well known that human clear cell renal cell carcinoma (ccRCC) is a highly immunogenic and chemo-resistant tumor. Recently, emerging data suggest that the immune checkpoint blockade therapy is an important breakthrough in the treatment against ccRCC. HHLA2, a recently reported member of B7 family, is uniquely expressed in humans but not in mice, and it plays an important role in the functional inhibition of CD4 and CD8 T cells. Herein, we aimed to study the clinical implications of HHLA2 expression in human ccRCC and its potential regulatory role in the biological functions of the cancer cells.MethodsIn the present study, we examined HHLA2 expression in human ccRCC tissues and analyzed the clinical implications as well as prognostic value. The intervention of HHLA2 in human ccRCC cell lines ACHN and 786-O was performed and its effect on the cellular function of the cells was also analyzed. We also identified the differentially expressed genes upon HHLA2 knockdown in ccRCC cell lines by using gene microarray analysis.ResultsWe found that higher HHLA2 mRNA expression level in human ccRCC tissues compared with that in adjacent normal tissues based on TCGA data, and the HHLA2 expression at mRNA level was positively and significantly correlated with PD-L1, PD-L2, B7-H6, but negatively and significantly correlated with B7-H3. Moreover, our immunohistochemistry study showed that the staining intensity of HHLA2 in human ccRCC tissues was significantly higher than that in the adjacent normal tissues, and the overall survival rate of ccRCC patients with higher HHLA2 expression was significantly poorer than that of the patients with lower HHLA2 expression. Higher expression of HHLA2 in ccRCC tissues was positively and significantly associated with larger tumor size and advanced TNM stage. The COX model revealed that the parameters including patient’s age, TNM stage and HHLA2 expression level could be used as the independent risk factors respectively for the prognostic prediction of the patients. Our cellular study showed that upon knockdown of HHLA2 expression in human ccRCC cell lines, the cell viability, the migration and the invasion ability were significantly inhibited, while the cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, according to the microarray data, the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly changed after knockdown of HHLA2 expression.ConclusionsOur findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy.