Renal Cell Carcinoma Cell Lines Research Articles

Curcumin Inhibits Vasculogenic Mimicry via Regulating ETS-1 in Renal Cell Carcinoma.

Metastatic renal cell carcinoma (RCC) poses a huge challenge once it has become resistant to targeted therapy. Vasculogenic mimicry (VM) is a novel blood supply system formed by tumor cells that can circumvent molecular targeted therapies. As one of the herbal remedies, curcumin has been demonstrated to play antineoplastic effects in many different types of human cancers; however, its function and mechanism of targeting VM in RCC remains unknown. Here, in the work, we explored the role of curcumin and its molecular mechanism in the regulation of VM formation in RCC. RNA-sequencing analysis, immunoblotting, and immunohistochemistry were used to detect E Twenty Six-1(ETS-1), vascular endothelial Cadherin (VE-Cadherin), and matrix metallopeptidase 9 (MMP9) expressions in RCC cells and tissues. RNA sequencing was used to screen the differential expressed genes. Plasmid transfections were used to transiently knock down or overexpress ETS-1. VM formation was determined by tube formation assay and animal experiments. CD31-PAS double staining was used to label the VM channels in patients and xenograft samples. Our results demonstrated that VM was positively correlated with RCC grades and stages using clinical patient samples. Curcumin inhibited VM formation in dose and time-dependent manner in vitro. Using RNA-sequencing analysis, we discovered ETS-1 as a potential transcriptional factor regulating VM formation. Knocking down or overexpression of ETS-1 decreased or increased the VM formation, respectively and regulated the expression of VE-Cadherin and MMP9. Curcumin could inhibit VM formation by suppressing ETS-1, VE-Cadherin, and MMP9 expression both in vitro and in vivo. Our finding might indicate that curcumin could inhibit VM by regulating ETS-1, VE-Cadherin, and MMP9 expression in RCC cell lines. Curcumin could be considered as a potential anti-cancer compound by inhibiting VM in RCC progression.

ASPSCR1::TFE3-mediated upregulation of insulin receptor substrate 2 (IRS-2) activates PI3K/AKT signaling and promotes malignant phenotype

The ASPSCR1::TFE3 fusion gene, resulting from chromosomal translocation, is detected in alveolar soft part sarcoma (ASPS) and a subset of renal cell carcinomas (RCC). The ASPSCR1::TFE3 oncoprotein, functioning as an aberrant transcription factor, contributes to tumor development and progression by inappropriately upregulating target genes. Here, we identified insulin receptor substrate 2 (IRS-2), a cytoplasmic adaptor protein, as a novel transcriptional target of ASPSCR1::TFE3. Ectopic expression of ASPSCR1::TFE3 led to increased IRS-2 mRNA and protein levels. Chromatin immunoprecipitation and luciferase assays demonstrated that ASPSCR1::TFE3 bound to the IRS-2 promoter region and enhanced its transcription. Moreover, IRS-2 was highly expressed in the ASPSCR1::TFE3-positive RCC cell line FU-UR1, while small interfering RNA-mediated depletion of ASPSCR1::TFE3 markedly decreased IRS-2 mRNA and protein levels. Functionally, IRS-2 knockdown attenuated activation of the PI3K/AKT pathway and reduced proliferation, migration, invasion, adhesion, and clonogenicity in FU-UR1 cells. Pharmacological inhibition of IRS-2 also reduced AKT activation as well as cell viability, migration, invasion, and adhesion. These findings suggest that IRS-2, regulated by ASPSCR1::TFE3, promotes tumor progression by activating PI3K/AKT signaling and enhancing the malignant phenotype.

Fc receptor-like A promotes malignant behavior in renal cell carcinoma and correlates with tumor immune infiltration.

Our study aims to investigate the mechanisms through which Fc receptor-like A (FCRLA) promotes renal cell carcinoma (RCC) and to examine its significance in relation to tumor immune infiltration. The correlation between FCRLA and data clinically related to RCC was explored using The Cancer Genome Atlas (TCGA), then validated using Gene Expression Omnibus (GEO) gene chip data. Enrichment and protein-protein interaction (PPI) network analyses were performed for FCRLA and its co-expressed genes. FCRLA was knocked down in RCC cell lines to evaluate its impact on biological behavior. Then the potential downstream regulators of FCRLA were determined by western blotting, and rescue experiments were performed for verification. The relevance between FCRLA and various immune cells was analyzed through GSEA, TIMER, and GEPIA tools. TIDE and ESTIMATE algorithms were used to predict the effect of FCRLA in immunotherapy. Fc receptor-like A was associated with clinical and T stages and could predict the M stage (AUC = 0.692) and 1-3- and 5-year survival rates (AUC = 0.823, 0.834, and 0.862) of RCC patients. Higher expression of FCLRA predicted an unfavorable overall survival (OS) in TCGA-RCC and GSE167573 datasets (p = 0.03, p = 0.04). FCRLA promoted the malignant biological behavior of RCC cells through the pERK1/2/-MMP2 pathway and was associated with tumor immune microenvironment in RCC. Fc receptor-like A is positively correlated with poor outcomes in RCC patients and plays an oncogenic role in RCC through the pERK1/2-MMP2 pathway. Patients with RCC might benefit from immunotherapy targeting FCRLA.

DEPDC1 as a metabolic target regulates glycolysis in renal cell carcinoma through AKT/mTOR/HIF1α pathway

Renal cell carcinoma (RCC) is considered a “metabolic disease” characterized by elevated glycolysis in patients with advanced RCC. Tyrosine kinase inhibitor (TKI) therapy is currently an important treatment option for advanced RCC, but drug resistance may develop in some patients. Combining TKI with targeted metabolic therapy may provide a more effective approach for patients with advanced RCC. An analysis of 14 RCC patients (including three needle biopsy samples with TKI resistance) revealed by sing-cell RNA sequencing (scRNA-seq) that glycolysis played a crucial role in poor prognosis and drug resistance in RCC. TCGA-KIRC and glycolysis gene set analysis identified DEPDC1 as a target associated with malignant progression and drug resistance in KIRC. Subsequent experiments demonstrated that DEPDC1 promoted malignant progression and glycolysis of RCC, and knockdown DEPDC1 could reverse TKI resistance in RCC cell lines. Bulk RNA sequencing (RNA-seq) and non-targeted metabolomics sequencing suggested that DEPDC1 may regulate RCC glycolysis via AKT/mTOR/HIF1α pathway, a finding supported by protein-level analysis. Clinical tissue samples from 98 RCC patients demonstrated that DEPDC1 was associated with poor prognosis and predicted RCC metastasis. In conclusion, this multi-omics analysis suggests that DEPDC1 could serve as a novel target for TKI combined with targeted metabolic therapy in advanced RCC patients with TKI resistance.

ADAMTS16 Suppression by MicroRNA-25 Leads to Oncogenic Properties in Renal Cell Carcinoma.

Increasing evidence indicates that microRNAs (miRNAs) play a crucial role in modulating tumor growth. This study is centered on investigating the contribution of miR-25 to the progression of Renal Cell Carcinoma (RCC). The investigators examined the expression levels of miR-25 and ADAMTS16 in RCC samples and cell lines. The association between miR-25 and ADAMTS16 was validated via a luciferase reporter assay. Cell viability, apoptosis, migration, and invasion were evaluated utilizing CCK-8 and flow cytometry techniques, while the expression levels of ADAMTS16, β-catenin, GSK-3β, and p-GSK-3β were assessed through western blot analysis. The investigation revealed elevated expression levels of miR-25 in RCC tissues. Subsequently, ADAMTS16 was identified as a target of miR-25. Increased miR-25 levels were associated with decreased expression of ADAMTS16, resulting in enhanced cell viability and diminished apoptosis. Conversely, inhibition of miR-25 led to decreased cell viability, proliferation, and migration. Additionally, the researchers observed that miR-25 triggered the phosphorylation of GSK-3β and β-catenin while leaving the total GSK-3β level unaffected. This study suggests that miR-25 regulates the expression of ADAMTS16 through the Wnt/β-catenin signaling pathway, providing new insights into the cause and potential treatment of RCC.

The role of cyclin-dependent kinase inhibitor 3 in the proliferation and migration of renal cell carcinoma

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, is over expressed in renal cell carcinoma (RCC). However, the cell biology functions of RCC are not well understood. The present study aimed to verify the ability of CDKN3 to promote the proliferation and migration of RCC through in vitro experiments. Subsequently, the clinical prognostic effects were analyzed using The Cancer Genome Atlas (TCGA; https://www.cancer.gov/) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The chelators, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an analogue of the anti-tumor agent, were screened through bioinformatics analysis. The expression of CDKN3 is positively correlated with the IC50 of Dp44mT. In two RCC cell lines, 786-0 and Caki-1, we conducted small interfering RNA (siRNA) knockdown of CDKN3 and overexpression of CDKN3 by transfection plasmid. Subsequently, we administered Dp44mT to examine the resulting alterations in cell proliferation, migration, and apoptosis, thereby elucidating the role of CDKN3 and Dp44mT in these processes. The results of the experiment revealed a positive association between CDKN3 expression and the proliferation of RCC cell lines. Down-regulating CDKN3 significantly increased the apoptosis rate and inhibited cell migration in 786-0 and Caki-1 cells. Furthermore, bioinformatics analysis revealed a high expression of CDKN3 in RCC and a negative association between CDKN3 expression and survival. Gene set enrichment analysis (GSEA) revealed a significant association between high CDKN3 expression and the cell cycle pathway. Furthermore, we identified Dp44mT as a drug highly correlated with CDKN3 through the database. Subsequent addition of Dp44mT resulted in similar findings to those observed upon CDKN3 knockdown. Our findings have important implications for the diagnosis and treatment of CDKN3 in RCC. Additionally, Dp44mT is likely to be a promising candidate for future clinical applications.

Tetraspanin immunoassay for the detection of extracellular vesicles and renal cell carcinoma

AbstractHalf of patients with renal cell carcinoma (RCC) develop metastases. New and noninvasive biomarkers are needed for the diagnosis of RCC. The study aims to develop an EV‐based assay for the detection of RCC using a highly sensitive nanoparticle‐aided time‐resolved fluorescence immunoassay (NP‐TRFIA). To confirm the presence of tetraspanins on EVs, size exclusion chromatography is used to separate EV‐ and PE‐fractions from RCC4, 786‐O, and HEK293 cell lines. EV‐ and PE‐fractions are quantified using NP‐TRFIA assays established for CD9, CD63, CD81, and CD151. Tetraspanins are measured from RCC CCM and serum samples of RCC (n = 14), benign (n = 17), and healthy (n = 9) individuals. Among the tetraspanins, CD63 exhibits 3‐5‐fold higher expression on RCC4 and 786‐O CCM compared to HEK293. A sandwich CD63‐CD63 assay demonstrates significant discrimination of RCC patients from benign (p = 0.0003), and healthy (p = 0.005) individuals, respectively. Similarly, the CD81‐CD81 assay also enables significant separation of RCC patients compared to benign (p = 0.014), and healthy (p = 0.003) controls, respectively. This suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63‐ and CD81‐EVs compared to controls. Detection of these EVs using NP‐TRFIA approach may play a vital role in the detection of RCC.

A Potent Solution for Tumor Growth and Angiogenesis Suppression via an ELR+CXCL-CXCR1/2 Pathway Inhibitor.

CXCR1/2 biomolecules play vital roles in cancer cell proliferation, tumor inflammation, and angiogenesis, making them attractive drug targets. In clear cell renal cell carcinoma (RCC) and head and neck squamous cell carcinoma (HNSCC), where CXCR1/2 is overexpressed, inhibition studies are limited. Building upon previous research efforts, we investigated new N,N'-diarylurea analogues as ELR+CXCL-CXCR1/2 inhibitors. Evaluations on RCC and HNSCC cell lines and 3D spheroid cultures identified compound 10 as a lead molecule, exhibiting significant inhibition of invasion, migration, and neo-angiogenesis. It demonstrated strong interference with the signaling pathway, with high selectivity toward kinases. In vivo studies on zebrafish embryos and RCC xenografted mice showed notable anticancer, antimetastatic, and antiangiogenic effects after oral administration and minimal toxicity. Compound 10 emerges as a promising candidate for further preclinical development as an oral anticancer and antiangiogenic drug targeting the ELR+CXCL-CXCR1/2 pathway.

TRIB3 promotes the progression of renal cell carcinoma by upregulating the lipid droplet-associated protein PLIN2

Abnormal lipid metabolism and lipid accumulation are characteristic hallmarks of renal cell carcinoma (RCC). While there is prior evidence closely linking such lipid accumulation within RCC cells and consequent tumorigenesis, the mechanisms underlying this process remain incompletely understood. In this study, a series of bioinformatics analyses were initially performed by screening RCC databases and gene sets, ultimately leading to the identification of TRIB3 as an oncogene that functions as a central regulator of lipid metabolism. TRIB3 overexpression was observed in both RCC patient tumor tissues and cell lines, and this upregulation was correlated with a worse RCC patient prognosis. When TRIB3 was knocked down, this resulted in a reduction in lipid accumulation and the consequent induction of endoplasmic reticulum (ER) stress-related apoptotic cell death. At the molecular level, interactions between TRIB3 and PLIN2 were found to abrogate TEB4-mediated PLIN2 ubiquitination and consequent degradation, thus maintaining higher PLIN2 expression levels. This simultaneously helps facilitate the accumulation of lipids while preserving ER homeostasis, thus driving accelerated RCC tumor progression. This TRIB3-PLIN2 axis thus represents a promising new target for efforts to treat 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.

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 μM Pazopanib, respectively. Cellular senescence was measured using senescence-associated β-galactosidase (SA-β-Gal) staining. Western blot analysis and real-time polymerase chain reactionwere used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress by increasing the production of reactive oxygen species,reducing the levels of glutathione peroxidase,and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Abstract 5670: CRISPR-Mediated knockout of Ceruloplasmin (CP) and UCHL1 reveals angiogenic role in renal cell carcinoma (RCC) cell survival and proliferation

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma, accounting for approximately 70% of all cases. Therapeutic treatment for metastatic ccRCC involve tyrosine kinase inhibitors or mTOR inhibiters. While these drugs extend patient survival, it also introduces a range of adverse side effects. Utilizing snRNA sequencing, snATACseq, and spatial transcriptomics, two potential ccRCC tumor specific markers have been identified: ceruloplasmin (CP) and UCHL1. These markers are notably overexpressed in a majority of ccRCC tumor cells and elevated CP levels have been correlated with reduced patient survival. To investigate the roles of CP and UCHL1 in ccRCC, CRISPR-based knockout experiments were conducted on two RCC cell lines, namely RCC4-VHL- and Caki-1. Through cell line characterization assays, we find that the absence of CP and UCHL1 impairs the cells' capabilities to migrate, proliferate, and adhere. Additionally, Cell-Derived Xenografts (CDX) of the knockout cell lines in nude mice revealed that cells lacking UCHL1 exhibited slower tumor growth compared to their parental lines. Bulk RNA sequencing further suggested a potential interrelationship between CP and UCHL1, as CP knockout cells showed increased UCHL1 expression relative to the parental line. These findings open new avenues for potential therapeutic strategies and biomarkers in the treatment and screening of ccRCC and further elevates our understandings of the roles of CP and UCHL1 in RCC tumor progression. Citation Format: Wagma Caravan, Yige Wu, Preet Lal, Kazu Sato, Yize Li, Nataly Naser Al Deen, Siqi Chen, Ateih Abedin, Feng Chen, Li Ding. CRISPR-Mediated knockout of Ceruloplasmin (CP) and UCHL1 reveals angiogenic role in renal cell carcinoma (RCC) cell survival and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5670.

Abstract 3980: Investigating the role of host obesity in promoting immunotherapy resistance

Abstract Obesity is linked to an increased risk of various cancers, including renal cell carcinoma (RCC). Many RCC patients have obesity at diagnosis and treatment initiation, and our lab has found that this significantly impairs immunotherapy outcomes. Both preclinical and clinical reports, from our lab and others, have demonstrated that obesity reduces the efficacy of CD8+ T cell-dependent immunotherapy by increasing pro-tumorigenic IL-1β. Combining anti-PD-1 based therapy with anti-IL-1β triggers the recruitment of CD8+ T cells and improves the efficacy of anti-PD-1 based immunotherapy. However, the mechanistic links between obesity and increased IL-1β production remains unknown. Our objective is to identify mechanisms promoting IL-1 β-driven immunosuppression within renal tumors of obese mice. In our in vivo studies, we utilized a BALB/c mouse model of diet-induced obesity (DIO) and lean controls in combination with the Renca-Luc RCC cell line. Using flow cytometry and western blot, we respectively assessed leukocyte populations producing IL-1β and tumor energetics. Flow cytometry revealed that intra-tumoral IL-1β is produced primarily by tumor associated macrophages (TAMS). Immunoblotting demonstrated that increased IL-1β production is linked to altered mitochondrial function and ATP production. These early findings support the hypothesis that obesity driven IL-1β expression is mediated by tumor-associated macrophages and that mitochondrial dysfunction drives immune suppression. Understanding these molecular and immune-related mechanisms may pave the way for novel strategies to reduce immunosuppression in obese RCC patients, thus improving immunotherapeutic efficacy and overall clinical outcomes. Citation Format: Henry Nnaemeka Ogbonna, Zachary Roberts, Haley Kvarnberg, Francesca Dempsey, Patrick N. Song, Anna G. Sorace, Lyse Norian. Investigating the role of host obesity in promoting immunotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3980.

Abstract 4296: Investigating metabolic sensitivity and organization of renal cell carcinoma: To enhance effective therapies and patient survival

Abstract Background: Renal cell carcinoma (RCC) is the 6th most common cancer in men and the 9th most common in women in the United States. RCC tissues are metabolically dysfunctional, often resulting from loss of von-Hippel Lindau (VHL) and consequent upregulation of hypoxia-inducible factor (HIF1/2). HIF1a is a transcription factor that upregulates glycolytic gene expression. Current therapies, including immune checkpoint inhibitors and tyrosine kinase inhibitors, have shown improvements in the outcomes of RCC patients, however, there are no biomarkers clinically available to predict the efficacy of these drugs. While genomic data and transcriptomic signatures have been investigated, they are not used in clinical practice due to lack of prospective validation. Understanding additional pathways pertaining to metabolic features of RCC, is critical. Our bulk gene expression analysis of primary RCC samples from TCGA-KIRC show a predominance of genes expressing mitochondrial ribosomal proteins and electron transport chain in tumors that relapsed early after nephrectomy, warranting further validation. Aim: We aim to investigate changes in metabolic adaptation in RCC cells from kidney cancer cell lines (VHL mutant) and primary cells from human-matched primary and metastatic kidney cancer sites. Using a novel biosensor-based technique developed in the Albeck lab, we aim to identify the dependence of live RCC cells on specific metabolic pathways and test the effect of standard-of-care (SOC) pathway inhibitors such as tyrosine kinase, HIF pathway, metabolic pathway inhibitors impacting tumorigenicity. Methods: To examine functional metabolic behavior in single cells, we generated immortalized cell lines (MCF10A, 184A1, FL83B and HBE-1) stably transfected with biosensors for the activity of AKT, glycolysis, mTOR and AMPK pathway. Data from Live-cells treated with metabolic inhibitors were collected and analyzed using our MATLAB-based pipeline. We have extended the analysis to RCC cell lines (786-O cell lines) and primary cells collected from nephrectomy specimens and matched metastatic biopsy samples, using current SOC pathway inhibitors (including sorafenib, sunitinib, belzutifan) and investigational metabolic drugs (metformin, IACS-010759, niclosamide). Results & Conclusion: Our preliminary results show an association of high OX-PHOS with primary tumors from early relapse after nephrectomy than the relapsed late tumors. Our biosensor single cell data show that even genetically homogeneous cell populations can vary in their usage of OXPHOS and glycolysis to supply ATP for cell growth. Tumor gene expression profile coupled with biosensor data, will address mechanism behind this metabolic shift in RCC tumors in primary cells isolated from primary and metastatic sites. Our research will improve tumor prognosis and allow designing therapeutic trials. Citation Format: Madhura Patankar, John Albeck, Shuchi Gulati. Investigating metabolic sensitivity and organization of renal cell carcinoma: To enhance effective therapies and patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4296.

Nesprin1 Deficiency Is Associated with Poor Prognosis of Renal Cell Carcinoma and Resistance to Sunitinib Treatment

Introduction: Nuclear envelope spectrin repeat protein (Nesprin) 1 encoded by SYNE1, crucially regulates the morphology and functions of the cell. Mutations in the SYNE1 gene are associated with various diseases; however, their significance in renal cell carcinoma (RCC) remains unknown. In this study, we have investigated the association of SYNE1/Nesprin1 with the progression and prognosis of clear cell RCC (ccRCC). Methods: In silico analyses of publicly available datasets of patients with RCC were performed. Based on the cohort data, Nesprin1 expression in nephrectomized tissue samples acquired from patients with ccRCC was analyzed using immunohistochemical staining. The invasion, migration, and proliferation of the SYNE1-knockdown human RCC cell lines were analyzed in vitro; moreover, RNA sequencing and gene set enrichment analysis were conducted to study the molecular mechanism underlying the association of SYNE1/Nesprin1 with prognosis of RCC. Results: Patients with RCC-associated SYNE1 gene mutations exhibited significantly worse overall and progression-free survivals. Patients with Nesprin1-negative ccRCC tumors exhibit significantly poorer overall, cancer-specific, and recurrence-free survival rates than those recorded in the Nesprin1-positive group. SYNE1 knockdown enhanced the invasion and migration of RCC cells; however, it did not influence the proliferation of cells. RNA sequencing and gene set enrichment analysis revealed that SYNE1 knockdown significantly altered the expression of genes associated with oxidative phosphorylation. Consistently, patients with RCC exhibiting low SYNE1 expression, who were treated with the vascular endothelial growth factor receptor inhibitor sunitinib, had worse progression-free survival. Conclusions: The results indicate that the expression of SYNE1/Nesprin1 and SYNE1 mutations in patients with RCC are closely linked to their prognosis and responsiveness to sunitinib treatment.

Nicotine-derived nitrosamines promote renal cell carcinoma cell migration via binding to nicotinic acetylcholine receptors

Several risk factors such as smoking, obesity, diabetes, and hypertension are related to the incidence of kidney cancer. Renal cell carcinoma (RCC) originates from the renal tubular epithelium and is the major form of kidney cancer. Notably, the relative risk of RCC in smokers compared to non-smokers is 1.38 while revealing a dose-dependent risk reduction in smoking cessation. Carcinogenic substances in tobacco, specifically nicotine-derived nitrosamines like 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), were known to induce the formation of DNA adducts, mutating genes that potentially initiate cancers. In addition to carcinogenesis, nicotine-derived nitrosamines may also contribute to modify the malignant features, which accelerates the cancer progression. This study was aimed to evaluate the effects of NNK in the modulation RCC cell proliferation and motility, besides, the possible underlying mechanisms were also studied. Our findings showed differential expression of nicotinic acetylcholine receptor (nAChR) mRNA subunits between normal human renal tubular cell (HK-2) and RCC cells (Achn), suggesting that RCC cells may have the ability in respond to the stimuli of NNK. While NNK didn't significantly show an influence in RCC cell viability, it enhanced cell motility and migration ability. Utilizing specific inhibitors, it was inferred that NNK stimulates RCC cell migration via nAChR activation and subsequent calcium channel opening. Our research underscores the necessity of smoking cessation in RCC management, even as further studies on diverse RCC cell lines and real-world data are required.

Fumarate induces LncRNA-MIR4435-2HG to regulate glutamine metabolism remodeling and promote the development of FH-deficient renal cell carcinoma

Fumarate hydratase (FH) deficient renal cell carcinoma (RCC) is a type of tumor with definite metabolic disorder, but the mechanism of metabolic remodeling is still unclear. LncRNA was reported to closely correlate with cancer metabolism, however the biological role of LncRNA in the development of progression of FH-deficent RCC was not well studied either. FH-deficient RCC samples were collected in my hospital and used for RNA-sequencing and Mass spectrometry analysis. FH-deficient RCC cell line UOK262 and control pFH cells were used for in vitro experiments, including proliferation assay, transwell assay, western-blot, mass spectrometry and so on. PDX mouse model was used for further drug inhibition experiments in vivo. In this study, we analyzed the profiles of LncRNA and mRNA in FH-deficienct RCC samples, and we found that the LncRNA-MIR4435-2GH was specifically highly expressed in FH-deficient RCC compared with ccRCC. In vitro experiments demonstrated that MIR4435-2HG was regulated by Fumarate through histone demethylation, and the deletion of this gene could inhibit glutamine metabolism. RNA-pulldown experiments showed that MIR4435-2HG specifically binds to STAT1, which can transcriptionally activate GLS1. GLS1 inhibitor CB-839 could significantly suppress tumor growth in PDX tumor models. This study analyzed the molecular mechanism of MIR4435-2HG in regulating metabolic remodeling of FH-deficient RCC in clinical samples, cells and animal models by combining transcriptional and metabolic methods. We found that that GLS1 was a therapeutic target for this tumor, and MIR4435-2HG can be used as a drug sensitivity marker.

Tumorigenic role of tacrolimus through mTORC1/C2 activation in post-transplant renal cell carcinomas.

Kidney transplant recipients (KTRs) face an increased risk of renal cell carcinoma (RCC), in which the immunosuppressive regimen plays an important role. This study aimed to identify intracellular signalling alterations associated with post-transplant (post-tx) tumour formation. Expression of mTOR-related proteins were analysed in kidneys obtained from end-stage renal disease (ESRD) patients and RCCs developed in KTRs or non-transplant patients. The effects of tacrolimus (TAC) and rapamycin (RAPA) on mTOR activity, proliferation, and tumour growth were investigated through different in vitro and in vivo experiments. Elevated mTORC1/C2 activity was observed in post-tx RCCs and in kidneys of TAC-treated ESRD patients. In vitro experiments demonstrated that TAC increases mTOR activity in a normal tubular epithelial cell line and in the investigated RCC cell lines, moreover, promotes the proliferation of some RCC cell line. In vivo, TAC elevated mTORC1/C2 activity in ischaemic kidneys of mice and enhanced tumour growth in xenograft model. We observed significantly increased mTOR activity in ischaemic kidneys and post-tx RCCs, which highlights involvement of mTOR pathway both in the healing or fibrotic processes of kidney and in tumorigenesis. TAC-treatment further augmented the already elevated mTOR activity of injured kidney, potentially contributing to tumorigenesis during immunosuppression.

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.

Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure–activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the β-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the β-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.