Expression In Renal Cell Carcinoma Research Articles

HECTD2 as a target for veratric acid in the regulation of ferroptosis in renal cell carcinoma

Function of HECTD2 in renal cell carcinoma malignant progression is undefined. Molecular mechanism behind anti-cancer effects of veratric acid (VA) from traditional Chinese medicine (TCM) is underexplored. The Cancer Genome Atlas was leveraged to study HECTD2 expression in renal cell carcinoma and its relationship with histological grading. Kaplan–Meier survival analysis was performed. HECTD2 expression was detected in cancer cells and tissues via qRT-PCR and immunohistochemistry. GPX4 and SLC7A11 expression in tumor samples with high or low HECTD2 expression was examined by immunohistochemistry, cell viability by CCK8, cell proliferation by colony formation assay, lipid ROS and mitochondrial superoxide levels by flow cytometry, Fe2+ and MDA content by assay kits, and GPX4 and SLC7A11 proteins by western blot. SeeSAR software screened TCM small molecule compounds with highest affinity to HECTD2, confirmed with cellular thermal shift assay. VA IC50 was measured by CCK8. Xenograft model was developed and treated with VA. Tumor size and weight were monitored, with immunohistochemistry to detect HECTD2 expression in tumors and assess ferroptosis-related markers. HECTD2 was overexpressed in tumor tissues and cells, which positively correlated with histological grading. HECTD2 depletion inhibited cell vitality and proliferation, raised intracellular lipid ROS, mitochondrial superoxide, Fe2+, and MDA. HECTD2 was a target with highest VA affinity. In vitro and vivo experiments concurred that VA treatment hindered malignancy of renal cell carcinoma and enhanced its susceptibility to ferroptosis. HECTD2 supports ferroptosis resistance in renal cell carcinoma, but VA, through its targeting of HECTD2, initiates ferroptosis, showcasing its anti-cancer efficacy.

TIGIT expression in renal cell carcinoma infiltrating T cells is variable and inversely correlated with PD-1 and LAG3

PurposeImmune checkpoint inhibitors have revolutionized the treatment of renal cell carcinoma (RCC), but many patients do not respond to therapy and the majority develop resistant disease over time. Thus, there is increasing need for alternative immunomodulating agents. The co-inhibitory molecule T-cell immunoglobulin and ITIM domain (TIGIT) may play a role in resistance to approved immune checkpoint inhibitors and is being investigated as a potential therapeutic target. The purpose of this study was to quantify TIGIT positivity in tumor-infiltrating T cells in RCC.MethodsWe employed tissue microarrays containing specimens from primary RCC tumors, adjacent normal renal tissue, and RCC metastases to quantify TIGIT within tumor-infiltrating CD3+ T cells using quantitative immunofluorescent analysis. We also compared these results to TIGIT+ CD3+ levels in four other tumor types (melanoma, non-small cell lung, cervical, and head and neck cancers).ResultsWe did not observe significant differences in TIGIT positivity between primary RCC tumors and patient-matched metastatic samples. We found that the degree of TIGIT positivity in RCC is comparable to that in lung cancer but lower than that in melanoma, cervical, and head and neck cancers. Correlation analysis comparing TIGIT positivity to previously published, patient-matched spatial proteomic data by our group revealed a negative association between TIGIT and the checkpoint proteins PD-1 and LAG3.ConclusionOur findings support careful evaluation of TIGIT expression on T cells in primary or metastatic RCC specimens for patients who may be treated with TIGIT-targeting antibodies, as increased TIGIT positivity might be associated with a greater likelihood of response to therapy.

Prognostic Significance of VAV3 Gene Variants and Expression in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is characterized by high mortality and morbidity rates. Vav guanine nucleotide exchange factors (VAVs), crucial for signal transduction between cell membrane receptors and intracellular mediators, have been implicated in carcinogenesis. However, their potential prognostic value in RCC remains unclear. The impact of 150 common VAV polymorphisms on RCC risk and survival was investigated in a cohort of 630 individuals. Publicly available gene expression datasets were utilized to analyze VAV gene expression in relation to patient outcomes. The VAV3 rs17019888 polymorphism was significantly associated with RCC risk and overall survival after adjusting for false discovery rates. Expression quantitative trait loci analysis revealed that the risk allele of rs17019888 is linked to reduced VAV3 expression. Analysis of 19 kidney cancer gene expression datasets revealed lower VAV3 expression in RCC tissues compared to normal tissues, with higher expression correlating with better prognosis. Gene set enrichment analysis demonstrated that VAV3 negatively regulates the ubiquitin-proteasome system, extracellular matrix and membrane receptors, inflammatory responses, matrix metalloproteinases, and cell cycle pathways. Furthermore, elevated VAV3 expression was associated with increased infiltration of B cells, macrophages, and neutrophils into the RCC tumor microenvironment. Our findings suggest that VAV3 gene variants influence RCC risk and survival, contributing to a favorable prognosis in RCC.

Clinical Importance of Focal Adhesion Kinase (FAK)-Src and Paxillin Expression in Renal Cell Carcinoma.

The complex focal adhesion kinase (FAK)/Src and paxillin seem to play a key role in the pathogenesis and progression of cancer. The aim of this study is to evaluate the expression of these proteins in renal cell carcinomas (RCCs), considering the immunoreactive score (IRS), the positivity and the intensity, and to find any association with patients' clinical characteristics, histologic type and other pathological features that imply a possible pathophysiological or prognostic role of FAK/Src and paxillin in RCC. Patients with RCC who had undergone partial or radical nephrectomy from January 2009 to September 2010 were eligible for this retrospective cross-sectional study. The immunohistochemical expression of FAK, Src and paxillin proteins in formalin-fixed paraffin-embedded tumour tissue was analysed in association with various clinicopathological features. Out of ninety patients, 58 had clear cell renal carcinoma, 15 had papillary, 11 had chromophobe and six had unclassified RCC. FAK, Src and paxillin were expressed in 55.6%, 32.2% and 18.9% of all cases, respectively.In univariate analysis, FAK positivity and IRS were more likely in patients with papillary and chromophobe histologic type versus clear cell RCC (p<0.005), Src positivity and IRS presented more frequently in stage T3 versus T1 (p<0.005) and paxillin positivity was more likely in patients with stage T3 versus T2 (p=0.021) and grades 3-4 versus grade 2 (p=0.013). Paxillin-IRS was not associated with any clinicopathological features. The same associations were also reproduced in the multifactorial analysis for the FAK and Src positivity and IRS, while it was found that paxillin positivity and IRS were associated with the female gender (p=0.052, p=0.024), and were higher in grades 3-4 versus grade 2 (p=0.022, p=0.020). Our study suggests that RCC shows immunohistochemical expression of FAK, Src and paxillin proteins, and this expression varies in relation to the histologic type, the stage and the stage/grade/gender, respectively. These findings imply a possible involvement of the FAK/Src signalling pathway in the pathogenesis and progression of cancer in RCC, providing future perspectives for targeted therapies with inhibitors.

Programmed death ligand I (PDL-1) Expression in renal cell carcinoma: A retrospective cohort study

Background and objectives: Inhibition of programed death -1and programed death ligand-1 pathway enhances antitumor activity of T lymphocytes, therefore, provides a new strategy for tumor treatment utilizing immunotherapy. The aim of this study to assess the frequency of programed death ligand-1 expression in renal cell carcinoma by using the immunohistochemistry and to correlate the results the clinicopathologic parameters. Methods: This is a cross sectional retrospective study performed in Duhok City from 2017-2022 on fifty-four formalin fixed paraffin embedded blocks of nephrectomy specimens diagnosed as renal cell carcinoma and collected from central lab of Duhok and some private labs. Two sections were prepared from each block, one section was stained with hematoxylin and eosin for histological analysis and the other one was used for immunohistochemical assessment of programed death ligand-1then the results were correlated with various clinicopathological parameters. Results: Programed death ligand -1; membranous expression was positive in 19 cases (35.2%) of renal cell carcinoma out of 54 cases. There was no significant correlation between programed death ligand -1 expression and age (p= 0.991), gender (p= 0.272), multifocality (p= 0.607), tumor size (p= 0.796), histological subtype (p= 0.107), tumor stage (p= 0.546), nuclear grade (p= 0.781), surgical margins involvement (p= 0.119) and lymphovascular invasion (p= 0.4), but there was statistically significant correlation with nodal metastases (p= 0.039). Conclusion: Programmed death ligand -1/ Combined positive score was ?1 in about one third (35.2%) of renal cell carcinoma cases and this result can be utilized for the provision of immune checkpoint inhibitor (ICIs), regardless the age, gender, histological type, stage, nuclear grade, and the presence of lymphovascular invasion (LVI).

Characterization of FOLH1 Expression in Renal Cell Carcinoma.

Given the emergence of PSMA-targeted diagnostic agents and therapeutics, we sought to investigate patterns of FOLH1 expression in RCC and their impacts on RCC outcomes. We conducted a pooled multi-institutional analysis of patients with RCC having undergone DNA and RNA next-generation sequencing. FOLH1-high/low expression was defined as the ≥75th/<25th percentile of RNA transcripts per million (TPM). Angiogenic, T-effector, and myeloid expression signatures were calculated using previously defined gene sets. Kaplan-Meier estimates were calculated from the time of tissue collection or therapy start. We included 1,724 patients in the analysis. FOLH1 expression was significantly higher in clear cell (71%) compared to non-clear cell RCC tumors (19.0 versus 3.3 TPM, p < 0.001) and varied by specimen site (45% primary kidney/55% metastasis, 13.6 versus 9.9 TPM, p < 0.001). FOLH1 expression was correlated with angiogenic gene expression (Spearman = 0.76, p < 0.001) and endothelial cell abundance (Spearman = 0.76, p < 0.001). While OS was similar in patients with FOLH1-high versus -low ccRCC, patients with FOLH1-high clear cell tumors experienced a longer time on cabozantinib treatment (9.7 versus 4.6 months, respectively, HR 0.57, 95% CI 0.35-0.93, p < 0.05). We observed differential patterns of FOLH1 expression based on histology and tumor site in RCC. FOLH1 was correlated with angiogenic gene expression, increased OS, and a longer duration of cabozantinib treatment.

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.

IL-6 and PIM1 expression in renal cell carcinoma.

IL-6 and PIM1 expression in renal cell carcinoma.

Evaluation of ARK5 and SIRT3 expression in renal cell carcinoma and their clinical significance

BackgroundGlobally Renal Cell Carcinoma (RCC) represents 3% of malignant tumours in adults and 1.78% in Egypt. AMPK-related protein kinase 5 (ARK5) is mainly associated with a hypoxic microenvironment which is a feature of the major RCC subtypes. Additionally, it displays decreased mitochondrial respiration. SIRT3 is a mitochondrial deacetylase that modifies multiple mitochondrial proteins.Material and methodsFifty eight cases of RCC, and 30 non-neoplastic cases (of End-Stage Kidney Disease (ESKD) were subjected to immunohistochemistry by ARK5 and SIRT3. The results of IHC were correlated together and correlated with the available clinicopathologic and survival data.ResultsAlthough no significant difference was detected between RCC and ESKD groups regarding ARK5 expression, there was a significant association with RCC regarding H-score and nucleocytoplasmic expression (both P = 0.001). Also, SIRT3 was highly expressed in RCC in comparison to the ESKD group (H-score: P = 0.001). There were significant associations between nucleocytoplasmic ARK5 expression and higher tumour grade, low apoptotic and high mitotic indices, tumour extent, advanced tumour stage, and impaired response of tumours to chemotherapeutic drugs (P = 0.039, P = 0.001, P = 0.027, P = 0.011, P = 0.009, and P = 0.014 respectively). Moreover, the H score of ARK5 expression showed significant associations with tumour grade, apoptotic and mitotic indices, tumour extension, tumour stage, and response to therapy (P = 0.01, 0.035, 0.001, 0.004. 0.003 and 0.013). Regarding SIRT3 expression, it showed significant associations with apoptotic and mitotic indices, tumour extent, tumour stage and response to therapy (P = 0.022, 0.02, 0.042, 0.039 and 0.027). Interestingly, there was a highly significant correlation between the expression of ARK5 and SIRT3 (P = 0.009). Univariate survival analysis revealed a significant association between short survival duration and both nucleocytoplasmic expression of ARK5 and positive SIRT3 expression (P = 0.014 and 0.035).ConclusionARK5 and SIRT3 are overexpressed in RCC and associated with parameters of poor prognosis as well as short survival. Both seem to influence response to therapy in RCC. So, they could be new targets for therapy that may improve tumour response and patients’ survival. There is a postulated relationship that needs more extensive investigation.

High XB130 expression in renal cell carcinoma is strongly associated with poor prognosis

The aim of this study was to assess the prognostic value of XB130 expression in three major RCC subtypes, and its association with clinical outcomes and adverse clinicopathologic features. A total of 101 nephrectomy samples at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand, from 2007 to 2017 were included in the study. XB130 immunohistochemistry was performed on slides from a tissue microarray comprised of 71 clear cell RCCs, 23 papillary RCCs, and 7 chromophobe RCCs, and were scored using a Histoscore system on a 0–300 scale. High XB130 expression in clear cell RCC and papillary RCC patients was associated with poor prognosis (log-rank test, P = 0.013, and P = 0.001, respectively). WHO/ISUP grade (P = 0.001) and XB130 high expression (P = 0.019) were found to be independent risk factors for mortality in clear cell RCC using multivariate analysis. The high expression of XB130 in clear cell RCC patients was also associated with high WHO/ISUP grade (P = 0.011), distant metastasis (P = 0.036), TNM stage (P = 0.007), sarcomatoid/rhabdoid differentiation (P = 0.061), and urinary collecting system invasion (P = 0.002). Similarly, high XB130 expression (P = 0.038) was associated with poor prognosis among papillary RCC patients as well as with lymphovascular invasion (P = 0.022), TNM stage (P = 0.030), and sarcomatoid/rhabdoid differentiation (P = 0.044). Overall, our findings showed that high XB130 expression in clear cell RCC and papillary RCC patients are associated with a worse prognosis.

Abstract A005: Regulation of HHLA2 expression in renal cell carcinoma and myeloid cells

Abstract The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in many tumors, including clear cell renal cell carcinoma (ccRCC). Multiple therapeutics directed against HHLA2 or its inhibitory receptor KIR3DL3 are about to enter clinical trials in metastatic RCC; however, little is known about the regulation of HHLA2 expression. Better understanding the regulation of HHLA2 in tumor cells and within the tumor microenvironment will facilitate translation of these therapeutic agents into the clinic by assisting in patient selection and development of rational drug combinations. Using flow cytometry, immunohistochemistry (IHC), and RNA analysis, HHLA2 expression was examined in monocytes, primary nephrectomy specimens and the established ccRCC cell lines, A498 and 786-0. A panel of cytokines was screened for the induction of HHLA2 in vitro. In vivo expression of HHLA2 in A498 and 786-0 human kidney cancer xenograft mouse models was examined. Tumor cells obtained from these xenograft models (either freshly isolated or cultured ex vivo) were characterized by RNASeq and flow cytometry. Analysis of HHLA2 expression in monocytes shows that among a wide range of cytokines tested, only IL-10 and BMP4, and to a lesser extent, IL-1b and IL-6, modestly enhanced HHLA2 mRNA and protein expression. These combinations of cytokines failed to induce HHLA2 in both A498 and 786-0. HHLA2 was frequently expressed in primary ccRCC tumors by IHC. Interestingly, analysis of HHLA2 by flow cytometry in primary RCC tumors showed that HHLA2 is progressively lost over 4 weeks when cells are cultured ex vivo. Hypoxia alone did not change HHLA2 expression. While HHLA2 was not expressed on either A498 or 786O cells in vitro, both A498 and 786-0 cells expressed HHLA2 when grown as subcutaneous xenografts in NSG immunodeficient mice. A498 xenografts express significantly more HHLA2 than 786-0 xenografts. HHLA2 expression is differentially regulated in monocytes and kidney cancer epithelial cells. High concentrations of cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in ccRCC; however, the tumor microenvironment in NSG mice significantly induced HHLA2 expression in both A498 and 786-0 xenografts. Complementary whole genome CRISPR knock out and gain-of-function screens are underway to assess by an unbiased means both nonimmune and immunologic regulators or repressors that may be responsible for inducing HHLA2 in kidney cancer in vivo. Furthermore, the A498 xenograft mouse model is an excellent humanized HHLA2 model for testing regulation of HHLA2 and HHLA2-directed therapeutics. Citation Format: Tomonari Shigemura, Nahuel Perrot, Zimo Huang, Aseman Bagheri Sheshdeh, Nourhan El Ahmar, David McDermott, Sabina Signoretti, Gordon Freeman, Kathleen Mahoney. Regulation of HHLA2 expression in renal cell carcinoma and myeloid cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A005.

The prognostic significance of CDK6 expression in renal cell carcinoma treated by immune checkpoint plus tyrosine kinase inhibition.

Checkpoint inhibitor immunotherapy plus tyrosine kinase inhibitor (IO/TKI) has become the first-line treatment for metastatic renal cell carcinoma (RCC), despite the lack of biomarkers. Cyclin-dependent kinase 6 (CDK6) has shown a regulatory role in antitumour response. The study enrolled two cohorts of metastatic RCC treated by IO/TKI (Zhongshan Hospital [ZS]-MRCC, n = 45; JAVELIN-101, n = 726) and two cohorts of localized RCC (ZS-HRRCC, n = 40; TCGA-KIRC, n = 530). CDK6 was evaluated by RNA-sequencing. Progression-free survival (PFS) was the primary endpoint. The prognostic role of CDK6 was evaluated by survival analysis. The correlation between CDK6 and tumour microenvironment was assessed by immunohistochemistry and flow cytometry. The high-CDK6 group displayed a lower response rate (13.6%) than the low-CDK6 group (56.5%) (P = .002). High-CDK6 was associated with poor PFS in both the ZS-MRCC cohort (high-CDK6, median PFS 6.4 months; low-CDK6, median PFS not reached; P = .010) and JAVELIN-101 cohort (high-CDK6, median PFS 10.0 months; low-CDK6, median PFS 13.3 month; P = .033). High-CDK6 was associated with increased PD1+ CD8+ T cells (Spearman's ρ = .47, P < .001) and decreased Granzyme B+ CD8+ T cells (Spearman's ρ = -.35, P = .030). Finally, a random forest score (RFscore) was built by integrating CDK6 and immunologic genes, which was associated with survival benefits of IO/TKI (RFscore-low, TKI vs IO/TKI, HR = 2.47, 95% CI 1.82-3.35, P < .001; RFscore-high, TKI vs IO/TKI, HR = 0.99, 95% CI 0.75-1.32, P = .963). Elevated CDK6 expression indicated resistance and poor PFS under IO/TKI therapy, which was related to exhausted CD8+ T cells. Integrated RFscore could evaluate the benefits of IO/TKI.

Long noncoding RNA UFC1 acts as an oncogene via stimulating EZH2-induced inhibition of APC expression in renal cell carcinoma.

This study aimed to illustrate the biological functions of long noncoding RNA (lncRNA) UFC1 in the carcinogenesis and cancer development of renal cell carcinoma (RCC), and the potential molecular mechanism. UFC1 levels in RCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Diagnostic and prognostic potentials of UFC1 in RCC were assessed by depicting receiver operating characteristic (ROC) curves and Kaplan-Meier curves, respectively. After transfection with si-UFC1, proliferative and migratory changes in ACHN and A498 cells were detected by cell counting kit-8 (CCK-8) and transwell assay, respectively. Subsequently, chromatin immunoprecipitation (ChIP) was conducted to examine the enrichments of EZH2 (enhancer of zeste homolog 2) and H3K27me3 in the APC promoter region. Finally, rescue experiments were carried out to identify the co-regulation of UFC1 and APC on RCC cell behaviors. The results showed that UFC1 was highly expressed in RCC tissues and cell lines. ROC curves revealed the diagnostic potential of UFC1 in RCC. Besides, survival analysis showed that highly expressed UFC1 predicted poor prognosis in RCC patients. Knockdown of UFC1 in ACHN and A498 cells attenuated cell proliferative and migratory abilities. UFC1 was able to interact with EZH2, and the knockdown of UFC1 could upregulate APC. In addition, both EZH2 and H3K27me3 were enriched in the APC promoter region, which could be blocked by the knockdown of UFC1. Moreover, rescue experiments demonstrated that the silence of APC was able to abolish the inhibited proliferative and migratory abilities in RCC cells with UFC1 knockdown. LncRNA UFC1 inhibits APC level through upregulating EZH2, thus aggravating the carcinogenesis and cancer development of RCC.

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.

MP04-16 EPITHELIAL TO MESENCHYMAL TRANSITION PROMOTES PD-L1 EXPRESSION IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP04-16 EPITHELIAL TO MESENCHYMAL TRANSITION PROMOTES PD-L1 EXPRESSION IN RENAL CELL CARCINOMA Allison May, Jonathan Jiang, Tyler Robinson, and Evan Keller Allison MayAllison May More articles by this author , Jonathan JiangJonathan Jiang More articles by this author , Tyler RobinsonTyler Robinson More articles by this author , and Evan KellerEvan Keller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sarcomatoid dedifferentiation in renal cell carcinoma (RCC) is thought to occur when the parental cell type, such as clear cell RCC (ccRCC), undergoes an epithelial mesenchymal transition (EMT). The mesenchymal sarcomatoid cells have increased ability to invade and metastasize, and thus sarcomatoid RCC (sRCC) has a very poor prognosis. Interestingly however, there is encouraging evidence that sRCC responds at higher rates to PD-1/PD-L1 directed immunotherapy than ccRCC. We aim to test the hypothesis that EMT directly upregulates PD-L1 expression in sRCC and that PD-L1 may have positive feedback on induction of EMT. METHODS: Baseline expression of EMT markers, EMT transcription factors and PD-L1 were measured using Western Blot and qPCR in two clear cell RCC lines (Caki-1 and 786-O) and five sarcomatoid RCC lines (RCJ-41T1, RCJ-41T2, RCJ-41M, UOK-276, UOK-127). EMT was induced in the cells through treatment with multiple agents including TGFB, IFNg, HGF, SPP1, and CoCl2 and by over expression of Snail or Zeb1. Effect of EMT on PD-L1 expression was measured. To evaluate the role of PD-L1 on EMT, PD-L1 expression was decreased using siRNA and its effect on EMT related protein expression was measured using Western Blot. RESULTS: At baseline, sRCC cell lines generally had higher expression of EMT related markers and transcription factors than ccRCC lines. Baseline PD-L1 expression positively correlated with increasing EMT state in all cell lines. Treatment of cells with known EMT inducers, TGFB, IFNg, and hypoxia led to increased levels of mesenchymal marker N-cadherin, decreased epithelial marker E-cadherin, and increased levels of EMT transcription factors Zeb1, Snail, and Slug. Treatment of cells with HGF and SPP1, two molecules associated with sRCC based on our prior work, also induced EMT. Finally, over expression of known EMT transcription factors Snail and Zeb1 led to EMT as well. All methods of EMT induction concomitantly increased PD-L1 expression. Knockdown of PD-L1 was tested in UOK-276 and 786-O cells and led to decreased expression levels of EMT transcription factors and increased epithelial markers. CONCLUSIONS: These data support the theory that sRCC arises from EMT and that EMT directly leads to upregulation of PD-L1. Furthermore, these results suggest that PD-L1 maintains the mesenchymal state. These findings may help explain the enhanced response to PD-1/PD-L1 directed therapy in sRCC and suggest that markers of EMT state could represent a biomarker for immunotherapy response in RCC. 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: e39 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Allison May More articles by this author Jonathan Jiang More articles by this author Tyler Robinson More articles by this author Evan Keller More articles by this author Expand All Advertisement PDF downloadLoading ...

Characterization of FOLH1 expression in renal cell carcinoma (RCC).

713 Background: The FOLH1 gene encodes prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein that is highly expressed in prostate cancer cells and on endothelial cells in the neovasculature of solid tumors, including RCC. PSMA has been used as a target for diagnostic imaging and therapeutic radioligand therapy. We utilized a database of molecularly profiled RCC tumors to evaluate associations with FOLH1 expression. Methods: NextGen sequencing of DNA (592-gene/whole exome) and RNA (whole transcriptome) was performed for RCC patient specimens (n=1765) through Caris Life Sciences (Phoenix, AZ). FOLH1-High/Low expression were defined as ≥75th/&lt;25th-percentile of RNA transcripts per million (TPM). Angiogenic, T-effector, and Myeloid expression signatures were calculated using previously defined gene sets (McDermott, 2018). Immune cell infiltration in tumor microenvironments (TMEs) was estimated using MCP-Counter (Becht, 2016). Tumor cell PD-L1+ expression (≥2+, ≥%5; SP142) was assessed by IHC. Kaplan-Meier estimates were calculated from time of tissue collection or therapy start. Results: FOLH1 expression was similar between sexes (71% male/29% female, 11.2 vs. 11.3 median TPM, p=0.54) and was not correlated with patient age at time of profiling (median 63 years, range 1-90+, spearman = 0.02, P=0.42). FOLH1 expression was significantly higher in clear cell RCC (ccRCC; 71.1% prevalence) compared to non-ccRCC tumors (19.0 vs 3.3 TPM, P&lt;0.001). FOLH1 expression varied by specimen site (45% kidney/55% metastatic, 13.6 vs. 9.9 TPM, P&lt;0.001), with notably lower expression in lymph nodes (5.3 TPM, P&lt;0.001, 8.2% prevalence). FOLH1 expression was strongly correlated with angiogenic gene expression compared to T-effector and myeloid signatures (spearman = 0.76 vs 0.33 and 0.20, respectively, each P&lt;0.001), with similar correlation strength observed for endothelial cell abundance in TMEs (spearman = 0.76 vs. 0.04-0.50 for immune cell types, P&lt;0.001). PD-L1+ IHC frequency was numerically lower yet not significantly different in FOLH1-High compared to -Low tumor among ccRCC (10 vs. 17%, P=0.07), but was similar among non-ccRCC (31 vs 32%, P=0.95). For patients stratified by median FOLH1 expression, no difference in overall survival from time of tissue sampling was observed for ccRCC (HR 1.2, P=0.57) or non-ccRCC cohorts (HR 0.77, P=0.59), while FOLH1-High was associated with numerically longer cabozantinib time-on-treatment (223 vs. 61 days, HR 0.60, P=0.08). Conclusions: We observed differential patterns of FOLH1 expression by histology and tumor site. FOLH1 expression was strongly correlated with angiogenic gene expression and distinct differences in TME composition, including endothelial cell abundance . FOLH1 gene expression was positively correlated with increased duration of anti-angiogenic treatment. Additional studies are needed to test the efficacy of PSMA-based diagnostics/therapeutics in RCC.

P53 and survivin expression in renal cell carcinoma.

Mutation of p53 is detected in more than 50% of human cancers, expression of p53 has a potential prognostic value in patients with renal cell carcinoma (RCC). Survivin is a member of the inhibitor of apoptosis protein family, its overexpression is observed in many malignancies, including RCC. The aim of the study was to estimate a correlation between survivin and p53 expression in tumor samples and the histologic type of a tumor, tumor stage, tumor grade, and survival of patients. Tumor samples were collected from surgical specimens of 90 patients who underwent radical or partial nephrectomy for RCC between November 2017 and July 2020. Tumors were staged according to the UICC (The Union for International Cancer Control) TNM classification system and histopathologically graded according to Fuhrman nuclear grade system. Histopathological diagnosis was confirmed with standard light microscopic evaluation, using hematoxylin and eosin staining and standard p53 and survivin antibodies. Positive p53 staining was observed in 36.7% of tumor specimens and 24.4% were survivin positive. There was a statistically significant correlation between p53 or survivin expression and histologic subtype of clear cell RCC as well as Type I and II of papillary RCC. There was a statistically significant correlation between p53 expression and tumor size, stage, and grade. The p53 or survivin expression was related to lower overall survival. The results of this study suggest that p53 overexpression and survivin positivity in RCC patients could be associated with poor prognosis. Thus, these proteins could be used as prognostic markers in RCC.

MiRNA-148a inhibits cell growth and drug resistance by regulating WNT10a expression in renal cell carcinoma.

BackgroundWe aimed to explore miR-148a exerts a tumor suppressor effect and arsenic trioxide (As2O3) sensitivity on renal cell carcinoma (RCC).MethodsWe performed polymerase chain reaction (PCR) on 42 pairs of tumor and paracancerous samples collected from RCC patients to investigate the miR-148a expression; meanwhile, we analyzed the interplay between clinical indicators and miR-148a expression of RCC. Then, the influence of miR-148a overexpression on the functions of RCC cells were analyze using transwell migration assay, Cell Counting Kit-8 (CCK-8), and cell wound healing assay. Furthermore, the ability of miR-148a to sensitize Caki-1 cells treated with As2O3 were detected using flow cytometry. Finally, the relevant mechanism of miR-148a on the downstream gene Wnt family member 10A (WNT10a) was explored by cell reverse method.ResultsThe results from RCC patients indicated a significantly lower miR-148a level than adjacent tissues. The low miR-148a expression increased prevalence of distant metastasis and decreased survival rate compared to those with high expression in patients. In the RCC cell lines, the proliferation and metastasis ability of the miR-148a mimic group was remarkably lower than the miR-NC group. At the same time, it was verified that WNT10a was remarkably higher cell lines and RCC tissues; and negatively related to miR-148a expression. In addition, miR-148a mimics were found to remarkably reduce the protein expression of WNT10a. In the cell reverse experiment, overexpression of WNT10a was confirmed to offset the miR-148a mimics effect on metastasis and proliferation of RCC cells. In addition, an increase in relative apoptosis was detected in As2O3 treated with/without miR-148a mimics for 48 hours, and apoptosis was significantly reduced after transfection with WNT10a in the Caki-1 cell line and significantly reduced after combined treatment.ConclusionsThe study revealed that miR-148a is associated with distant metastases and leads to poor prognosis in RCC patients. Moreover, miR-148a inhibit the malignant progression and increase the sensitivity of RCC cells to As2O3 by regulating WNT10a.

Abstract ND07: JNJ-78306358: A first-in-class bispecific T cell redirecting HLA-G antibody

Abstract JNJ-78306358 is a first-in-class bispecific antibody (bsAb), engineered using the Zymeworks Azymetric™ platform, to treat advanced stage solid tumors. Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility class I molecule with an immune tolerance role at the maternal-fetal interface. HLA-G has limited normal tissue expression, mainly detected in placenta and pituitary gland. However, HLA-G is expressed in multiple human cancers, with a potential role in cancer immune evasion. Comprehensive immunohistochemistry analysis of patient-derived tumors revealed high prevalence of HLA-G expression in renal cell carcinoma (RCC, 75%), ovarian (61%), colon (64%) and rectal cancers (40%), and moderate HLA-G expression prevalence in lung adenocarcinoma, endometrial, and pancreatic cancer. JNJ-78306358 induces HLA-G-expressing tumor cell killing via T cell redirection. This bsAb features an anti-HLA-G single-chain fragment variable (scFv) domain that binds with high affinity (KD ~ 13 pM) to HLA-G on tumor cells and a Fab domain that binds with weaker affinity (KD ~22 nM) to the epsilon subunit of the cluster of differentiation 3 (CD3ε). The immunoglobulin (Ig)G1 heavy chains feature Fc region mutations that disrupt interaction with Fcγ receptors. JNJ-78306358 demonstrated potent PBMC- and T cell-mediated in vitro cytotoxicity (EC50 10.4 - 442.3 pM) against endogenous membrane HLA-G-expressing tumor cell lines and absence of killing against cancer cells lacking HLA-G membrane expression, highlighting the specificity against antigen-expressing tumor cells. JNJ-78306358 exhibited hallmarks of T cell engagement in vitro, including T cell proliferation and cytokine release. In addition, JNJ-78306358 showed HLA-G-expression-dependent anti-tumor activity in mice (humanized with human donor CD3+ pan-T cells or human umbilical cord-blood-derived CD34+ hematopoietic stem cells [HSCs]) bearing cell line- and patient-derived tumors. In these xenograft models, a dose-dependent increase in CD4+ and CD8+ T cell infiltration into tumors was observed with complete tumor regressions at low doses of JNJ-78306358 (0.03 mg/kg). JNJ-78306358’s safety, tolerability and preliminary anti-tumor activity are currently being evaluated in a first-in-human phase I study in advanced stage solid tumors with high prevalence of HLA-G protein expression (NCT04991740). This antigen-targeting approach may address an unmet medical need in patients with tumors expressing HLA-G. Citation Format: Nataša Obermajer, Adam Zwolak, Kelly Van De Ven, Shana Versmissen, Aleksandra Brajic, Ted Petley, Dan Weinstock, Jason Aligo, Fang Yi, Stephen Jarantow, Keith Schutsky, Ken Tian, Angelilo Lorraine, Diana Alvarez Arias, Kristel Buyens, Vince Torti, Krista Menard, Katharine Rogers, Brian Geist, Marjolein Van Heerden, Gerald Chu, Bie Verbist, Maté Ongenaert, Julien Hasler, Kathryn Packman, Jacintha Shenton, Dirk Brehmer, Josh Lauring, Regina J. Brown, James Greger, Daphne Salick Ryan, Sanjaya Singh, Matthew V. Lorenzi, Laurie Lenox, Sylvie Laquerre. JNJ-78306358: A first-in-class bispecific T cell redirecting HLA-G antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND07.

In Silico, In Vitro, and Clinical Investigations of Cathepsin B and Stefin A mRNA Expression and a Correlation Analysis in Kidney Cancer.

The cysteine protease Cathepsin B (CtsB) plays a critical role in multiple signaling pathways, intracellular protein degradation, and processing. Endogenous inhibitors regulate its enzymatic activity, including stefins and other cystatins. Recent data proved that CtsB is implicated in tumor extracellular matrix remodeling, cell invasion, and metastasis: a misbalance between cathepsins and their natural inhibitors is often considered a sign of disease progression. In the present study, we investigated CtsB and stefin A (StfA) expression in renal cell carcinoma (RCC). mRNA analysis unveiled a significant CTSB and STFA increase in RCC tissues compared to adjacent non-cancerogenic tissues and a higher CtsB expression in malignant tumors than in benign renal neoplasms. Further analysis highlighted a positive correlation between CtsB and StfA expression as a function of patient sex, age, tumor size, grade, lymph node invasion, metastasis occurrence, and survival. Alternative overexpression and silencing of CtsB and StfA confirmed the correlation expression between these proteins in human RCC-derived cells through protein analysis and fluorescent microscopy. Finally, the ectopic expression of CtsB and StfA increased RCC cell proliferation. Our data strongly indicated that CtsB and StfA expression play an important role in RCC development by mutually stimulating their expression in RCC progression.