Human Renal Cell Carcinoma Samples Research Articles

Abstract 3457: Distinct signatures of tumor-associated macrophage subpopulations predict survival in renal cell carcinoma

Abstract Background: Comprising 90% of renal cancer cases in adults, renal cell carcinoma (RCC) is marked by significant heterogeneity in its tumor microenvironment. This study aims to test the hypothesis that tumor-associated macrophages (TAMs) play a role in modulating RCC progression and patient response to treatment. We explored the prognostic implications of TAM signatures on RCC survival, leveraging immunomics to decipher TAM-related alterations in the tumor microenvironment. Methods: Utilizing independent single-cell RNA-seq data from human RCC samples, we crafted 8 distinct RCC TAM signatures reflective of TAM presence. A LASSO Cox regression model was developed to prognosticate survival, tested against the TCGA dataset and independently validated across multiple RCC cohorts. Model performance was corroborated through Kaplan-Meier survival plots, receiver operating characteristic (ROC) curves, principal component analysis, and t-SNE analyses. Results: We identified two TAM signatures that correlate positively with patient survival, indicative of the abundance of specific subsets of TAMs in RCC. These signatures showed a strong association of specific TAM markers and macrophage infiltration. Survival analysis demonstrated that specific TAM signature gene expressions are significant prognostic markers. Forest plots underscored their prognostic relevance. A 32-gene risk score model was established, successfully stratifying patients into distinct risk categories, with low-risk patients exhibiting markedly improved overall survival. Conclusions: The study underscores the positive association between abundance of specific TAM subsets of and RCC patient survival. The precision of our LASSO Cox regression model, informed by novel abundance markers of specific TAM subsets, advances our understanding of TAM roles in TIME and RCC progression. These TAM signatures could potentially provide additional insights and targets to enhance the efficacy of RCC treatments. Keywords: Tumor associated macrophage, renal cell carcinoma, prognosis, tumor microenvironment, immunomics. Citation Format: Youngsoo Han, Aidan Shen, Zhaohui Wang, Aliesha Garrett, Chongming Jiang. Distinct signatures of tumor-associated macrophage subpopulations predict survival 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 3457.

FOXA2 activates HIF2α expression to promote tumor progression and is regulated by the E3 ubiquitin ligase VHL in renal cell carcinoma

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted renal cancer cell proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2α expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel‒Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2α expression, and promote renal cancer cell proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in RCC patients positively correlated with the expression of HIF2α and was associated with shorter overall and disease-free survival. Together, these findings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

S100 Calcium-Binding Protein A8 Functions as a Tumor-Promoting Factor in Renal Cell Carcinoma via Activating NF-κB Signaling Pathway

Background Renal cell carcinoma (RCC), arising from the renal tubular epithelium, is one of the most common types of genitourinary malignancies. Based on the Gene Expression Omnibus (GEO) database (GSE100666), S100 calcium-binding protein A8 (S100A8) was highly expressed in RCC tissues. S100A8, an inflammatory regulatory factor, has emerged as an important mediator associated with the occurrence and development of cancer. Materials and Methods The Gene Expression Omnibus (GEO) database was used to identify the key genes and investigate the main signaling pathways in RCC. Human RCC samples and corresponding adjacent normal tissues were collected in our hospital. The expression of S100A8 in human RCC samples was detected using western blotting and immunohistochemical analysis. S100A8 overexpression or knockdown was mediated by using Lipofectamine 3000 in human renal cell carcinoma cell line 786-O and ACHN cells. Basic experiments, including MTT and cell apoptosis assays, were utilized for investigating the function of S100A8 in RCC. Furthermore, the levels of inflammation were also evaluated in 786-O and ACHN cells. Results In the current study, we found that downregulation of S100A8 inhibited proliferation and promoted apoptosis in 786-O and ACHN RCC cells. Of note, S100A8 silencing downregulated the phosphorylation of NF-κB p65, thereby decreasing the levels of TNF-α, cleaved caspase1, and MMP9. By contrast, S100A8 upregulation could increase these expressions. Conclusion Overall, S100A8 knockdown restrained RCC malignant biological properties, which was associated with the deactivation of the NF-κB signaling pathway. This present study demonstrates new insights that S100A8 may be a potential therapeutic target in RCC.

PET Imaging of the Natural Killer Cell Activation Receptor NKp30.

Redirecting the immune system in cancer treatment has led to remarkable responses in a subset of patients. Natural killer (NK) cells are innate lymphoid cells being explored as they engage tumor cells in different mechanisms compared with T cells, which could be exploited for treatment of nonresponders to current immunotherapies. NK cell therapies are monitored through measuring peripheral NK cell concentrations or changes in tumor volume over time. The former does not detect NK cells at the tumor site, and the latter is inaccurate for immunotherapies because of pseudoprogression. Therefore, new imaging methods are required as companion diagnostics for optimizing immunotherapies. Methods: In this study, we developed and completed preclinical in vivo validation of 2 antibody-based PET probes specific for NKp30, an activation natural cytotoxicity receptor expressed by human NK cells. Quantitative, multicolor flow cytometry during a variety of NK cell activation conditions was completed on primary human NK cells and the NK92MI cell line. Human renal cell carcinoma (RCC) tumors were stained for the NK cell receptors CD56, NKp30, and NKp46 to determine expression on tumor-infiltrating NK cells. An NKp30 antibody was radiolabeled with 64Cu or 89Zr and evaluated in subcutaneous xenografts and adoptive cell transfer mouse models. Results: Quantitative flow cytometry showed consistent expression of the NKp30 receptor during different activation conditions. NKp30 and NKp46 costained in RCC samples, demonstrating the expression of these receptors on tumor-infiltrating NK cells in human tumors, whereas tumor cells in one RCC sample expressed the peripheral NK marker CD56. Both PET tracers showed high stability and specificity in vitro and in vivo. Notably, 89Zr-NKp30Ab had higher on-target contrast than 64Cu-NKp30Ab at their respective terminal time points. 64Cu-NKp30Ab delineated NK cell trafficking to the liver and spleen in an adoptive cell transfer model. Conclusion: The consistent expression of NKp30 on NK cells makes it an attractive target for quantitative imaging. Immunofluorescence staining on human RCC samples demonstrated the advantages of NKp30 targeting versus CD56 for detection of tumor infiltrating NK cells. This work advances PET imaging of NK cells and supports the translation of imaging agents for immunotherapy monitoring.

Patterns of Expression of H2S-Producing Enzyme in Human Renal Cell Carcinoma Specimens: Potential Avenue for Future Therapeutics.

Renal cell carcinoma (RCC) is the most common cancer of the kidney. The most common histotype is clear-cell (cc) RCC. Hydrogen sulfide (H2S) is an angiogenic and anti-apoptotic gasotransmitter that is elevated under pseudohypoxic conditions. H2S is endogenously produced by three enzymes: Cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST). Seeing as increased expression of these enzymes has been observed in other human cancer types, this study aimed to quantify H2S-producing enzyme expression in human RCC samples and evaluate whether it correlated with clinical outcomes. Eighty-eight human kidney tissue specimens, with healthy and cancerous tissue components, were immunohistochemically stained for CSE, CBS, and MPST. The mean pixel intensity of positively stained areas was quantified. A retrospective analysis was conducted to obtain patient demographics, rates of metastasis/recurrence, and prognostic characteristics. Statistical correlations between enzyme expressions and subsequent patient outcomes were evaluated. There was significantly greater expression of CSE, CBS, and MPST in cc-RCC compared to paired healthy tissue (p<0.0001). The difference in expression of CSE in cancerous versus normal tissue was significantly greater than that for CBS and MPST (p<0.0001 and p<0.01, respectively). Enzyme expression patterns in cancerous versus normal tissue did not correlate with nuclear grade, stage, histological type or cancer recurrence/metastasis. To our knowledge, this is the first report of the differential increase in expression of CSE, CBS, and MPST in human RCC. Although these patterns do not appear to correlate with cancer recurrence, metastasis, size or nuclear grade, their differential increase suggests a potential therapeutic target.

RBCK1 promotes p53 degradation via ubiquitination in renal cell carcinoma

Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies, and the incidence of RCC continues to rise worldwide. Although RCC can be treated with surgery at an early stages, the five-year survival rates have been observed to decline dramatically in patients with advanced disease. Most patients with RCC treated with cytotoxic or targeted drugs will develop resistance at some point during therapy. Thus, it is necessary to identify novel therapeutic targets for RCC. Here, we found that RANBP2-type and C3HC4-type zinc finger-containing 1 (RBCK1) expression was upregulated in human RCC samples. Analysis of multiple public databases revealed the correlation between RBCK1 expression and poor prognosis in RCC patients. Subsequently, we performed RBCK1 depletion experiments in RCC cells that severely affected the in vivo and in vitro proliferation of renal cancer cells. The effects of RBCK1 on cell proliferation could be rescued with p53 expression knockdown in two cell lines expressing wild-type p53. Further experiments demonstrated that RBCK1 could facilitate p53 poly-ubiquitination and degradation by direct interaction with p53. Together, our results show that RBCK1 may serve as a promising target for RCC therapy by restoring p53 functions.

High expression of fructose-bisphosphate aldolaseA induces progression of renal cell carcinoma.

Aldolase A (fructose-bisphosphate aldolaseA, ALDOA) is a glycolytic enzyme that catalyzes reversible conversion of fructose‑1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. ALDOA has been revealed to be related with many carcinomas, but its expression and function in renal cell carcinoma (RCC) remain unknown. This study aimed to detect expression of ALDOA in human RCC tissue samples and to explore its function in RCC cell lines. Reverse transcription-polymerase chain reaction was used to quantify ALDOA in human RCC samples. A total of 139RCC tissue samples obtained after surgery were analyzed in tissue microarray for ALDOA immunohistochemistry-based protein expression. Assays for cell cycle, viability, migration, and invasion were performed to assess phenotypic changes in RCC cells after ALDOA knockdown by small interfering RNA-mediated gene silencing approach and ALDOA upregulation by overexpression plasmids. Western blot analysis was used to identify alterations in markers for epithelial-mesenchymal transition (EMT), which affects metastasis and the Wnt/β‑catenin signaling pathway that influences RCC cell growth. ALDOA was upregulated in RCC samples and RCC cell lines (P<0.01). Expression of ALDOA was significantly associated with metastasis (P=0.020) and survival (P=0.0341). Downregulation of ALDOA suppressed proliferation (P<0.05) by triggering G0/G1 cell cycle arrest (P<0.05) and also inhibited migration (P<0.05) and invasion (P<0.01). Upregulation of ALDOA promoted proliferation (P<0.05) and enhanced migration (P<0.001) and invasion (P<0.001). Low expression of ALDOA could reverse EMT and inactivate the Wnt/β‑catenin signaling pathway. Our data revealed that ALDOA functions as a tumor promoter, plays a prominent role in proliferation, migration, and invasion of RCC cells with high expression, and may promote EMT and activate the Wnt/β‑catenin signaling pathway.

MiR-488 inhibits cell growth and metastasis in renal cell carcinoma by targeting HMGN5.

PurposemicroRNAs are thought to play crucial roles in tumorigenesis. Dysregulation of miR-488 has been implicated to be involved in several cancer progressions. However, the biological functions of miR-488 in renal cell carcinoma (RCC) remain unclear. This study aimed to explore the molecular mechanism underlying the role of miR-488 in RCC development.Materials and methodsThe expression levels of miR-488 were detected in 38 paired RCC tumor samples and cell lines by quantitative real-time polymerase chain reaction method. miR-488 was upregulated by mimics transfection in RCC cell lines. MTT, colony formation, transwell assay, flow cytometry assay, and a xenograft model were performed to determine cell proliferation, invasion, migration, epithelial-to-mesenchymal transition, and apoptosis in vitro and in vivo. Moreover, the potential target of miR-488 was verified by dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blot. The correlation between miR-488 expression and its target gene expression was confirmed by Spearman’s correlation analysis in 38 selected RCC tissue samples.ResultsWe found that miR-488 was remarkably downregulated in human RCC samples and cell lines compared with paired normal tissues and cell lines. Functional investigations revealed that overexpression of miR-488 significantly suppressed cell proliferation, invasion, and migration, and promoted cell apoptosis in RCC cells. Nucleosome binding protein 1 (high-mobility group nucleosome binding domain 5 [HMGN5]) was identified as a direct target of miR-488, and an inverse relationship was found between miR-488 expression and HMGN5 mRNA levels in RCC specimens. Rescue experiments suggested that restoration of HMGN5 partially abolished miR-488-mediated cell proliferation and invasion inhibition in RCC cells through regulating phosphatidylinositol 3-kinase/protein kinase B/the mammalian target of rapamycin and epithelial-to-mesenchymal transition signaling pathways.ConclusionThese data indicated that miR-488 acted as a tumor suppressor in RCC proliferation and invasion by targeting HMGN5, which might provide potential therapeutic biomarker for RCC patients.

MALDI Orbitrap Mass Spectrometry Profiling of Dysregulated Sulfoglycosphingolipids in Renal Cell Carcinoma Tissues.

Matrix-assisted laser desorption/ionization coupled with Orbitrap mass spectrometry (MALDI-Orbitrap-MS) is used for the clinical study of patients with renal cell carcinoma (RCC), as the most common type of kidney cancer. Significant changes in sulfoglycosphingolipid abundances between tumor and autologous normal kidney tissues are observed. First, sulfoglycosphingolipid species in studied RCC samples are identified using high mass accuracy full scan and tandem mass spectra. Subsequently, optimization, method validation, and statistical evaluation of MALDI-MS data for 158 tissues of 80 patients are discussed. More than 120 sulfoglycosphingolipids containing one to five hexosyl units are identified in human RCC samples based on the systematic study of their fragmentation behavior. Many of them are recorded here for the first time. Multivariate data analysis (MDA) methods, i.e., unsupervised principal component analysis (PCA) and supervised orthogonal partial least square discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples to reveal the most up- and downregulated lipids in tumor tissues. Obtained results are closely correlated with MALDI mass spectrometry imaging (MSI) and histologic staining. Important steps of the present MALDI-Orbitrap-MS approach are also discussed, such as the selection of best matrix, correct normalization, validation for semiquantitative study, and problems with possible isobaric interferences on closed masses in full scan mass spectra. Graphical Abstract ᅟ.

Infiltrating mast cells promote renal cell carcinoma angiogenesis by modulating PI3K\u2192AKT\u2192GSK3\u03b2\u2192AM signaling

The recruitment of vascular endothelial cells from the tumor microenvironment (TME) to promote angiogenesis plays key roles in the progression of renal cell carcinoma (RCC). The potential impact of immune cells in the TME on RCC angiogenesis, however, remains unclear. Here, we found that recruitment of mast cells resulted in increased RCC angiogenesis in both in vitro cell lines and in vivo mouse models. Mechanistic analyses revealed that RCC recruited mast cells by modulating PI3K→AKT→GSK3β→AM signaling. A clinical survey of human RCC samples also showed that higher expression of the PI3K→AKT→GSK3β→AM signaling pathway correlated with increased angiogenesis. Interruption of PI3K→AKT→GSK3β→AM signaling via specific inhibitors led to decreased recruitment of mast cells, and targeting this infiltrating mast cell-related signaling via an AKT-specific inhibitor suppressed RCC angiogenesis in xenograft mouse models. Together, these results identified a novel role of infiltrating mast cells in RCC angiogenesis and metastasis and suggest a new strategy for treating RCC by targeting this newly identified signaling pathway.

ATF2 predicts poor prognosis and promotes malignant phenotypes in renal cell carcinoma.

BackgroundActivating transcription factor 2 (ATF2) is a basic helix-loop-helix transcription factor, which has been shown to participate in the pathobiology of numerous cancers. However, the role of ATF2 in renal cell carcinoma (RCC) remains unclear.MethodsATF2 knockdown and overexpression studies were performed in RCC cells to evaluate changes in cell viability, cell cycle, apoptosis, migration and invasion. Xenograft models were used to examine the tumorigenic and metastatic capability of RCC cells upon ATF2 suppression. The expression of ATF2 in human RCC samples was determined using immunohistochemistry on a tissue microarray.ResultsATF2 knockdown in RCC cells reduced their proliferative and metastatic potentials, whereas ATF2 overexpression enhanced these properties. Mechanistic studies revealed that the transcription of CyclinB1, CyclinD1, Snail and Vimentin was directly regulated by ATF2 in RCC cells. Moreover, ATF2 was shown to be highly expressed in RCC tissues, especially in tumors with metastases. High expression of ATF2 correlated with aggressive clinico-pathological characteristics and predicted poor prognosis of RCC patients.ConclusionsATF2 exerts an oncogenic role in RCC and could serve as an important prognostic biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0383-2) contains supplementary material, which is available to authorized users.

Decreased apoptosis repressor with caspase recruitment domain confers resistance to sunitinib in renal cell carcinoma through alternate angiogenesis pathways

Apoptosis repressor with caspase recruitment domain (ARC), an endogenous inhibitor of apoptosis, is upregulated in a number of human cancers, thereby conferring drug resistance and giving a rationale for the inhibition of ARC to overcome drug resistance. Our hypothesis was that ARC would be similarly upregulated and targetable for therapy in renal cell carcinoma (RCC). Expression of ARC was assessed in 85 human RCC samples and paired non-neoplastic kidney by qPCR and immunohistochemistry, as well as in four RCC cell lines by qPCR, Western immunoblot and confocal microscopy. Contrary to expectations, ARC was significantly decreased in the majority of clear cell RCC and in three (ACHN, Caki-1 and 786-0) of the four RCC cell lines compared with the HK-2 non-cancerous human proximal tubular epithelial cell line. Inhibition of ARC with shRNA in the RCC cell line (SN12K1) that had shown increased ARC expression conferred resistance to Sunitinib, and upregulated interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). We therefore propose that decreased ARC, particularly in clear cell RCC, confers resistance to targeted therapy through restoration of tyrosine kinase-independent alternate angiogenesis pathways. Although the results are contrary to expectations from other cancer studies, they were confirmed here with multiple analytical methods. We believe the highly heterogeneous nature of cancers like RCC predicate that expression patterns of molecules must be interpreted in relation to respective matched non-neoplastic regions. In the current study, this procedure indicated that ARC is decreased in RCC.

MiR-134 functions as a tumor suppressor in cell proliferation and epithelial-to-mesenchymal Transition by targeting KRAS in renal cell carcinoma cells.

Aberrant microRNAs (miRNAs) are reported to contribute to the pathogenesis of most human malignancies. The miRNA, miR-134, has been found to be downregulated in renal cell carcinoma (RCC), but its function in the disease is unknown. The aims of this study were to detect the expression of miR-134 in human RCC samples and explore its function in RCC cell lines. Real-time qualitative polymerase chain reaction (qPCR) was used to quantify miR-134 in human RCC samples. Assays for cell cycle, viability, migration, and invasion were performed to assess the phenotypic changes in RCC cells. A luciferase reporter assay was carried out to confirm whether KRAS (Kirsten rat sarcoma viral oncogene homolog) is a direct target of miR-134. Western blot was used to identify the potential signaling pathways that had an impact on RCC cell growth and alterations of markers for epithelial-mesenchymal transition (EMT), which affected metastasis by miR-134. miR-134 was found to be downregulated in RCC samples (p<0.05), while overexpression of miR-134 suppressed proliferation (p<0.05) by triggering G1/G0 cell cycle arrest (p<0.05). Forced expression of miR-134 could also inhibit migration (p<0.05) and invasion (p<0.05) by blocking EMT in RCC cell lines. KRAS was identified as a target of miR-134, and miR-134 may act as a tumor suppressor through the KRAS-related MAPK/ERK pathway other than PI3K/AKT signaling. Thus, miR-134 may function as a tumor suppressor in cell proliferation and EMT by targeting KRAS in RCC cells.

Endoplasmic Reticulum Protein ERp46 in Renal Cell Carcinoma

An established inverse clinical correlation between serum adiponectin levels and renal cell carcinoma (RCC) aggressiveness exists. We have recently demonstrated that adiponectin suppresses clear cell RCC (ccRCC) progression through interaction with its receptor, adiponectin receptor 1 (AdipoR1). ERp46 has been shown to inhibit adiponectin signaling via interaction with AdipoR1 in HeLa cells. However, the expression of ERp46 in RCC has not been described thus far. The objectives of this study were to investigate ERp46 in RCC, its expression, its effects on RCC growth in a mouse model and whether it interacts with AdipoR1. We demonstrated a higher ERp46/AdipoR1 expression ratio in metastatic compared to non-metastatic ccRCC, as determined by immunohistochemistry of tissue microarrays and subsequent image analysis. When ERp46 was stably knocked down using shRNA or overexpressed in murine RCC RAG cells, RCC growth after subcutaneous injection in BALB/c nude mice was inhibited and accelerated, respectively. In vitro analysis to determine the molecular interaction between AdipoR1 and ERp46 included co-immunoprecipitation using human ccRCC 786-O cells and a bacterial adenylate cyclase-based two hybrid system and demonstrated no sustained AdipoR1-ERp46 interaction. This is the first report to suggest a role for ERp46 as a potential therapeutic target in RCC given its expression profile in human RCC samples and its effect on in vivo RCC growth. Since a stable interaction with AdipoR1 could not be established, we suggest that the tumorigenic properties of ERp46 in RCC cells are not related to an inhibitory modulation of AdipoR1.

Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10(-8) for clear cell; and p = 3.6 x 10(-4) for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

Von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR

We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription-polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-beta. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.

Loss of heterozygosity and protein expression of APC gene in renal cell carcinomas.

This study evaluated the potential contribution of the APC gene to malignant transformation in patients with renal cell carcinoma. We tested 36 human renal cell carcinoma samples and 18 adjacent normal kidney tissues for the expression of APC protein, both wild and truncated types, by western blot using antibodies that recognize either the carboxy or the amino epitope of the APC protein. The same tumor samples together with autologous peripheral blood were also analyzed at the DNA level. Using specific oligonucleotide primers for exons 11 and 15, gene instability was followed by polymerase chain reaction/loss of heterozygosity (LOH) (on the basis of restriction fragment length polymorphism). Molecular data were also compared to pathohistological diagnosis, TNM stage, and patient's age using multivariate statistical methods. All normal renal tissues revealed expression of the wild-type APC protein. Neither wild nor mutant type proteins were found in 36% (13/36) of tumor samples; the rest of tumor tissues expressed the wild-type protein (312 kDa). Mutated APC protein, with a molecular weight of 117 kDa, was found in only one tumor sample. From 36 tumor samples 16 (44.4%) were informative for RsaI exon 11 polymorphic site, while only half of these (8/16) demonstrated LOH. From 13 tumor samples that had no detectable protein product by western blot analysis eight were homozygous for the exon 11 polymorphism and were tested for another polymorphic site, MspI/exon 15. The overall proportion of LOH cases for both polymorphisms tested was 52.9% (9/17). Pathohistological diagnosis and molecular data showed no correlation. However, multivariate analysis determined a stage strong positive correlation of age and TNM with the presence of LOH and the absence of the wild-type APC protein. Out results suggest that the APC tumor suppressor gene plays a role in renal carcinogenesis. Alterations in this gene are responsible for tumor evolution and progression, but cannot be considered as a first event in tumor initiation.