RCC Cell Research Articles

Formin-related protein 1 facilitates proliferation and aggressive phenotype of clear cell renal cell carcinoma through MAPK/MMP2 pathway

BackgroundFormin-related protein-1(FRL1) has reportedly been overexpressed in a variety of malignancies, such as clear cell renal cell carcinoma (ccRCC). However, the clinical value and molecular mechanisms underlying ccRCC tumorigenesis and progression in association with FRL1 remain poorly understood. MethodsImmunohistochemical analysis was performed on 119 paraffin-embedded RCC tissue samples to detect FRL1 expression and analyze its prognostic value. Colony formation, the CCK-8 assay, flow cytometry, and in vivo nude mice subcutaneous experiments were used to identify the effects of FRL1 on growth and proliferation. In vitro tests for wound healing, migration, and invasion were used to assess the involvement of FRL1 in invasion and metastatic potential. The process of epithelial-mesenchymal transition process (EMT) and the MMP2 expression were detected in stably transfected RCC cells via western blotting, as well as in tumor tissue paraffin sections from xenograft model. ResultsBoth FRL1 mRNA and protein levels were noticeably elevated in ccRCC cell lines and samples. Aberrant overexpression of FRL1 was associated with unfavorable clinicopathological features of ccRCC and indicated poor prognosis. Ectopic overexpression of FRL1 increased the growth-promoting traits of ccRCC cells as well as the migratory and invasive capacity of RCC cells, whereas FRL1-silencing caused the opposite results. In addition, FRL1 promoted epithelial-mesenchymal transition (EMT) and upregulated the expression of matrix metalloproteinase 2 (MMP2). Finally, overexpression of FRL1 upregulated phosphorylation level of ERK1/2 with no effect on total level of ERK1/2 in the RCC cells. MAPK/ERK inhibitor reversed the promotional effects of FRL1. ConclusionFRL1 was overexpressed in ccRCC tissues and predicted poor prognosis. FRL1 contributes to invasion and aggressive phenotype of ccRCC by facilitating EMT through MAPK/MMP2 axis.

Insulin receptor expression to predict resistance to axitinib and elucidation of the underlying molecular mechanism in metastatic renal cell carcinoma.

The study aimed to examine the significance of insulin receptor (INSR) expression in predicting resistance to axitinib in clear cell renal cell carcinoma (ccRCC). Clinicopathological data were collected from 36 consecutive patients with metastatic RCC who received axitinib. Thirty-three primary tumours were obtained for immunohistochemistry. Patient-derived xenograft (PDX) models were created by transplanting primary tumours into immunodeficient mice, establishing axitinib-resistant PDX models. RCC cell lines were co-cultured with human renal glomerular endothelial cells (HGECs) treated with siRNA of INSR (HGEC-siINSR). Gene expression alteration was analysed using microarray. The patients with low INSR expression who received axitinib had a poorer outcome. Multivariate analysis showed that INSR expression was the independent predictor of progression-free survival. INSR expression decreased in axitinib-resistant PDX tumours. RCC cell lines showed upregulated interferon responses and highly increased interferon-β levels by co-culturing with HGEC-siINSR. HGECs showed decreased INSR and increased interferon-β after axitinib administration. RCC cell lines co-cultured with HGEC-siINSR showed high programmed death-ligand 1 (PD-L1) expression, which increased after interferon-β administration. Decreased INSR in RCC could be a biomarker to predict axitinib resistance. Regarding the resistant mechanism, vascular endothelial cells with decreased INSR in RCC may secrete interferon-β and induce PD-L1.

Free-fatty acid receptor-1 (FFA1/GPR40) promotes papillary RCC proliferation and tumor growth via Src/PI3K/AKT/NF-κB but suppresses migration by inhibition of EGFR, ERK1/2, STAT3 and EMT

BackgroundPapillary renal cell carcinoma (pRCC) is a highly metastatic genitourinary cancer and is generally irresponsive to common treatments used for the more prevalent clear-cell (ccRCC) subtype. The goal of this study was to examine the novel role of the free fatty-acid receptor-1 (FFA1/GPR40), a cell-surface expressed G protein-coupled receptor that is activated by medium-to-long chained dietary fats, in modulation of pRCC cell migration invasion, proliferation and tumor growth.MethodsWe assessed the expression of FFA1 in human pRCC and ccRCC tumor tissues compared to patient-matched non-cancerous controls, as well as in RCC cell lines. Using the selective FFA1 agonist AS2034178 and the selective FFA1 antagonist GW1100, we examined the role of FFA1 in modulating cell migration, invasion, proliferation and tumor growth and assessed the FFA1-associated intracellular signaling mechanisms via immunoblotting.ResultsWe reveal for the first time that FFA1 is upregulated in pRCC tissue compared to patient-matched non-cancerous adjacent tissue and that its expression increases with pRCC cancer pathology, while the inverse is seen in ccRCC tissue. We also show that FFA1 is expressed in the pRCC cell line ACHN, but not in ccRCC cell lines, suggesting a unique role in pRCC pathology. Our results demonstrate that FFA1 agonism promotes tumor growth and cell proliferation via c-Src/PI3K/AKT/NF-κB and COX-2 signaling. At the same time, agonism of FFA1 strongly inhibits migration and invasion, which are mechanistically mediated via inhibition of EGFR, ERK1/2 and regulators of epithelial–mesenchymal transition.ConclusionsOur data suggest that FFA1 plays oppositional growth and migratory roles in pRCC and identifies this receptor as a potential target for modulation of pathogenesis of this aggressive cancer.

Connexin43 is associated with the progression of clear cell renal carcinoma and is regulated by tangeretin to sygergize with tyrosine kinase inhibitors

BackgroundThe roles of Connexin43 (Cx43) in clear cell renal cell carcinoma (ccRCC) microenviroment remains to be poorly defined. MethodsThe expression profile, prognosis and immune analysis of Cx43 in various cancers, particularly in ccRCC were performed using TCGA database, and various biological function assays were applied to explore the physiological role of Cx43 and tangeretin in ccRCC. Western blot were applied to examine the protein expression and Kunming mice were used to evaluate preliminary safety or anti-tumor activity of tangeretin and sunitinib. ResultsCompared with the normal group, higher expression levels of Cx43 in ccRCC, and distinct associations between Cx43 expression and ccRCC prognosis or immune infiltration, were found. Notably, the expression of Cx43 was found to be highly correlated with that of receptor tyrosine kinases (RTKs), particularly with VEGFR1, VEGFR2 and VEGFR3. The expression of Cx43 and EGFR was also found to be higher in ccRCC than that in the para-cancerous specimens. Knocking down Cx43 expression decreased RCC cell viability, cell migration, p-EGFR, MMP-9 and survivin expression. Using 14 Chinese medicine monomers, tangeretin was screened and found to inhibit tumor cell viability and Cx43 expression. Tangeretin also enhanced the sensitivity of RCC cells to tyrosine kinase inhibitors (TKIs) sunitinib and sorafenib. However, the same concentration of tangeretin exerted a less prominent effect on normal renal cell viability. ConclusionsCx43 is strongly associated with RTK expression and ccRCC progression, while tangeretin can inhibit RCC cell malignancy by inhibiting Cx43 expression and enhance the sensitivity of RCC cells to TKIs.

E2F2 serves as an essential prognostic biomarker and therapeutic target for human renal cell carcinoma by presenting “E2F2/miR-16–5p/SPTLC1” schema

BackgroundRenal cell carcinoma (RCC) is a common malignant tumor of the urinary system with high mortality and morbidity. Although E2F2, a classical transcription factor implicated in cell cycle, has been shown to foster tumorigenesis in several human cancers, it could not draw a satisfy answer referring to precise downstream signaling axis in RCC development yet. MethodsBased on the publicly available data from TCGA database, expression patterns of E2F2, SPTLC1 and miR-16–5p were identified, either with the ability to predict the prognosis of patients with RCC, which was further validated in 38 paired RCC tissues and matched adjacent tissues by RT-qPCR and Western blot, respectively. Their cellular biofunctions were evaluated using MTT, EdU, Colony formation and transwell assays. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay were employed to certain the exquisite core transcription regulatory circuitry of E2F2/miR-16–5p/SPTLC1 in RCC progression, which was also determined in xenograft tumor model. ResultsConsistent with the public TCGA database, E2F2 was significantly increased in RCC tissues and cells, indicating shorter overall survival. Mechanistically, E2F2 served as a transcriptional activator of miR-16–5p, thus accounting for its negative regulation on SPTLC1 expression. E2F2 knockdown-mediated suppressive biofunctions on RCC cells were rescued by miR-16–5p mimics, while this effect was abolished again by SPTLC1 overexpression. Role of E2F2 on RCC tumorigenesis via the miR-16–5p/SPTLC1 axis was verified both in vitro and in vivo. ConclusionE2F2 promoted RCC progression via the miR-16–5p/SPTLC1 axis, which may represent a novel prognostic and therapeutic biomarker for RCC.

Integrated single-cell and bulk characterization of cuproptosis key regulator PDHB and association with tumor microenvironment infiltration in clear cell renal cell carcinoma.

Renal clear cell carcinoma (ccRCC) is one of the most prevalent cancers worldwide. Accumulating evidence revealed that copper-induced cell death played a vital role in various tumors. However, the underlying mechanism of cuproptosis with molecular heterogeneity and tumor microenvironment (TME) in ccRCC remains to be elucidated. The present study aimed to discover the biological function of cuproptosis regulators with the potential to guide clinical therapy. Using Single-cell RNA-seq, bulk transcriptome and other multi-omics datasets, we identify essential cuproptosis-related hub gene PDHB for further study. The dysregulation of PDHB in ccRCC was characterized, together with survival outcomes, pathway enrichment and immune infiltration among tumor microenvironments. The functional significance and clinical association of PDHB was validated with loss of function experiments and surgical removal specimens. PDHB mRNA and protein expression level was significantly downregulated in ccRCC tissues compared with normal and paired normal tissues. Clinicopathological parameters and tissue microarray (TMA) indicated that PDHB was identified as a prognostic factor for survival outcomes among ccRCC patients. Additionally, low PDHB was negatively correlated with Treg cells, indicating an immunosuppressive microenvironment. Mechanistically, knockdown PDHB appeared to promote the RCC cells proliferation, migration, and invasion potentials. Subsequent studies showed that copper-induced cell death activation could overcome sunitinib resistance in RCC cells. This research illustrated a cuproptosis-related hub gene PDHB which could serve as a potential prognostic marker and provide therapeutic benefits for clinical treatment of ccRCC patients.

A novel hypoxia-related lncRNA risk score model for prognosis evaluation of clear cell renal cell carcinoma.

Renal cell carcinoma is the most common aggressive tumor of the genitourinary system. The main pathological subtype is clear cell renal cell carcinoma (ccRCC), and its treatment options are very limited. Therefore, identifying specific biomarkers of ccRCC is of great significance for diagnosis and prognosis. First, we obtained transcriptome data and clinical data of 611 patients with renal clear cell carcinoma to analyze the relationship between hypoxia-related lncRNAs and overall survival (OS). We screened hypoxia-related lncRNAs through Pearson correlation and Cox regression analysis. Univariate and multivariate regression analysis were applied to assess survival-related risk factors. According to the median risk score, patients were divided into two groups. Next, a nomogram map was built, and GSEA was used for gene function annotation. RT-qPCR, Western Blot, and Flow Cytometry were used to determine the role of SNHG19 in RCC cells. By analyzing the co-expression of hypoxia genes and lncRNAs, 310 hypoxia-related genes were obtained. Four sHRlncRs (AC011445.2, PTOV1-AS2, AP004609.3, and SNHG19) with the highest prognostic values were included in the group to construct the HRRS model. The high-risk group had a shorter OS than the low-risk group. HRRS was considered to be an independent prognostic factor and associated with OS. The two groups showed different pathways in GSEA. Experiments showed that SNHG19 plays essential roles in the autophagy and apoptosis of RCC cells. We constructed and validated a hypoxia-related lncRNA model for ccRCC patients. This study also provides new biomarkers for the poor prognosis of ccRCC patients.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing

BackgroundEarly-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined.MethodsHere, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes.ResultsAnalysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of gammaH2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased gammaH2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities.ConclusionsTogether, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

A mathematical modeling technique to understand the role of decoy receptors in ligand-receptor interaction

The ligand-receptor interaction is fundamental to many cellular processes in eukaryotic cells such as cell migration, proliferation, adhesion, signaling and so on. Cell migration is a central process in the development of organisms. Receptor induced chemo-tactic sensitivity plays an important role in cell migration. However, recently some receptors identified as decoy receptors, obstruct some mechanisms of certain regular receptors. DcR3 is one such important decoy receptor, generally found in glioma cell, RCC cell and many various malignant cells which obstruct some mechanism including apoptosis cell-signaling, cell inflammation, cell migration. The goal of our work is to mathematically formulate ligand-receptor interaction induced cell migration in the presence of decoy receptors. We develop here a novel mathematical model, consisting of four coupled partial differential equations which predict the movement of glioma cells due to the reaction-kinetic mechanism between regular receptors CD95, its ligand CD95L and decoy receptors DcR3 as obtained in experimental results. The aim is to measure the number of cells in the chamber’s filter for different concentrations of ligand in presence of decoy receptors and compute the distance travelled by the cells inside filter due to cell migration. Using experimental results, we validate our model which suggests that the concentration of ligands plays an important role in cell migration.

A group of novel VEGF splice variants as alternative therapeutic targets in renal cell carcinoma.

The efficacy of anti-angiogenic treatment by targeting VEGF/VEGF receptors in metastatic clear cell renal cell carcinoma (ccRCC) varies from patient to patient. Discovering the reasons behind this variability could lead to the identification of relevant therapeutic targets. Thus, we investigated the novel splice variants of VEGF that are less efficiently inhibited by anti-VEGF/VEGFR targeting than the conventional isoforms. By in silico analysis, we identified a novel splice acceptor in the last intron of the VEGF gene resulting in an insertion of 23 bp in VEGF mRNA. Such an insertion can shift the open-reading frame in previously described splice variants of VEGF (VEGFXXX ), leading to a change in the C-terminal part of the VEGF protein. Next, we analysed the expression of these alternatively spliced VEGF new isoforms (VEGFXXX/NF ) in normal tissues and in RCC cell lines by qPCR and ELISA, and we investigated the role of VEGF222/NF (equivalent to VEGF165 ) in physiological and pathological angiogenesis. Our in vitro data demonstrated that recombinant VEGF222/NF stimulated endothelial cell proliferation and vascular permeability by activating VEGFR2. In addition, VEGF222/NF overexpression enhanced proliferation and metastatic properties of RCC cells, whereas downregulation of VEGF222/NF resulted in cell death. We also generated an in vivo model of RCC by implanting RCC cells overexpressing VEGF222/NF in mice, which we treated with polyclonal anti-VEGFXXX/NF antibodies. VEGF222/NF overexpression enhanced tumour formation with aggressive properties and a fully functional vasculature, while treatment with anti-VEGFXXX/NF antibodies slowed tumour growth by inhibiting tumour cell proliferation and angiogenesis. In a patient cohort from the NCT00943839 clinical trial, we investigated the relationship between plasmatic VEGFXXX/NF levels, resistance to anti-VEGFR therapy and survival. High plasmatic VEGFXXX/NF levels correlated with shorter survival and lower efficacy of anti-angiogenic drugs. Our data confirmed the existence of new VEGF isoforms that could serve as novel therapeutic targets in patients with RCC that are resistant to anti-VEGFR therapy.

Abstract 3705: L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer

Abstract The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of renal cell carcinoma-RCC (clear cell histology) and promotes tumor progression. L-2HG is structurally similar to α-ketoglutarate (α-KG). Therefore, L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH), resulting in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line has revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. The findings were consistent in the patient samples where high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions than that of the low L-2HG renal tumors and the patient-matched normal kidneys. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type (WT) mice revealed less serine content in the absence of L2HGDH, further confirming that high L-2HG suppresses serine biosynthesis in vivo. We found that L-2HG-mediated inhibition of the α-KG-dependent histone demethylase KDM4C silences ATF4 transcription. ATF4 is a master regulator of amino acid biosynthetic genes including PHGDH and PSAT1. Using ATF4 gain of function analysis, we confirmed that high L-2HG causes the suppression of PHGDH and PSAT1 in an ATF4-dependent manner. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. Using mutational analysis, we demonstrate that L-2HG promotes m6A accumulation at a specific site within the 3’UTR of PSAT1 that silences its translation. In accord with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, Sunil Sudarshan. L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer. [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 3705.

Abstract 5016: IL-6 influences PIM1 expression in renal cell carcinoma

Abstract Renal cell carcinoma (RCC) is the most common kidney neoplasm, accounting for over 430,000 new cases worldwide and 179,000 deaths annually. The overexpression of the proviral integration site for moloney murine leukemia virus 1 (PIM1) kinase is associated with poor clinical outcomes in patients with RCC. PIM1 is a constitutively active effector serine/threonine kinase with roles in tumor progression including cell proliferation, apoptosis, invasion, and migration. Yet, the mechanisms underlying PIM1 expression and its function in RCC are not fully delineated. IL-6 is a pleiotropic cytokine involved in the activation of the JAK/STAT signaling cascade. High serum IL-6 levels are implicated in the poor prognosis of RCC patients and are hypothesized to contribute to RCC invasion and metastasis. PIM1 is shown to be transcriptionally regulated downstream of JAK/STAT signaling and an IL-6/STAT3/PIM1 axis exists in pancreatic cancer. We thus hypothesize that this pathway also regulates expression of PIM1 in RCC. Here, we explore the levels of PIM1 in RCC and identify potential signaling pathways influencing PIM1 expression. Our results show five RCC cell lines, 769-P, 786-O, ACHN, CAKI-1, and RCC-4 cells overexpress PIM1 relative to normal renal proximal tubule epithelial cells. In RCC cell lines, IL-6 secretion correlates with PIM1 protein levels. Treatment with ruxolitinib, a JAK1/2 inhibitor, leads to a dose and concentration dependent decrease in PIM1 levels. We then tested whether incubation with an IL-6 neutralizing antibody is sufficient to regulate PIM1 protein levels. Indeed, use of an IL-6 antibody resulted in decreased pJAK2 protein and PIM1 protein. Our initial studies suggest that differential expression of PIM1 among RCC cell lines may be linked to autocrine IL-6 signaling. Interestingly, 769-P cells demonstrate increased PIM1 levels but do not secrete IL-6 and neither ruxolitinib treatment nor incubation with an IL-6 neutralizing antibody appear to affect PIM1 levels. Therefore, an IL-6/JAK-independent pathway resulting in increased PIM1 protein levels likely exists in 769-P cells. Further investigation is required to elucidate the contexts of PIM1 regulatory pathways in order to develop novel targeted anti-cancer strategies in RCC. Citation Format: Kimberly S. Meza, Sheldon L. Holder. IL-6 influences PIM1 expression in renal cell carcinoma. [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 5016.

Abstract 3972: Novel renal cell carcinoma therapy targeting glutaminolysis

Abstract Objective: Metabolic reprogramming of cancer cells contributes to tumor development and provides a target for cancer therapy ASCT2 is a glutamine transporter on the plasma membrane. Glutamine is converted into the cell to glutamate by glutaminase (GLS) and utilized by the TCA cycle. This glutaminolysis has been implicated in tumor progression in various malignancies, including renal cell carcinoma. In the present study, we investigated whether the unique strategy that effectively targets glutamine addiction would be effective as a treatment for RCC. Methods: Tumor tissue (T) and normal renal tissue (N) from surgical specimens of 66 patients with RCC were collected, and metabolomic analysis using liquid chromatography-mass spectrometry (LC/MS) to calculate the T/N ratio of Gln and Glu, respectively, were performed. In addition, immunohistological (IHC) staining of paraffin-embedded sections of 106 metastatic RCC cases was used to compare GLS and ASCT2 expression in tumor and normal tissue. In vitro, cell viability of the human RCC cell lines 786-O and 769-P was assessed by WST-8 assays. The protein expression was detected by Western blotting. Intracellular concentrations of glutamine and glutamate were analyzed using LC/MS. V9302 was used as the specific ASCT2 inhibitor, and CB839 as the specific GLS inhibitor. As the xenograft model in vivo, athymic nude mice were injected subcutaneously with 786-O cells and given various doses of V9302 and CB839 intraperitoneally. Results: LC/MS assays using surgical specimens showed a notable increase in glutamine concentration in tumor tissue. IHC assays confirmed high expression of ASCT2 in tumor tissue, and patients with high expression of ASCT2 had a poorer prognosis than those with low expression. Treatment of V9302 with 786-O and 769-P resulted in a limited decrease in cell viability. LC/MS analysis confirmed that V9302 treatment reduced glutathione metabolites. V9302 administration was shown to increase intracellular glutamine levels, presumably by compensatory mechanisms, whereas the combination of V9302 and CB839 significantly decreased glutamine levels in 786-O cells. Western blot analysis showed that the expression levels of ASCT2 and GLS1 decreased after the combination therapy. The xenograft study confirmed that the combination of V9302 and CB839 significantly inhibited 786-O tumor growth compared with controls within three weeks after treatment (p <0.001). Conclusion: It has been shown that ASCT2 expression is a marker of poor prognosis in RCC and that suppression of ASCT2 has an antitumor effect by affecting glutathione metabolism. On the other hand, the administration of V9302 raised the issue that a compensatory mechanism increases intracellular glutamine levels. Our results suggest that a novel combination strategy using CB839 in addition to V9302 could overcome the tolerance for the monotherapy and effectively target glutamine addiction in RCC. Citation Format: Yoshinari Muto, Akihito Takeuchi, Kenji Zennami, Eiji Sugihara, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Novel renal cell carcinoma therapy targeting glutaminolysis. [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 3972.

Abstract 1611: Statins inhibit the collaborative metastasis mediated by VHL heterogeneity in clear cell renal cell carcinoma (ccRCC) via a non-cholesterol pathway

Abstract BACKGROUND: Despite the advancement of targeted therapies and immunotherapies, the outcome of clear cell renal cell carcinoma (ccRCC) remains poor at the metastatic stage, with a median survival of mere 7 months. This devastating outcome is partially due to an inadequate understanding of the metastatic mechanism of ccRCC. Previously, we found that two distinct cell populations co-exist in the malignancy: one with the functional von-Hippel Lindau (VHL) gene (VHL(+)) and the other without (VHL(-)). This heterogeneity in VHL plays an essential role, where the VHL(-) cells serve as the metastatic driver and promote VHL(+) cells to disseminate to distant organs such as the lungs. In this study, we sought to find small molecules that can selectively inhibit the VHL(-) cells' growth and survival. We hypothesize that these molecules will effectively inhibit ccRCC metastases by targeting the metastatic driver population. METHODS: CRISPR knockout was conducted to generate VHL(-) cells from a murine RCC cell line, RENCA. Both VHL(-) and (+) cells were then fluorescently labeled and went through high-throughput screening (HTS) with 2,530 FDA-approved drugs. We followed a ratiometric black-box approach to identify selective modulators of cell growth and survival of VHL(-) cells without affecting VHL(+) counterparts. CRISPR knockin was conducted on human ccRCC cells, as well, generating VHL(+) and (-) pairs of 786O, RCC4, and a patient-derived primary cell line. RESULTS: HTS yielded 9 hits that selectively inhibited the survival of the metastatic drivers VHL(-) cells. 4 out of 9 hits belonged to the same drug family called statins. Statins bind to HMG-CoA reductase (HMGCR) and help lower cholesterol, thus are widely prescribed to high-risk patients for cardiovascular diseases. Interestingly, not all statins showed the VHL(-) suppressing effect, resulting in IC50 varying from 0.6uM to >50mM. Atorvastatin and rosuvastatin, the two most potent and prescribed statins for cholesterol-lowering with the highest HMGCR affinity, did not show as much efficacy as other statins. This observation also held true in human ccRCC cell lines, suggesting an involvement of a non-cholesterol/HMGCR pathway in statins’ inhibitory effect on VHL(-) cells. Animal studies were conducted using fluvastatin, which had the lowest IC50 in our model. Intraperitoneal injection of 15mg/kg fluvastatin Q.O.D for 2 weeks successfully suppressed the growth of primary tumors, especially by eliminating VHL(-) cells, and most importantly, prevented lung metastases in the treatment group, whereas all of the control group developed metastases. CONCLUSIONS: Translational potential of this project is enormous. By investigating the mechanism of action further, we could potentially reposition the FDA-approved, cheap, and widely available small molecules as a novel anti-metastatic therapeutic in ccRCC. Citation Format: Moe Ishihara, Junhui Hu, Celine Cano-Ruiz, Maia Jackson, Aimee Wu, Stuart Conway, Robert D. Damoiseaux, Lily Wu. Statins inhibit the collaborative metastasis mediated by VHL heterogeneity in clear cell renal cell carcinoma (ccRCC) via a non-cholesterol pathway [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 1611.

Abstract 4088: A neovasculature-inducible CA9 CAR resistant to FASL and TGFb mediated suppression for the treatment of ccRCC

Abstract Clinically effective CAR-T cell therapy for solid tumors, such as clear cell renal cell carcinoma (ccRCC), will require substantial T cell engineering to increase their specificity and potency. We have developed an Integrated Circuit T cell (ICT) that encodes multiple synthetic “modules” in order to overcome diverse barriers to efficacy in ccRCC; ICT cells are generated via CRISPR-mediated, targeted knock-in of a single large transgene into the novel GS94 safe-harbor locus. Both primary and metastatic sites of ccRCC are highly vascularized, with the majority of tumor cells expressing elevated levels of carbonic anhydrase IX (CA9), suggesting CA9 may be an excellent CAR target. However, CA9 is also expressed in healthy bile ducts and stomach tissue which has led to on-target, off-tumor toxicities in patients treated with constitutive CA9 CAR T cells. To improve the therapeutic index of CA9 CAR T cells, we developed an “AND” logic gated ICT cell that requires the presence of two antigens to trigger tumor cell killing, thereby enhancing tumor specificity. Induction of the CA9 CAR is gated on the expression of PSMA found on the tumor neovasculature of ccRCC. Importantly, PSMA and CA9 are not co-expressed in normal tissues. When the anti-PSMA priming receptor (PrimeRTM) binds PSMA, PrimeRTM engagement triggers proteolytic release of a chimeric, fully human transcription factor that induces expression of a CA9 CAR. We confirmed the feasibility of vascular priming using a transwell assay where ICTs were primed by a PSMA expressing endothelial cell line and then migrated across the transwell membrane to kill CA9 expressing RCC cells. In addition, a dual flank xenograft model was used to show logic gated circuits selectively kill tumors that express both CA9 and PSMA, and not tumors that express CA9 alone. Transforming growth factor beta (TGFb) is an immunosuppressive cytokine known to be highly expressed in ccRCC. To further increase the potency and persistence of the ICT cells an shRNA cassette was developed targeting both FAS and TGFBR2, a receptor required for TGFB signaling in T cells. Addition of FAS/TGFBR2 shRNA enhanced antitumor activity of PSMAxCA9 logic gate expressing T cells during in vitro chronic stimulation assays conducted in the presence of exogenous TGFb. Furthermore, FAS/TGFBR shRNA containing ICTs demonstrated enhanced antitumor activity in multiple xenograft RCC models. Collectively, these results demonstrate that PSMAxCA9 ICT cells can (i) selectively target antigens that cannot be safely targeted by conventional CARs and (ii) overcome multiple suppressive mechanisms in the tumor microenvironment. Citation Format: Angela C. Boroughs, Irene Scarfo, Nickolas Attanasio, Thomas Gardner, Jenessa B. Smith, Jennifer McDevitt, Laura Lim, Nishant Mehta, Suchismita Mohanty, James Zhang, Eric Cui, Vibhavari Sail, Amanda Fearon, Samuel Williams, Stephen Santoro, W. Nicholas Haining, Levi Gray-Rupp. A neovasculature-inducible CA9 CAR resistant to FASL and TGFb mediated suppression for the treatment of ccRCC. [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 4088.

PD17-12 SPATIAL MOLECULAR IMAGING IN SARCOMATOID RENAL CELL CARCINOMA REVEALS CROSSTALK BETWEEN IMMUNE CELLS AND TUMOR CELLS

You have accessJournal of UrologyCME1 Apr 2023PD17-12 SPATIAL MOLECULAR IMAGING IN SARCOMATOID RENAL CELL CARCINOMA REVEALS CROSSTALK BETWEEN IMMUNE CELLS AND TUMOR CELLS Allison May, Claire Williams, Stephanie The, Sean Kim, Nathan Schurman, Greg Shelley, Tyler Robinson, Simpa Salami, Rohit Mehra, Aaron Udager, and Evan Keller Allison MayAllison May More articles by this author , Claire WilliamsClaire Williams More articles by this author , Stephanie TheStephanie The More articles by this author , Sean KimSean Kim More articles by this author , Nathan SchurmanNathan Schurman More articles by this author , Greg ShelleyGreg Shelley More articles by this author , Tyler RobinsonTyler Robinson More articles by this author , Simpa SalamiSimpa Salami More articles by this author , Rohit MehraRohit Mehra More articles by this author , Aaron UdagerAaron Udager More articles by this author , and Evan KellerEvan Keller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sarcomatoid renal cell carcinoma (sRCC) is thought to arise by epithelial to mesenchymal transition (EMT) of the parental tumor in diverse RCC including clear cell RCC (ccRCC). sRCC is also known to be highly immunomodulatory. Cells undergoing EMT and immune cells can have reciprocal feedback on each other; however, the role of such crosstalk in sRCC is unknown. Here, we evaluate the heterogeneity of EMT within sRCC in correlation with the immune microenvironment and explore mechanisms of crosstalk. METHODS: A sRCC nephrectomy specimen was subjected to single cell spatial transcriptomics of 1000 mRNA targets (NanoString CosMX). Semi-supervised clustering was performed and cell types were assigned by reference to scRNAseq data and then mapped spatially. Differential gene expression and cell to cell distance analysis was performed. In-vitro assays included treatment of 786-O and Caki-1 ccRCC lines with recombinant SPP1 and protein and mRNA analysis. RESULTS: Histopathologic evaluation revealed a ccRCC zone, a transition zone (tz) between the ccRCC and sRCC in which the ccRCC cells gained an increased mesenchymal morphology, and a well-developed sRCC zone. Transcriptomic data revealed four distinct tumor cell populations: a ccRCC, a tzRCC, and two sRCC populations. Importantly, a subset of the histologically-categorized ccRCC had a tzRCC transcriptional signature, demonstrating this signature is identifiable prior to development of morphologic features of sRCC (Figure 1c). CCL20, a promoter of macrophage recruitment, was highly expressed in tzRCC. Distance analysis showed macrophages were highly spatially correlated with tzRCC and sRCC cells. Macrophages near tzRCC differentially expressed SPP1. In-vitro treatment of ccRCC cells with recombinant SPP1 led to induction of EMT and further upregulation of CCL20 expression. CONCLUSIONS: We report a unique tumor cell population characterized by ccRCC morphology but harboring a transcriptome suggestive of progression towards sRCC. This provides rationale for the development of a novel molecular signature to subclassify ccRCC. We identified key genes, CCL20 and SPP1, that mediate crosstalk between RCC cells and macrophages that may serve as targets to inhibit progression to sRCC. Source of Funding: Clarke Family Fellowship for Kidney Cancer Research © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e500 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Allison May More articles by this author Claire Williams More articles by this author Stephanie The More articles by this author Sean Kim More articles by this author Nathan Schurman More articles by this author Greg Shelley More articles by this author Tyler Robinson More articles by this author Simpa Salami More articles by this author Rohit Mehra More articles by this author Aaron Udager More articles by this author Evan Keller More articles by this author Expand All Advertisement PDF downloadLoading ...

PD24-08 SEQUENTIAL USAGE OF MTOR INHIBITOR FOLLOWED BY IMMUNOTHERAPIES FOR CABOZANTINIB-RESISTANT METASTATIC CLEAR CELL RENAL CELL CARCINOMAS

You have accessJournal of UrologyCME1 Apr 2023PD24-08 SEQUENTIAL USAGE OF MTOR INHIBITOR FOLLOWED BY IMMUNOTHERAPIES FOR CABOZANTINIB-RESISTANT METASTATIC CLEAR CELL RENAL CELL CARCINOMAS Jee Soo Park, Myung Eun Lee, Won Sik Jang, Jongchan Kim, Se Mi Park, and Won Sik Ham Jee Soo ParkJee Soo Park More articles by this author , Myung Eun LeeMyung Eun Lee More articles by this author , Won Sik JangWon Sik Jang More articles by this author , Jongchan KimJongchan Kim More articles by this author , Se Mi ParkSe Mi Park More articles by this author , and Won Sik HamWon Sik Ham More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003302.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cabozantinib, a multitargeted tyrosine kinase inhibitor (TKI), is an important option for the treatment of advanced clear cell renal cell carcinoma (accRCC), used as the first-line setting for intermediate- or poor-risk metastatic ccRCC, when immunotherapy is not available. We explored the underlying signaling pathway associated with the cabozantinib resistance and provided the optimal sequencing. METHODS: Cabozantinib-resistant RCC (CaR) cell lines were established by long-term treatment with cabozantinib. The mRNA expression profiles of PD-L1 and PI3K/AKT/mTOR signaling pathway proteins were investigated and compared in CaR and cabozantinib-sensitive (CaS) cell lines. Pulmonary metastatic orthotopic murine mRCC models have been developed with CaR Renca and CaS Renca. Mice were randomized and treated as 4 groups; 1) control (PBS treatment), 2) combination immunotherapy (anti-PD-1 and anti-CTLA-4 antibodies), 3) everolimus, and 4) sequential treatment of everolimus and followed by combination immunotherapy. RESULTS: All CaR cell lines demonstrated significant decreased in PD-L1 compared to CaS cell lines (p<0.05). Furthermore, the mRNA expression of major PI3K/AKT/mTOR pathway members, including PIK3CB, AKT3, and mTORC2 were increased in CaR Renca compared to CaS Renca. Everolimus treatment significantly increased PD-L1 expression in CaR Renca compared to CaS Renca. For murine mRCC models, CaR Renca model demonstrated significantly increased primary tumor growth, but decreased pulmonary metastasis compared to CaS Renca model. The best therapeutic benefits were obtained in group 4. CONCLUSIONS: We showed that cabozantinib decrease PD-L1 expression, while increase PI3K/AKT/mTOR signaling. Furthermore, mTOR inhibitor increased the PD-L1 expression in CaR cell line demonstrating that everolimus followed by combination immunotherapy can be the best subsequent treatment in CaR accRCC. Source of Funding: This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Ministry of Health & Welfare, Republic of Korea [grant number: HI17C1095], and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [grant number: 2019R1A2C1002863 and 2022R1A2C2003831] © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e725 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jee Soo Park More articles by this author Myung Eun Lee More articles by this author Won Sik Jang More articles by this author Jongchan Kim More articles by this author Se Mi Park More articles by this author Won Sik Ham More articles by this author Expand All Advertisement PDF downloadLoading ...

DIFFERENTIATION OF SOLID RENAL TUMOURS WITH MULTIPARAMETRIC MR IMAGING WITH HISTOLOGICAL CORRELATION

OBJECTIVES: To identify the radiologic ndings of the most common solid renal masses found in adult age group, illustrate imaging and histology correlates, and draw a clear diagnostic approach. 62 MATERIALS &amp; METHODS: patients suspected to have solid renal masses were evaluated with mp-MRI. T2 SIR, ADC value and ADC ratio on DWI, SII on chemical shift imaging and wash in and wash out indices on dynamic contrast enhanced imaging were calculated for each tumor and the values were compared among different histologically proven renal tumors. ccRCC showed maximum T2 SIR wherea RESULTS: s pRCC and lp-AML showed lowest mean T2 SIR. There is signicant difference in SII of clear cell RCC (15.03±7.62) and non-clear cell renal cortical tumor (1.54±10.67), p&lt;0.001. We found signicant difference in mean tumor ADC of benign vs malignant lesions (p &lt;0.006), higher stage (III &amp; IV) vs lower stage (stage I &amp;II) p &lt;0.001. We found signicant differences between clear cell and non-clear cell renal tumors in wash in indexes in all phases (p = 0.001) and late wash out index (p&lt; 0.001). There is signicant difference of papillary RCCs and other renal tumors in arterial and parenchymal WiI (p&lt;0.001), early WoI (p=0.018) as well as late WoI (p&lt;0.001). There is also signicant difference between chromophobe RCCs and oncocytomas for arterial WiI (p=0.009), parenchymal and late WiI (p= 0.006); Multiparametric renal MRI is useful CONCLUSION: for differentiating solid renal tumors, predict their histologic grading and thus guide further management

Fibroblast growth factor receptor type 4 as a potential therapeutic target in clear cell renal cell carcinoma

BackgroundSeveral clear cell renal cell carcinoma (ccRCC) cases harbour fibroblast growth factor receptor 4 (FGFR4) gene copy number (CN) gains. In this study, we investigated the functional contribution of FGFR4 CN amplification in ccRCC.MethodsThe correlation between FGFR4 CN determined via real-time PCR and protein expression evaluated using western blotting and immunohistochemistry was assessed in ccRCC cell lines (A498, A704, and 769-P), a papillary RCC cell line (ACHN), and clinical ccRCC specimens. The effect of FGFR4 inhibition on ccRCC cell proliferation and survival was assessed via either RNA interference or using the selective FGFR4 inhibitor BLU9931, followed by MTS assays, western blotting, and flow cytometry. To investigate whether FGFR4 is a potential therapeutic target, a xenograft mouse model was administered BLU9931.Results60% of ccRCC surgical specimens harboured an FGFR4 CN amplification. FGFR4 CN was positively correlated with its protein expression. All ccRCC cell lines harboured FGFR4 CN amplifications, whereas ACHN did not. FGFR4 silencing or inhibition attenuated intracellular signal transduction pathways, resulting in apoptosis and suppressed proliferation in ccRCC cell lines. BLU9931 suppressed tumours at a tolerable dose in the mouse model.ConclusionFGFR4 contributes to ccRCC cell proliferation and survival following FGFR4 amplification, making it a potential therapeutic target for ccRCC.

Possible role of combined therapy targeting MET and pro-HGF activation for renal cell carcinoma: analysis by human HGF-producing SCID mice

MET is a high-affinity receptor tyrosine kinase of HGF (hepatocyte growth factor). HGF is secreted as an inactive single-chain precursor (pro-HGF), which requires proteolytic activation for conversion to an active form. HGF activator inhibitor (HAI)-2 is a transmembrane Kunitz-type serine protease inhibitor, which inhibits all pro-HGF-activating enzymes. In RCC, increased expression of MET and decreased expression of HAI-2 were reported to be poor prognostic factors. In the current study, we tried to inhibit the growth of RCC cells by dual inhibition of both MET phosphorylation and pro-HGF-activation using MET inhibitor and HAI-2 overexpression. A transgenic mouse model which expressed human HGF (HGF mouse) was used for in vivo analysis to evaluate the HGF/MET signaling axis accurately. Initially, doxycycline-induced HAI-2 overexpression RCC cells (786-O-HAI2) were prepared. The cells were cultured with pro-HGF, and inhibitory effect of MET inhibitor (SCC244) and HAI-2 was evaluated by phosphorylation of MET and cell proliferation. Next, the cells were subcutaneously implanted to HGF mice and the growth inhibition was determined by SCC244 and HAI-2. Single use of each inhibitor showed significant inhibition in MET phosphorylation, migration and proliferation of 786-O-HAI2 cells; however, the strongest effect was observed by combined use of both inhibitors. Although in vivo analysis also showed apparent downregulation of MET phosphorylation and growth inhibition in combined treatment, statistical significance was not observed compared with single use of MET inhibitor. Combined treatment with MET-TKI and HAI-2 suggested to consider as a candidate for new strong therapy for RCC.