RCC Cell Research Articles

The prognostic implications and oncogenic role of NSUN5 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC), a predominant form of urinary malignancy, requires the identification of reliable biomarkers to enhance both prognostic outcomes and therapeutic developments specific to ccRCC. NSUN5, a member of the NOL1/NOP2/SUN domain (NSUN) family, plays a critical role in RNA stabilization and exhibits widespread expression across various tumor types. However, the exact function of NSUN5 in ccRCC remains insufficiently understood. Data were collated from cohorts of ccRCC patients who underwent nephrectomy, including those from the Cancer Genome Atlas (TCGA) and the Sun Yat-sen University Cancer Center (SYSUCC), to evaluate the clinical relevance of NSUN5. Integrative models based on NSUN5 expression were subsequently developed to predict the prognosis of ccRCC within the TCGA and SYSUCC cohorts. Furthermore, the impact of NSUN5 on RCC cells and its association with cellular senescence were corroborated through in vitro experimental analyses. NSUN5 exhibited elevated expression in both ccRCC patients and renal cancer cell lines, whose upregulation significantly correlated with age, tumor size, TNM stage, WHO/International Society of Urological Pathology (ISUP) grade, presence of necrosis, and a poor prognosis. An accessible nomogram, incorporating NSUN5 along with various clinicopathological parameters, was adept at predicting outcomes for ccRCC patients. Additionally, in vitro findings indicated that reduced expression of NSUN5 enhanced tumor cell senescence and simultaneously inhibiting cell proliferation and migration. These observations suggest that elevated NSUN5 expression is linked to poorer overall survival (OS) and progression-free survival (PFS), positioning NSUN5 as a viable diagnostic and prognostic biomarker in ccRCC.

Prognostic Significance of Elevated UCHL1, SNRNP200, and PAK4 Expression in High-Grade Clear Cell Renal Cell Carcinoma: Insights from LC-MS/MS Analysis and Immunohistochemical Validation.

Recent advancements in proteomics have enhanced our understanding of clear cell renal cell carcinoma (CCRCC). Utilizing a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by immunohistochemical validation, we investigated the expression levels of UCHL1, PAK4, and SNRNP200 in high-grade CCRCC samples. Our analysis also integrated Reactome pathway enrichment to elucidate the roles of these proteins in cancer-related pathways. Our results revealed significant upregulation of UCHL1 and SNRNP200 and downregulation of PAK4 in high-grade CCRCC tissues compared to non-cancerous tissues. UCHL1, a member of the ubiquitin carboxy-terminal hydrolase family, showed variable expression across different tissues and was notably involved in the Akt signaling pathway, which plays a critical role in cellular survival in various cancers. SNRNP200, a key component of the RNA splicing machinery, was found to be essential for proper cell cycle progression and possibly linked to autosomal dominant retinitis pigmentosa. PAK4's role was noted as critical in RCC cell proliferation and invasion and its expression correlated significantly with poor progression-free survival in CCRCC. Additionally, the expression patterns of these proteins suggested potential as prognostic markers for aggressive disease phenotypes. This study confirms the upregulation of UCHL1, SNRNP200, and PAK4 as significant factors in the progression of high-grade CCRCC, linking their enhanced expression to poor clinical outcomes. These findings propose these proteins as potential prognostic markers and therapeutic targets in CCRCC, offering novel insights into the molecular landscape of this malignancy and highlighting the importance of targeted therapeutic interventions.

FOXC1 transcriptionally suppresses ABHD5 to inhibit the progression of renal cell carcinoma through AMPK/mTOR pathway

BackgroundIncreased activity of the transcription factor FOXC1 leads to elevated transcription of target genes, ultimately facilitating the progression of various cancer types. However, there are currently no literature reports on the role of FOXC1 in renal cell carcinoma.MethodsBy using RT-qPCR, immunohistochemistry and Western blotting, FOXC1 mRNA and protein expression was evaluated. Gain of function experiments were utilized to assess the proliferation and metastasis ability of cells. A nude mouse model was created for transplanting tumors and establishing a lung metastasis model to observe cell proliferation and spread in a living organism. Various techniques including biological analysis, CHIP assay, luciferase assay, RT-qRCR and Western blotting experiments were utilized to investigate how FOXC1 contributes to the transcription of ABHD5 on a molecular level. FOXC1 was assessed by Western blot for its impact on AMPK/mTOR signaling pathway.ResultsFOXC1 is down-regulated in RCC, causing unfavorable prognosis of patients with RCC. Further experiments showed that forced FOXC1 expression significantly restrains RCC cell growth and cell metastasis. Mechanically, FOXC1 promotes the transcription of ABHD5 to activate AMPK signal pathway to inhibit mTOR signal pathway. Finally, knockdown of ABHD5 recovered the inhibitory role of FOXC1 overexpression induced cell growth and metastasis suppression.ConclusionIn general, our study demonstrates that FOXC1 exerts its tumor suppressor role by promoting ABHD5 transcription to regulating AMPK/mTOR signal pathway. FOXC1 could serve as both a diagnostic indicator and potential treatment focus for RCC.

Comprehensive analysis of MAPK genes in the prognosis, immune characteristics, and drug treatment of renal clear cell carcinoma using bioinformatic analysis and Mendelian randomization

Mitogen-activated protein kinase (MAPK) signalling is vitally important in tumour development and progression. This study is the first to comprehensively analyse the role of MAPK-family genes in the progression, prognosis, immune-cell infiltration, methylation, and potential therapeutic value drug candidates in ccRCC. We identified a novel prognostic panel of six MAPK-signature genes (MAP3K12, MAP3K1, MAP3K5, MAPK1, MAPK8, MAPK9), and introduced a robust MAPK-signature risk model for predicting ccRCC prognosis. Model construction, evaluation, and external validation using datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database demonstrated its stability, as well as high sensitivity and specificity. Enrichment analysis suggested the participation of immune-mediated mechanism in MAPK dysregulation in ccRCC. Immune-infiltration analysis confirmed the relationship and revealed that the MAPK-signature risk model might stratify immunotherapy response in ccRCC, which was verified in drug sensitivity analysis and validated in external ccRCC immunotherapy dataset (GSE67501). Potential therapeutic drug predictions for key MAPKs using DSigDB, Network Analyst, CTD, and DGIdb were subsequently verified by molecular docking with AutoDock Vina and PyMol. Mendelian randomization further demonstrated the possibilities of the MAPK-signature genes as targets for therapeutic drugs in ccRCC. Methylation analysis using UALCAN and MethSurv revealed the participation of epigenetic modifications in dysregulation and survival difference of MAPK pathway in ccRCC. Among the key MAPKs, MAP3K12 exhibited the highest significance, indicating its independent prognostic value as single gene in ccRCC. Knockout and overexpression validation experiments in vitro and in vivo found that MAP3K12 acted as a promoter of tumour progression in RCC, suggesting a pivotal role for MAP3K12 in the proliferation, migration, and invasion of RCC cells. Our findings proposed the potential of MAPK-signature genes as biomarkers for prognosis and therapy response, as well as targets for therapeutic drugs in ccRCC.

Fangchinoline targets human renal cell carcinoma cells through modulation of apoptotic and non‑apoptotic cell deaths

The process of apoptosis is one of the essential processes involved in maintenance of homeostasis in the human body. It can aid to remove misfolded proteins or cellular organelles. This sequence is especially necessary in cancer cells. However, specifically targeting already apoptotic pathways can induce drug resistance in cancer cells and hence drugs can induce cell death by alternative mechanism. We investigated whether fangchinoline (FCN) can target renal carcinoma cells by inducing multiple cell death mechanisms. Both paraptosis, autophagy, and apoptosis were induced by FCN through stimulation of diverse molecular signaling pathways. FCN induced ROS production with GSH/GSSG imbalance, and ER stress. In addition, formation of autophagosome and autophagy related markers were stimulated by FCN. Moreover, FCN induced cell cycle arrest and PARP cleavage. Except for blocking protein synthesis, these three cell death pathways were found to be complementarily working together with each other. FCN also exhibited synergistic effects with paclitaxel in inducing programmed cell death in RCC cells. Our data indicates that FCN could induce apoptotic cell death and non-apoptotic cell death pathways and can be con-tribute to development of novel cancer prevention or therapy.

Metabolomics Reveals Tyrosine Kinase Inhibitor Resistance-Associated Metabolic Events in Human Metastatic Renal Cancer Cells.

The development of resistance to tyrosine kinase inhibitors (TKIs) is a major cause of treatment failure in metastatic renal cell carcinoma (mRCC). A deeper understanding of the metabolic mechanisms associated with TKI resistance is critical for refining therapeutic strategies. In this study, we established resistance to sunitinib and pazopanib by exposing a parental Caki-1 cell line to increasing concentrations of sunitinib and pazopanib. The intracellular and extracellular metabolome of sunitinib- and pazopanib-resistant mRCC cells were investigated using a nuclear magnetic resonance (NMR)-based metabolomics approach. Data analysis included multivariate and univariate methods, as well as pathway and network analyses. Distinct metabolic signatures in sunitinib- and pazopanib-resistant RCC cells were found for the first time in this study. A common metabolic reprogramming pattern was observed in amino acid, glycerophospholipid, and nicotinate and nicotinamide metabolism. Sunitinib-resistant cells exhibited marked alterations in metabolites involved in antioxidant defence mechanisms, while pazopanib-resistant cells showed alterations in metabolites associated with energy pathways. Sunitinib-resistant RCC cells demonstrated an increased ability to proliferate, whereas pazopanib-resistant cells appeared to restructure their energy metabolism and undergo alterations in pathways associated with cell death. These findings provide potential targets for novel therapeutic strategies to overcome TKI resistance in mRCC through metabolic regulation.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe

BackgroundRenal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation.Experimental designWe slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization.ResultsEY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy.ConclusionTaken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Radix Actinidiae chinensis induces the autophagy and apoptosis in renal cell carcinoma cells

BackgroundRenal cell carcinoma (RCC) is a malignant tumor. Radix Actinidiaechinensis (RAC) is the root of Actinidia arguta (Sieb. et Zucc) Planch. ex Miq. In clinical research, RAC was confirmed to have a certain anti-tumor effect, including liver cancer and cholangiocarcinoma. This study investigated the anticancer effect and mechanism of RAC in RCC cells.MethodsThe 786-O and A498 cells were intervened with varying concentrations of RAC (0–100 mg/mL) to detect the half maximal inhibitory concentration (IC50) of RAC. The cells were then co-cultured with 0–50 mg/mL RAC for 0–72 h to assess the effect of RAC on cell viability using the cell counting kit-8. The effects on cell proliferation, cell cycle or apoptosis, migration or invasion, and autophagy were detected using cloning, flow cytometry, Transwell, AOPI assay and Western blot. The number of autophagolysosomes was quantified using a transmission electron microscope. PI3K/AKT/mTOR pathway-related proteins were detected by Western blot. Additionally, an autophagy inhibitor 3-MA was used to explore the underlying mechanism of RAC.ResultsIC50 values of RAC in 786-O and A498 were 14.76 mg/mL and 13.09 mg/mL, respectively. RAC demonstrated the ability to reduce the cell malignant phenotype of RCC cells, blocked the S phase of cells, promoted apoptosis and autophagy in cells. Furthermore, RAC was observed to increase autophagy-related proteins LC3II/I and Beclin-1, while decreasing the level of P62. The expression of apoptosis-related proteins was increased, while the ratios of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR, p-P38/P38 and p-ERK/ERK were reduced by RAC. However, the addition of 3-MA reduced the apoptosis and autophagy- promotion effects of RAC on RCC cells.ConclusionRAC induced the apoptosis and autophagy, to inhibit the progression of RCC cells. This study may provide a theoretical and experimental basis for clinical anti-cancer application of RAC for RCC.

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.

MIER2/PGC1A elicits sunitinib resistance via lipid metabolism in renal cell carcinoma

IntroductionRenal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system and accounts for more than 90 % of all renal tumors. Resistance to targeted therapy has emerged as a pivotal factor that contributes to the progressive deterioration of patients with advanced RCC. Metabolic reprogramming is a hallmark of tumorigenesis and progression, with an increasing body of evidence indicating that abnormal lipid metabolism plays a crucial role in the advancement of renal clear cell carcinoma. ObjectivesClarify the precise mechanisms underlying abnormal lipid metabolism and drug resistance. MethodsBioinformatics screening and analyses were performed to identify hub gene. qRT-PCR, western blot, chromatin immunoprecipitation (ChIP) assays, and other biological methods were used to explore and verify related pathways. Various cell line models and animal models were used to perform biological functional experiments. ResultsIn this study, we identified Mesoderm induction early response 2 (MIER2) as a novel biomarker for RCC, demonstrating its role in promoting malignancy and sunitinib resistance by influencing lipid metabolism in RCC. Mechanistically, MIER2 facilitated P53 deacetylation by binding to HDAC1. Acetylation modification augmented the DNA-binding stability and transcriptional function of P53, while deacetylation of P53 hindered the transcriptional process of PGC1A, leading to intracellular lipid accumulation in RCC. Furthermore, Trichostatin A (TSA), an inhibitor of HDAC1, was found to impede the MIER2/HDAC1/P53/PGC1A pathway, offering potential benefits for patients with sunitinib-resistant renal cell cancer. ConclusionOur findings highlight MIER2 as a key player in anchoring HDAC1 and inhibiting PGC1A expression through the deacetylation of P53, thereby inducing lipid accumulation in RCC and promoting drug resistance. Lipid-rich RCC cells compensate for energy production and sustain their own growth in a glycolysis-independent manner, evading the cytotoxic effects of targeted drugs and ultimately culminating in the development of drug resistance.

Interaction of immune cells with renal cancer development: Mendelian randomization (MR) study

BackgroundRenal cell carcinoma (RCC) is a prevalent and extensively immune-infiltrated malignancy of the urinary system. Immune cells play a crucial role in both the progression and therapeutic interventions targeting RCC. Nevertheless, the interplay between RCC and immune cells remains understudied, lacking substantial evidence supporting their causal relationship.MethodsFor the purpose of investigating the causal connection between RCC and immune cell characteristics, a two-way two-sample Mendelian randomization (MR) analysis was carried out in this study. The aim was to determine whether specific immune cell traits have a causal impact on the risk of RCC. In order to achieve this, publicly accessible genetic data was utilized to examine and establish the potential relationship between 731 immune cell characteristics and the likelihood of developing RCC. Additionally, various techniques were applied to verify the reliability, variability, and presence of horizontal pleiotropy in the outcomes.ResultsWe found a bidirectional causal relationship between RCC and immune cells according to the MR analysis results. It should be noted that CD4-CD8-T cells (OR = 1.61, 95%CI = 1.02–2.55, P = 4.07 × 10–2) pose a risk for RCC, whereas BAFF-R (OR = 0.69, 95%CI = 0.53–0.89, P = 5.74 × 10–3) and CD19 (OR = 0.59, 95%CI = 1.02–2.55, P = 4.07 × 10–2) on B cells act as protective factors. Furthermore, the presence of RCC reduces the levels of B cells (OR = 1.05, 95%CI = 1.01–1.09, P = 1.19 × 10–2) and CD8 + T cells (OR = 1.04, 95%CI = 1.00–1.08, P = 2.83 × 10–2).ConclusionsOur research illustrates the intricate correlation between immune cells and RCC, presenting novel insights for the prospective safeguarding against RCC risk and the exploration of fresh therapeutic targets.

Targeting STING elicits GSDMD-dependent pyroptosis and boosts anti-tumor immunity in renal cell carcinoma.

While Stimulator-of-interferon genes (STING) is an innate immune adapter cruicial for sensing cytosolic DNA and modulating immune microenvironment, its tumor-promoting role in tumor survival and immune evasion remains largely unknown. Here we reported that renal cancer cells are exceptionally dependent on STING for survival and evading immunosurveillance via suppressing ER stress-mediated pyroptosis. We found that STING is significantly amplified and upregulated in clear cell renal cell carcinoma (ccRCC), and its elevated expression is associated with worse clinical outcomes. Mechanically, STING depletion in RCC cells specifically triggers activation of the PERK/eIF2α/ATF4/CHOP pathway and activates cleavage of Caspase-8, thereby inducing GSDMD-mediated pyroptosis, which is independent of the innate immune pathway of STING. Moreover, animal study revealed that STING depletion promoted infiltration of CD4+ and CD8+ T cells, consequently boosting robust antitumor immunity via pyroptosis-induced inflammation. From the perspective of targeted therapy, we found that Compound SP23, a PROTAC STING degrader, demonstrated comparable efficacy to STING depletion both in vitro and in vivo for treatment of ccRCC. These findings collectively unveiled an unforeseen function of STING in regulating GSDMD-dependent pyroptosis, thus regulating immune response in RCC. Consequently, pharmacological degradation of STING by SP23 may become an attractive strategy for treatment of advanced RCC.

Abstract 627: IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is a top ten malignancy in the U.S. Overexpression of the proviral integration site for moloney murine leukemia virus 1 (PIM1) kinase is associated with poor clinical outcomes in RCC patients. PIM1 is a constitutively active serine/threonine kinase promoting cell proliferation, apoptosis resistance, invasion, and migration. The mechanisms underlying PIM1 expression and its function in RCC are not fully delineated. IL-6 is a pleiotropic cytokine that activates the JAK/STAT signaling cascade. High serum IL-6 levels are associated with the poor prognosis of RCC patients and may contribute to RCC invasion and metastasis. STAT3/5 binds directly to the PIM1 promoter inducing PIM1 expression. An IL-6/STAT3/PIM1 axis exists in pancreatic and breast cancer. We previously reported that PIM1 is overexpressed in a panel of human RCC cell lines relative to normal and immortalized renal proximal tubule epithelial cells. We also identified that RCC cells secrete IL-6. Our prior studies suggest that differential expression of PIM1 may be linked to autocrine IL-6 signaling. We thus hypothesize that an IL-6/JAK/STAT pathway regulates the expression of PIM1 in RCC. To understand how IL-6 signaling through the JAK/STAT pathway may regulate PIM1 expression in RCC cells, we examined whether IL-6 blockade using anti-IL-6 antibody or tocilizumab, would modulate PIM1 expression. Similarly, we assessed whether ruxolitinib, and LLL12, a STAT3 inhibitor could regulate PIM1 expression. We then evaluated the effect of ruxolitinib treatment on cell viability. In RCC cell lines, IL-6 blockade through either anti-IL-6 antibody or tocilizumab was sufficient to decrease PIM1 protein levels. Treatment with ruxolitinib leads to a dose and time-dependent decrease in PIM1 levels. Incubation with a STAT3 inhibitor also resulted in decreased PIM1 levels in RCC cells. Treatment with ruxolitinib also appears to decrease cell viability in a dose-dependent manner. These results suggest that differential expression of PIM1 in RCC may be linked to autocrine IL-6 signaling via a JAK/STAT/PIM1 axis. Multiple FDA-approved agents are available that target this pathway. Further investigation is required to determine the efficacy of these agents in pre-clinical models and even clinical trials. Citation Format: Kimberly S. Meza, Kimberly Seymour, Sheldon L. Holder. IL-6 signaling via JAK/STAT axis influences PIM1 expression in renal cell carcinoma [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 627.

Abstract 3283: Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance

Abstract Introduction: Despite numerous available treatments, metastatic renal cell carcinoma (mRCC) remains highly lethal, with over 90% of patients succumbing within 5 years. Sorafenib (SF), a second-line treatment for mRCC, exhibits modest efficacy, and the underlying molecular basis for its failure is elusive. Hyaluronic acid (HA), implicated in promoting cell proliferation, invasion, and motility through CD44 and RHAMM receptors, represents a potential player in SF resistance. Hymecromone (HC), a non-toxic dietary supplement, is known to inhibit HA synthesis and downregulate CD44 and RHAMM mRNA levels. This study investigates the elusive mechanisms of SF resistance and evaluates the effectiveness of the HC+SF combination as a targeted therapy for advanced RCC, providing insights into a novel and potentially impactful treatment approach. Methods: HA levels were measured in normal and tumor kidney specimens using ELISA-like assay and IHC. Similarly, HAS3 and UGT1A9 (A9) levels and SF glucuronidation were measured by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination was examined in A9 and HAS3 knockdown and overexpression cell lines (transfectants) and primary RCC spheroids by proliferation, apoptosis, motility, and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in subcutaneous and orthotopic xenograft mouse models, with bioluminescence imaging. Results: SF failure in mRCC was linked due to its glucuronidation and inactivation by A9. Tumor microsomes from patients who developed mRCC glucuronidated SF 5-fold higher than from non-mRCC patients. A9 levels were elevated (8-10 folds) in mRCC patients’ tumor along with HA and HAS3 (5-10 folds) as compared to non-mRCC patients and normal kidney tissues. HC treatment downregulated A9 promoter activity and inhibited SF-glucuronidation in RCC cells. SF (5uM) and HC (20, 40ug/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold) and downregulated HAS3 expression (>80%), and HA synthesis in RCC cells. While A9 and HAS3 transfectants both were resistant to SF+HC treatment, A9 and HAS3 - KO transfectants were sensitive to SF. Endothelial cells co-cultured with either A9 or HAS3 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in A9 or HAS3 transfectants. HC (100 or 200mg/kg) and SF (30mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. HC+SF treatment decreased A9, HA and HAS3 levels, Ki67 index and microvessel density. Both A9 and HAS3 overexpressing tumors were resistant to treatment. Conclusion: This study demonstrates the mechanism of SF failure in mRCC and proposes a safe, oral adjunct combination for effective treatment. Citation Format: Anuj Kumar Sharma, Karina Aguilar, Chandramukhi S. Panda, Vinata B. Lokeshwar. Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib 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 3283.

Abstract 6066: An inhibitor of deubiquitinating enzyme UCHL5/USP14, VLX1570, enhances the cytotoxic effects of temsirolimus by suppressing PCNA in uuman renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is a common type of kidney cancer. The overall 5-year survival rate is 74%, but it drops to only 8% for Stage 4 cancers. Several treatments are available for metastatic RCC, including tyrosine kinase inhibitors, mTOR inhibitors, and immune checkpoint inhibitors. However, these treatments have limited success. VLX1570 is an inhibitor of deubiquitylating enzymes UCHL5/USP14 that is currently in Phase I clinical trials. It has shown promise in various cancer models, but its effects on RCC have not been reported. This study investigated the impact of UCHL5/USP14 expression on tumor progression and patient survival in a clinical cohort. The results showed that VLX1570 induced apoptosis and cell cycle arrest in RCC cells, leading to reduced cell viability. Additionally, VLX1570 synergized with temsirolimus in vitro, likely by inhibiting PCNA. Moreover, the combination treatment suppressed tumor growth in vivo more effectively than either VLX1570 or temsirolimus alone. These findings suggest that UCHL5/USP14 plays a role in RCC progression and prognosis, making it a promising therapeutic target for overcoming drug resistance in this cancer. Citation Format: Kuan-Lin Kuo, Po-Ming Chow, Chen-Hsun Hsu, Shih-Ming Liao, Yen-Ling Chiu, Jun-Ren Dong, Tzu-Yao Ke, Kuo-How Huang. An inhibitor of deubiquitinating enzyme UCHL5/USP14, VLX1570, enhances the cytotoxic effects of temsirolimus by suppressing PCNA in uuman renal cell carcinoma [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 6066.

Abstract 1710: DPF3 over-expression oncogenic effect in renal cancer is through SWI/SNF BAF complex

Abstract Renal Cancer (RCC) is the seventh most common type of cancer in the United States, and most part of these tumors do not respond to radiation and chemotherapy treatments. A Genome Wide Association Study (GWAS) identified 13 regions that are related to the risk of developing RCC, and one of this regions is the 14q24 region, a region that has already been shown to have a Single Nucleotide Polymorphism (SNP) involved in the development of RCC, by which the presence of the risk allele is capable of increase the expression of DPF3 and this increase is capable of regulated the RCC cell line proliferation. It is very published the oncogenic effect of DPF3 in RCC, however, the mechanism by which DPF3 is acting is not very knew. Our objective is to understand the mechanisms by which DPF3 regulate renal cancer development. For this, we did a co-immunoprecipitation of DPF3 with nuclear extract of RCC cell line that overexpress DPF3 and analyze the interactions between DPF3 and other proteins of SWI/SNF Complex. We did a proteomic analyzes in Targeted Mass Spectrometry (MRM) for see the proteins levels. We could see an increase in expression of other SWI/SNF BAF Complex proteins in RCC cell line that overexpress DPF3, such like SMARCA2, SMARCE1 and ARID1A, suggesting a relation between DPF3 and SWI/SNF BAF Complex and evidencing that DPF3 is acting through BAF Complex. To extend the evaluation of DPF3 we inhibit the expression of the SNP associated with the 14q24 region, rs4903064, in renal cancer cell lines (UOK121, SN12C and 786-O) using the CRISPRi technic and analyze the role of this SNP in DPF3, CEMIP and IL23R expression. Our initial results demonstrate that in the UOK121 and SN12C lineage, the inhibition of the SNP was able to decrease the expression of DPF3, and in the SN12C cell line we also see a decrease in CEMIP expression. When we analyze the 786-O cell line, we see that the blockade of SNP does not change the expression of DPF3, nor of the other genes, indicating that the action of the SNP on DPF3 occurs only in the presence of the risk allele for RCC. We also use a murine (Renca) RCC cell line that overexpress DPF3 under regulation of TetOn promoter and we did an in vivo experiment in mouse (Balb/c and Balb/c Nude), for analyze the function of DPF3 in tumor growth, migration and proliferation. When we inject (s.c) in Balb/c Nude we could see an increase in tumor growth, in tumor volume and weight in the group treated with doxycycline diet when compare with de control group, suggesting that DPF3 is capable of increase RCC growth. We also analyze the DPF3 function in RCC invasion and migration, and when we inject (intrarenal) in Balb/c we could see a metastatic signal in the lung. The lung of the animals treated with doxycycline diet presents an increase in volume and weight when compare with the control group. In this way, the study of genetic and proteomic behind DPF3 function and RCC development is essential for a biomarker discovery and help in the patient prognosithes and treatment. Citation Format: Leticia Andrade Costa, Aline Gomes Souza, Vírginia Campos Silvestrini, Timothy Myers, Vitor Marcel Faça, Stephen Chanock, Leandro Machado Colli. DPF3 over-expression oncogenic effect in renal cancer is through SWI/SNF BAF complex [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 1710.

Abstract 3177: Angiopoietin-like 4 suppress clear cell renal cell carcinoma via inhibition lysosomal acid lipase

Abstract Renal cell carcinomas(RCC) account for about 76,000 new cancer cases annually in the U.S. and 403,000 cases world wide. Clear cell RCC (ccRCC) is both the most common and aggressive subtype, accounting for 75-85% of cases with a 5-year survival rate of 50-69% for all cases and only 10% for metastatic disease. In our study, we report thatANGPTL4 is highly upregulated in ccRCC compared to other types of cancers and normal kidney tissues. However, we identified a cohort of patients with significantly lower expression of ANGPTL4 which had a worse prognosis compared to rest of the patients with higher expression of ANGPTL4. This subset of patients had a lower frequency of mutated VHL and were enriched for genes related to lipid metabolism. In transplant tumor models using RCC cell lines with wild-type VHL, we found that ANGPTL4 suppresses tumor growth. Moreover, loss of ANGPTL4 in ccRCC cell lines reduces colony formation in a lysosomal acid lipase (LAL) dependent manner, a phenotype that is rescued by expression of nANGPTL4. This data indicates that ANGPTL4 suppresses ccRCC tumors in a cancer cell intrinsic manner via regulation of LAL activity. This cohort of patients with low ANGPTL4 may be more dependent on lipid metabolism and less likely to respond to anti-angiogenic TKI plus immune checkpoint inhibitor therapy. Citation Format: Zeng Jin, Umasankar De, Tanzia Tithi, Jeremy Kleberg, Akhila Nataraj, Elena Jolley, Brandon Davies, Weizhou Zhang, Ryan Kolb. Angiopoietin-like 4 suppress clear cell renal cell carcinoma via inhibition lysosomal acid lipase [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 3177.

Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.

Interaction between Wnt/β-catenin signaling pathway and EMT pathway mediates the mechanism of sunitinib resistance in renal cell carcinoma

BackgroundTargeted drugs are the main methods of RCC treatment. However, drug resistance is common in RCC patients, in-depth study of the drug-resistant mechanism is essential.MethodsWe constructed sunitinib resistant and Twist overexpressed A498 cells, and studied its mechanisms in vitro and in vivo.ResultsIn cell research, we found that either sunitinib resistance or Twist overexpression can activate Wnt/β-catenin and EMT signaling pathway, and the sunitinib resistance may work through β-catenin/TWIST/TCF4 trimer. In zebrafish research, we confirmed the similarity of Twist overexpression and sunitinib resistance, and the promoting effect of Twist overexpression on drug resistance.ConclusionsSunitinib resistance and Twist overexpression can activate Wnt/β-catenin signaling pathway and EMT to promote the growth and metastasis of RCC cells.

Retracted: Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation.

[This retracts the article DOI: 10.1155/2022/9139338.].