Human Renal Cell Carcinoma Tissues Research Articles

Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

AURKA promotes renal cell carcinoma progression via regulation of CCNB1 transcription

AURKA is a member of the serine/threonine kinase family and its kinase activity is crucial for the progression of mitosis. Recent studies have highlighted the therapeutic significance of AURKA inhibition in multiple cancer types. However, the specific mechanisms by which AURKA contributes to the progression of renal cell carcinoma (RCC) have not been fully elucidated. In this study, AURKA expression level was identified in human RCC tissues by immunohistochemical (IHC) staining. The function of AURKA on cell malignant phenotypes was evaluated in vitro after AURKA inhibition. The subcutaneous xenograft was conducted to confirm the in vivo effect of AURKA knockdown on growth of RCC cells. Finally, Co-IP, luciferase assay and ChIP experiments were performed to reveal the regulatory mechanism of AURKA on CCNB1. Our results showed a significant upregulation of AURKA in RCC tissues and cell lines, and a high AURKA expression was associated with poor prognosis. AURKA knockdown inhibited RCC cell proliferation and migration, induced cell apoptosis, and led to G1/G2 phase arrest. This effect was further confirmed by the use of an AURKA inhibitor. Mechanistically, AURKA interacted with E2F1, and subsequently recruited it to the promoter region of CCNB1. CCNB1 expression was essential for AURKA-induced RCC progression. Collectively, our results suggested that AURKA plays an important role in development of RCC via regulating CCNB1 transcription.

Expression and Prognostic Significance of Ferroptosis-related Proteins SLC7A11 and GPX4 in Renal Cell Carcinoma.

The ferroptosis inhibitory gene solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) inhibit ferroptosis in carcinoma cells. However, whether SLC7A11 and GPX4 serve as an oncogene in renal cell carcinoma (RCC) remains unclear. Immunohistochemistry (IHC) assays were performed to assess the expression of SLC7A11 and GPX4 in human RCC tissues. Clinical-pathological analysis was performed to explore the correlation between SLC7A11 and GPX4 expression. Kaplan-Meier survival analysis was performed to characterise the associations between protein expression and patient progressionfree survival (PFS). The upregulation of SLC7A11 and GPX4 was detected by IHC in RCC tissues compared with that in normal renal tissues. Meanwhile, the expression level of SLC7A11 and GPX4 was correlated with tumour diameter and distant metastasis (P<0.05). Kaplan-Meier survival analysis indicated that patients with high SLC7A11 and GPX4 expression levels exhibited worse PFS than those with low SLC7A11 and GPX4 expression levels (P<0.05). The upregulation of SLC7A11 and GPX4 expression was associated with poor prognosis in patients with RCC. SLC7A11 and GPX4 may serve as diagnostic and prognostic biomarkers for patients with RCC.

Abstract 672: A genetic defect in phenylalanine hydroxylase (PAH) affects the immune system in kidney

Abstract PAH, one of the genes associated with inborn error of metabolism (IEM), codes for phenylalanine hydroxylase and is specifically expressed in the liver and kidney. When PAH is dysfunctional, phenylalanine (Phe) cannot be converted to tyrosine (Tyr), and it produces toxic metabolites such as phenylpyruvate by a metabolic shift. We previously analyzed germline variants of IEM-related genes using public cancer multi-omics data. As a result, there was a high frequency of mutations in the PAH gene, and germline variants were particularly high in renal cell carcinoma (RCC). Based on this data, we suggest that kidney-specific expressed PAH mutations could change the kidney tissue microenvironment, which makes them more vulnerable to chronic kidney disease (CKD) and affects cancer development. We used the PAH enu2 mice model, which has a missense mutation in the PAH gene. PAH homozygous mutant mice display phenylketonuria (PKU) when not maintained on a Phe-free diet. PAH enu2 mice were maintained a normal diet. The concentration of phenylalanine in the mice's serum was measured each week. Then, the proportions of immune cells were analyzed in peripheral blood and kidney. Next, Jurkat cells, which are well-known as human T-cell lines, were applied to the in vitro experiment. Jurkat cells were cultured in a medium containing Phe or PPA and were activated by PHA/PMA or anti-CD3/CD28. Then after, the expression of immune-related markers was evaluated. Additionally, the expression of the PAH enzyme in human RCC and normal tissue was assessed by immunohistochemistry (IHC). The concentration of Phe in mice serum was higher when the PAH gene was defective; homozygous mutant, heterozygous mutant. As PAH-defective mice got older, the T cell population decreased in the blood and kidney immune cells. When Jurkat T cells were cultured with Phe or PPA, the expression of IL-2, a cytokine related to T cell expansion, decreased in the PPA-treated and activated group. There were more significant changes when treated with PPA rather than Phe. IHC data shows the expression of PAH was significantly decreased in RCC tissues compared to non-tumor tissue. These results suggest that the immune system could be changed by PAH dysfunction bearing a germline mutation. Moreover, the kidney tissue microenvironment with PAH defects is vulnerable to the development of kidney disease, which can lead to RCC. This is a novel discovery that can explain the importance of the role PAH gene in kidney disease. Citation Format: Seojeong Kim, Chaelin Kang, Jihyun Park, Seung Seok Han, Youngil Koh, Sung-Soo Yoon. A genetic defect in phenylalanine hydroxylase (PAH) affects the immune system in kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 672.

Effect of AKT silence on malignant biological behavior of renal cell carcinoma cells

BackgroundAs the most common malignant tumor of primary renal tumor, renal cell carcinoma (RCC) is the highly invasive disease with high mortality. AKT is a serine/threonine kinase that play a critical role in the phosphoinositide 3-kinase (PI3K) signaling pathway, and it is an attractive target for RCC treatment. The aim of present study was to investigate the effect of AKT silence on malignant behavior of renal cell carcinoma cells.MethodsAKT expression was quantified by immunohistochemistry in tumor tissues and normal tissues. The human RCC cell lines Caki-2 cell were chosen for this study. The optimal silencing siRNA was subsequently selected by RT-qPCR and western blot. The effect of AKT silence on RCC cells was investigated by CCK8 assay, transwell assay, scratch test and flow cytometry. The AKT1 expression in human renal cell carcinoma tissue was detected by immunohistochemical staining.ResultsThe AKT in Caki-2 cells was silenced successfully. The results shown AKT silence could inhibit cell proliferation, invasion, and, migration. In addition, AKT silence could promote Caki-2 cell apoptosis with prevention of RCC cells move from G1 phase to S phase. Immunohistochemical staining revealed significant difference of expression of AKT1 in RCC tissues and normal renal tissues. Taken together, AKT family members might involve in malignant growth of RCC, and might be a potential therapeutic target.ConclusionOur data show that AKT silence inhibited cell proliferation, invasion, and, migration of Caki-2 cell, and promoted Caki-2 cell apoptosis. Moreover, AKT silence prevented RCC cells move from G1 phase to S phase. Therefore, AKT may act as an effective therapeutic target for RCC.

The atypical sphingosine 1-phosphate variant, d16:1 S1P, mediates CTGF induction via S1P2 activation in renal cell carcinoma.

Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term 'S1P' mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.

MicroRNA-501-3p inhibits the proliferation of kidney cancer cells by targeting WTAP.

BackgroundEmerging evidence suggests that miR‐501‐3p plays an important role in the pathogenesis and progression of various carcinomas. However, its role and underlying mechanisms in renal cell carcinoma (RCC) remain to be elucidated.MethodsQuantitative RT‐PCR, western blot, and bioinformatics methods were used to evaluate the expression of miR‐501‐3p and Wilms’ tumor 1‐associating protein (WTAP) in RCC cell lines and clinical tissues. The effects of miR‐501‐3p on the proliferation of RCC cells were investigated using flow cytometric, colony formation, and CCK8 assays. The target gene of miR‐501‐3p was confirmed by western blotting, qRT‐PCR, and dual‐luciferase reporter assays. The levels of RNA methylation with N6‐methyladenosine (m6A) following miR‐501‐3p overexpression or knockdown of its target gene were quantified using a dot‐blot assay.ResultsmiR‐501‐3p expression was significantly downregulated in human RCC cell lines and tissues. In contrast, its overexpression markedly inhibited cancer cell proliferation in vitro by inducing G1 phase arrest. Moreover, WTAP was verified as a direct target gene of miR‐501‐3p. WTAP gene knockdown alone efficiently produced the same cancer‐inhibiting effects as miR‐501‐3p overexpression, with the level of m6A in RCC cells being decreased under both scenarios. The intermolecular interaction between miR‐501‐3p and WTAP was further substantiated by rescue experiments.ConclusionRCC progression is regulated via the miR‐501‐3p/WTAP/CDK2 axis and is inhibited by the overexpression of miR‐501‐3p.

SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

Renal cell carcinoma (RCC) cells have increased lipogenesis and cholesterol synthesis. Sterol regulatory element-binding protein-1 (SREBP1) is cleaved by site 1 protease (S1P) to release the transcriptionally active amino-terminal domain. PF-429242 is a potent and competitive S1P inhibitor. We here tested its activity in RCC cells. In established and primary human RCC cells, PF-429242 potently inhibited cell proliferation, migration, and invasion. The S1P inhibitor provoked apoptosis activation in RCC cells. Furthermore, shRNA-mediated S1P silencing or CRISPR/Cas9-induced S1P knockout led to RCC cell growth inhibition and apoptosis activation. Conversely, ectopic overexpression of SREBP1 or S1P augmented RCC cell proliferation and migration. Daily i.v. injection of a single dose of PF-429242 robustly inhibited RCC xenograft growth in severe combined immunodeficiency mice. Additionally, intratumoral injection of S1P shRNA lentivirus inhibited RCC xenograft growth in mice. SREBP1, S1P, and its target gene low density lipoprotein receptor (LDLR) were significantly elevated in human RCC tissues. These results suggest that targeting S1P by PF-429242 inhibited RCC cell growth in vitro and in vivo.

Endogenous Retroviral-K Envelope Is a Novel Tumor Antigen and Prognostic Indicator of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is one of the ten most common cancers for men and women with an approximate 75% overall 5-year survival. Sixteen histological tumor subtypes exist and the most common are papillary, chromophobe and clear cell renal cell carcinoma (ccRCC) representing 85% of all RCC. Although epigenetically silenced, endogenous retroviral (ERV) genes become activated in tumors and function to ignite immune responses. Research has intensified to understand ERV protein function and their role as tumor antigens and targets for cancer (immune) therapy. ERV-K env is overexpressed and implicated as a therapeutic target for breast cancer, however studies in RCC are limited. In this investigation a human RCC tissue microarray (TMA) (n=374) predominantly consisting of the most common histological tumor subtypes was hybridized with an ERV-K env antibody and correlated with patient clinical data. TMA results showed the highest amount of ERV-K env protein expression and the strongest significant membrane expression in ccRCC versus other RCC subtypes. High ERV-K env total protein expression of all tumor subtypes significantly correlated with low tumor grading and a longer disease specific survival using multivariable analyses. Cell proliferation and invasion were assayed using the kidney cell lines HEK293 with wild-type p53 and a ccRCC cell line MZ1257RC mutated for p53. Transfecting these cell lines with a codon optimized ERV-K113 env overexpressing CMV vector was performed with or without 5’-Aza-2’-deoxycytidine (Aza) treatment to sustain promoter de-methylation. MZ1257RC showed induction of ERV-K113 expression and significantly increased both proliferation and invasion in the presence or absence of Aza. HEK293 cells demonstrated a restriction of ERV-K113 env expression and invasion with no changes in proliferation in the absence of Aza. However, in the presence of Aza despite increased ERV-K113 env expression, an inhibition of HEK293 proliferation and a further restriction of invasion was found. This study supports ERV-K env as a single prognostic indicator for better survival of RCC, which we propose represents a new tumor antigen. In addition, ERV-K env significantly regulates proliferation and invasion depending on p53 status and Aza treatment.

Role of SIRT1/AMPK signaling in the proliferation, migration and invasion of renal cell carcinoma cells.

Renal cell carcinoma (RCC) is a lethal urologic tumor commonly seen in men that best responds to partial nephrectomy. An enhanced understanding of the molecular pathogenesis of RCC can broaden treatment options and tumor prevention strategies. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that regulates several bioactive substances, and the present study aimed to identify the role of SIRT1/AMP-activated protein kinase (AMPK) signaling in RCC progression. SIRT1 expression was detected in 100 patients with RCC using tissue microarray immunohistochemistry. SIRT1-knockdown and overexpression were performed via RNA interference and plasmid transfection. Inhibition of AMPK was used for the phenotypic rescue assays to verify whether AMPK was a downstream target of SIRT1. Reverse transcription-quantitative PCR was performed to verify transfection efficiency. Transwell, MTT and flow cytometry apoptosis assays were performed to evaluate the migration, invasion, proliferation and early apoptosis level of RCC cells. SIRT1 and AMPK protein expression in human RCC tissues and cell lines (786-O and ACHN) was detected using western blotting and immunofluorescence staining. The current results, combined with data from The Cancer Genome Atlas database, revealed that SIRT1 expression in RCC tissues was downregulated compared with in adjacent normal tissues. Additionally, high SIRT1 expression was associated with an improved prognosis in patients with RCC. Overexpression of SIRT1 inhibited the proliferation, migration and invasion of RCC cell lines and induced apoptosis, while inhibition of SIRT1 expression had the opposite effects. Further experiments indicated that SIRT1 may serve an anticancer role by upregulating the expression levels of downstream AMPK, thus revealing a potential therapeutic target for RCC.

Expression of protease activated receptor-2 is reduced in renal cell carcinoma biopsies and cell lines.

Expression of the protease sensing receptor, protease activated receptor-2 (PAR2), is elevated in a variety of cancers and has been promoted as a potential therapeutic target. With the development of potent antagonists for this receptor, we hypothesised that they could be used to treat renal cell carcinoma (RCC). The expression of PAR2 was, therefore, examined in human RCC tissues and selected RCC cell lines. Histologically confirmed cases of RCC, together with paired non-involved kidney tissue, were used to produce a tissue microarray (TMA) and to extract total tissue RNA. Immunohistochemistry and qPCR were then used to assess PAR2 expression. In culture, RCC cell lines versus primary human kidney tubular epithelial cells (HTEC) were used to assess PAR2 expression by qPCR, immunocytochemistry and an intracellular calcium mobilization assay. The TMA revealed an 85% decrease in PAR2 expression in tumour tissue compared with normal kidney tissue. Likewise, qPCR showed a striking reduction in PAR2 mRNA in RCC compared with normal kidney. All RCC cell lines showed lower levels of PAR2 expression than HTEC. In conclusion, we found that PAR2 was reduced in RCC compared with normal kidney and is unlikely to be a target of interest in the treatment of this type of cancer.

MiR-6838-5p facilitates the proliferation and invasion of renal cell carcinoma cells through inhibiting the DMTF1/ARF-p53 axis.

Renal cell carcinoma (RCC) is one of the most common renal malignancies in the urinary system. Numerous studies have demonstrated that miRNAs can regulate tumorigenesis and progression. This study aims to investigate the role and regulatory mechanism of miR-6838-5p in RCC. Our study confirmed that miR-6838-5p was upregulated in human RCC tissues (30/42, 77.43%, P < 0.01) and RCC cell lines (P < 0.05) compared to adjacent non-neoplastic tissues and normal renal epithelial cells. In vitro, overexpression of miR-6838-5p enhanced cell proliferation and invasion in human RCC cell lines (ACHN and 786-O), which were detected by CCK-8, Transwell and Colony formation assays (P < 0.05), and knockdown of miR-6838-5p suppressed cell proliferation and invasion (P < 0.05). Results of Bioinformatics analysis combined with Dual-luciferase reporter gene assay demonstrated that miR-6838-5p could bind to Cyclin D binding myb-like transcription factor 1 (DMTF1). In addition, RT-qPCR and Western blotting confirmed that DMTF1 was downregulated in RCC tissues and cell lines. Meanwhile, it was demonstrated that overexpression of miR-6838-5p inhibited DMTF1 level in ACHN cells. Next, we confirmed that DMTF1 overexpression reversed the inhibitory effects of overexpression of miR-6838-5p on phosphatase and tensin homolog (PTEN), tumor protein 53(p53), murine double minute 2 (MDM2) and alternative reading frame (ARF) protein levels in the ARF-p53 signaling pathway. In conclusion, our research showed that miR-6838-5p enhanced the proliferation and invasion of RCC cells by inhibiting the DMTF1/ARF-p53 axis.

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.

Downregulation of long non-coding RNA DUXAP10 inhibits proliferation, migration, and invasion of renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common kidney malignancy that frequently leads to metastasis. Increasing evidence has shown that long non-coding RNAs (lncRNAs) play crucial roles affecting the progression of RCC. The role of lncRNA DUXAP10 in the evolution of RCC has not been defined yet. This project was designed to clarify the effects of DUXAP10 on the proliferation and tumorigenesis of RCC. We examined the expression of DUXAP10 in the Cancer Genome Atlas (TCGA) and ONCOMINE oncology databases. Then, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to evaluate DUXAP10 expression in human RCC tissues and cell lines. The correlation between the expression of DUXAP10 and clinical characteristics of RCC patients was analyzed by univariate and Kaplan-Meier analyses. To unveil the biological function of DUXAP10 in cell cycle progression, cell growth, and invasion of RCC, we conducted knockdown experiments in vitro. qRT- PCR and western blotting assays were performed to further investigate the function of DUXAP10 in cancer biology. The data from TCGA showed that the expression of DUXAP10 was upregulated in tissues of RCC compared with normal tissues. Moreover, ONCOMINE database analysis indicated that high DUXAP10 levels were correlated with high clinical stages, inferior TNM classification, and poor overall survival. Furthermore, the results indicated that knockdown of DUXAP10 remarkably inhibited the RCC cell growth, mobility, and invasion, in association with the upregulation of E-cadherin and downregulation of cyclin D, cyclin E, CDK4, N-cadherin, and vimentin. Our findings highlight the oncogenic role of DUXAP10 in RCC and that DUXAP10 may serve as a novel predictive biomarker and therapeutic target for RCC.

Expression of IGF-IEc Isoform in Renal Cell Carcinoma Tissues.

Insulin-like growth factor-I (IGF-I) regulates various aspects of cancer biology. There is a growing body of evidence regarding the potential distinct role of IGF-I isoforms, particularly of IGF-IEc, in the pathophysiology of various human cancer types, however, there are no studies which examined the expression of the different IGF-I isoforms in renal cell carcinoma (RCC). This study aimed to characterize the expression of IGF-IEc in human RCC tissues and investigated whether its expression is associated with the histopathological type of RCC as well as with the overall survival of patients. Formalin-fixed paraffin-embedded renal tissue samples from 94 patients (58 males and 36 females) were assessed for IGF-IEc expression by immunohistochemistry. RCC tissues showed mainly cytoplasmic IGF-IEc staining but immunoreactivity of IGF-IEc was also localized in the cell membrane. Significantly lower IGF-IEc expression was found in clear cell RCC vs. all other histological types (p=0.010), and this remained significant after adjusting for tumor size, grade, stage, and mitotic index (p<0.05). No association was found between IGF-IEc expression level and overall survival of patients with RCC. The differential expression of IGF-IEc isoform among the RCC histopathological types may indicate its histological type-specific regulation and possibly suggests a discrete biological role of this isoform in the pathophysiology of RCC.

Curcumin Negatively Regulates Cigarette Smoke-Induced Renal Cell Carcinoma Epithelial-Mesenchymal Transition Through the ERK5/AP-1 Pathway.

BackgroundSo far, little research has been conducted regarding the underlying mechanism of renal carcinogenesis at molecular level. Epithelial–mesenchymal transition (EMT) exerts an important part during tumor genesis as well as the development through mitogen-activated protein kinase (MAPK) pathways. Therefore, we hypothesized that EMT could promote renal cell carcinoma (RCC) progression via the ERK5/AP-1pathway.Materials and MethodsThe RCC cell lines were utilized to be the models with in vitro exposure to cigarette smoke extract (CSE). We used the curcumin for the EMT intervention study. In the present study, immunohistochemistry (IHC), Western blotting, and real-time quantitative reverse transcription PCR had been used to determine the experimental results. EMT phenotypic alterations were assessed by changes in cell morphology, invasion and transfer ability, as well as expression of epithelial and mesenchymal markers.ResultsIn human renal cell carcinoma tissue, E-cadherin expression within the smoking renal cancer patients was down-regulated compared with that among the non-smokers. However, Vimentin, N-cadherin, and TWIST levels increased (P<0.05). Significantly, we clarified that ERK5/AP-1 exerted positive regulation on the renal cell carcinoma EMT mediated by CS, which was suggested based on the results of CS activating the ERK5/AP-1 pathway, as well as ERK5 inhibition via XMD8-92 reversed AP-1 protein levels and the EMT process. Furthermore, curcumin showed the same inhibitory effect as XMD8-92 and significantly reversed CS-induced EMT through inhibiting the ERK5/AP-1 signaling pathway.ConclusionThe above results indicated that ERK5/AP-1 signaling pathway exerts a vital part for CS-associated RCC development and cancer intervention.

Abstract 6493: Optimized methods for studies of extracellular vesicles in kidney cancer

Abstract Background: Extracellular vesicles (EVs) are small membranous vesicles. All cells can secrete EVs into ECM and biofluids. The EV membrane helps to protect protein or nucleotide cargos, that can be transferred from the parental to recipient cells in an autocrine or paracrine manner. Thus, EVs are a mode of intercellular communication and valuable source of (cancer) biomarkers. The majority of human cancer EV research studies EVs from cell culture, blood or urine. Cell culture EVs are reproducible but lack sensitivity. Benign cells contribute to the total EV population found in blood and urine and thus lack tumor-specificity. We optimized EV isolation directly from renal cell carcinoma (RCC) tissue, which can aid RCC biomarker discovery. We isolated from human RCC and healthy kidney tissue. The isolated EV-product was evaluated in correspondence with the latest guidelines MISEV-guidelines (Minimal Information for Studies of Extracellular Vesicles). Methods: Following radical nephrectomy, three technical replicates and three biological replicates were obtained for both normal and clear cell RCC. The tissues were processed and used to incubate media. EVs were isolated using a combination of differential centrifugation, filtration, and ultracentrifugation. Vesicle yield and size distribution of each sample were measured using two methods; the conventional Nanoparticle Tracking Analysis (NanoSight), and the novel method Nano Flowcytometry (NanoFCM). Transmission Electron Microscopy (TEM) was used to determine presence of intact vesicles in the EV samples. Western blot was used to detect presence of EV protein markers (CD81, CD63, Flotillin-1), and absence of cellular debris (calnexin). Results: The normalized vesicle counts for the technical replicates of normal kidney EVs were 1.44 × 1010 ± 2.51 × 109 p/mL (mean ± SD) measured by NanoSight, and 1.67 × 1010 ± 6.89 × 109 p/mL (mean ± SD) measured by NanoFCM. Vesicle counts for replicates RCC EVs measured by NanoSight were 1.80 × 1010 ± 3.59 × 109 p/mL (mean ± SD), and they were 1.68 × 1010 ± 4.18 × 109 p/mL (mean ± SD) measured by NanoFCM. We observed an acceptable biological variance in EV counts of less than 10-fold among different patients. Comparing NanoSight with NanoFCM, the differences in measured vesicle counts of identical samples were less than 5-fold. TEM confirmed presence of small EVs in all technical and biological replicates. Protein analyses demonstrate presence of CD81, CD63, and Flotillin-1 in all samples. Furthermore, calnexin was absent in all samples. Conclusions: We optimized a method for EV isolation from human kidney cancer and normal kidney tissue. Our protocol consistently results in high vesicle counts, as we confirmed using a standard method and a new method. Particle concentrations of both methods were similar, but the NanoFCM performs better in assessing particle size distribution. We confirmed the presence of intact vesicles and EV proteins, and the absence of cellular contamination. This optimized protocol can contribute to RCC-biomarker discovery and aid biological kidney cancer research. Citation Format: Richard C. Zieren, Liang Dong, Phillip M. Pierorazio, Kenneth J. Pienta, Theo M. de Reijke, Sarah R. Amend. Optimized methods for studies of extracellular vesicles in kidney cancer [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 6493.

Serum response factor increases renal cell carcinoma migration and invasion through promoting epithelial-mesenchymal transition.

To explore the regulation and function of serum response factor in epithelial-mesenchymal transition in renal cell carcinoma. First, bioinformatics analysis of human renal cell carcinoma tissues was carried out. Then, the expression of serum response factor, mesenchymal markers (N-cadherin, vimentin and fibronectin) and epithelial markers (zonula occludens-1 and epithelial cadherin) was examined in 786-O cells (a human renal cell carcinoma cell line). Serum response factor was overexpressed with pcDNA-serum response factor plasmid, and suppressed by CCG-1423 (a small molecule inhibitor of serum response factor) to study how serum response factor affects epithelial-mesenchymal transition in renal cell carcinoma. A xenograft nude mouse model was established to explore whether serum response factor affected the tumorigenic ability of renal cell carcinoma cells. Serum response factor interacted with several important differentially expressed genes in renal cell carcinoma. In 786-O cells, serum response factor, N-cadherin, vimentin and fibronectin were upregulated, whereas zonula occludens-1 and epithelial cadherin were downregulated. Serum response factor upregulation in 786-O cells increased the Snail expression. Serum response factor suppression reduced Snail induction, and migration and invasion in renal cell carcinoma, which decreased the xenograft tumor volume. Serum response factor is a critical transcription factor in human renal cell carcinoma. Increased serum response factor activity induces epithelial-mesenchymal transition, migration and invasion in 786-O cells, and facilitates the progression of renal cell carcinoma. Targeting serum response factor with CCG-1423 might be an attractive therapeutic strategy for renal cell carcinoma.

Nuclear NADPH oxidase-4 associated with disease progression in renal cell carcinoma

Nuclear NADPH oxidase-4 (Nox4) is a key component of metabolic reprogramming and is often overexpressed in renal cell carcinoma (RCC). However, its prognostic role in RCC remains unclear. Here we examined the significance of nuclear Nox4 on disease progression and development of drug resistance in advanced RCC. We analyzed human RCC tissue from multiple regions in the primary index tumor, cancer-associated normal adjacent parenchyma, intravascular tumor in locally advanced cancer patients. We found that the higher nuclear Nox4 expression was significantly associated with progression and death. These findings were consistent after controlling for other competing clinical variables. In contrast, patients with lower nuclear Nox4, even in higher stage RCC had better prognosis. We identified a subset of patients with high nuclear Nox4 who had rapid disease progression or died within 6 months of surgery. In addition, higher nuclear Nox4 level correlated with resistance to targeted therapy and immunotherapy. Western blotting performed on fresh human RCC tissue as well as cell-lines revealed increased nuclear Nox4 expression. Our data support an important prognostic role of Nox4 mediated regulation of RCC independent of other competing variables. Nox4 localizes to the nucleus in high-grade, high-stage RCC. Higher nuclear Nox4 has prognostic significance for disease progression, poor survival, and development of drug resistance in RCC.

SP1/AKT/FOXO3 Signaling Is Involved in miR-362-3p-Mediated Inhibition of Cell-Cycle Pathway and EMT Progression in Renal Cell Carcinoma

Emerging evidence has indicated that dysregulation of miR-362-3p is involved in the initiation and progression of several types of human cancers. However, the molecular mechanism of miR-362-3p in renal cell carcinoma (RCC) is still not completely clear. In this study, we found that miR-362-3p was frequently down-regulated in human RCC tissues. Overexpression of miR-362-3p in RCC cells significantly suppressed the proliferation, cell cycle and motility in vitro and in vivo via regulating AKT/FOXO3 signaling. We further confirmed that SP1 was a direct target of miR-362-3p. Knockdown of SP1 expression by a small interfering RNA (siRNA) phenocopied the effect of miR-362-3p overexpression in RCC cells. In conclusion, the current results provide evidence for the role of miR-362-3p in the pathogenesis of RCC and thus miR-362-3p may serve as an attractive candidate for RCC therapy.