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Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin Jr WGInhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1: 237-246
Abstract
Inactivation of the von Hippel-Lindau tumor suppressor gene is linked to the development of hereditary (VHL Disease-associated) and sporadic clear cell carcinoma of the kidney. The VHL gene product, pVHL, targets the heterodimeric transcription factor HIF for polyubiquitination, and restoration of pVHL function in VHL(-/-) renal carcinoma cells suppresses their ability to form tumors in nude mice. Here we show that tumor suppression by pVHL can be overridden by a HIF variant that escapes pVHL control. These studies prove that HIF is a critical downstream target of pVHL and establish that activation of HIF target genes can promote tumorigenesis in vivo.
... In the past decades, renal cell carcinoma (RCC) incidence has been increasing steadily, although the reasons for this remain unknown. The von Hippel-Lindau (VHL) is the most frequently mutated gene in RCC, accounting for ∼80% of RCC samples (1). Numerous studies have revealed that pVHL could act as an E3 ligase component to regulate the expression of several substrates including hypoxia inducible factor α (HIF-α), Zinc Fingers And Homeoboxes 2 (ZHX2), and Scm-like with four malignant brain tumor domains 1 (SFMBT1) (1)(2)(3)(4)(5). ...
... The von Hippel-Lindau (VHL) is the most frequently mutated gene in RCC, accounting for ∼80% of RCC samples (1). Numerous studies have revealed that pVHL could act as an E3 ligase component to regulate the expression of several substrates including hypoxia inducible factor α (HIF-α), Zinc Fingers And Homeoboxes 2 (ZHX2), and Scm-like with four malignant brain tumor domains 1 (SFMBT1) (1)(2)(3)(4)(5). Among them, the VHL-HIF pathway is one of the most characterized mechanisms contributing to clear cell RCC (ccRCC) tumorigenesis, primarily by activating the expression of multiple target genes, such as VEGF, GLUT1, and EPO (6). ...
... Among them, the VHL-HIF pathway is one of the most characterized mechanisms contributing to clear cell RCC (ccRCC) tumorigenesis, primarily by activating the expression of multiple target genes, such as VEGF, GLUT1, and EPO (6). One of key HIF subunits, HIF2α, has been confirmed to promote the occurrence and development of kidney cancer in vitro and in vivo (1,7,8). Therefore, targeting HIF2α represents one of most important therapeutic strategies for treating RCC (9). ...
Clear cell renal cell carcinoma (ccRCC) is characterized by the loss of tumor suppressor Von Hippel Lindau (VHL) function. VHL is the component of an E3 ligase complex that promotes the ubiquitination and degradation of hypoxia inducible factor α (HIF-α) (including HIF1α and HIF2α) and Zinc Fingers And Homeoboxes 2 (ZHX2). Our recent research showed that ZHX2 contributed to ccRCC tumorigenesis in a HIF-independent manner. However, it is still unknown whether ZHX2 could be modified through deubiquitination even in the absence of pVHL. Here, we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and identified USP13 as a DUB that bound ZHX2 and promoted ZHX2 deubiquitination. As a result, USP13 promoted ZHX2 protein stability in an enzymatically dependent manner, and depletion of USP13 led to ZHX2 down-regulation in ccRCC. Functionally, USP13 depletion led to decreased cell proliferation measured by two-dimensional (2D) colony formation and three-dimensional (3D) anchorage-independent growth. Furthermore, USP13 was essential for ccRCC tumor growth in vivo, and the effect was partially mediated by its regulation on ZHX2. Our findings support that USP13 may be a key effector in ccRCC tumorigenesis.
... These cells demonstrate increased expression of HIF-2a and VEGF proteins. This cell line has been used in numerous studies including initial studies demonstrating the function of the VHL/HIF pathway (24,36,44). In a study by Iliopoulos et al, restoration of pVHL in 786-O cells effectively suppressed tumor growth in nude mice (24). ...
... Kondo et al. demonstrated that the tumor suppression gained by pVHL can be overridden by a HIF variant that escapes pVHL control. This effectively demonstrated HIF as a target of pVHL and that unsuppressed activation of HIF promotes tumorigenesis (24,44). Additionally, 786-O cells were used to demonstrate that inhibition of HIF-2a with shRNA suppressed tumor growth in 786-O cell derived xenograft models (44). ...
... This effectively demonstrated HIF as a target of pVHL and that unsuppressed activation of HIF promotes tumorigenesis (24,44). Additionally, 786-O cells were used to demonstrate that inhibition of HIF-2a with shRNA suppressed tumor growth in 786-O cell derived xenograft models (44). About 2 decades later, belzutifan, a small molecule HIF-2a inhibitor would receive FDA approval for treatment of patients with VHL disease (45). ...
Modeling renal cell carcinoma is critical to investigating tumor biology and therapeutic mechanisms. Multiple systems have been developed to represent critical components of the tumor and its surrounding microenvironment. Prominent in vitro models include traditional cell cultures, 3D organoid models, and microphysiological devices. In vivo models consist of murine patient derived xenografts or genetically engineered mice. Each system has unique advantages as well as limitations and researchers must thoroughly understand each model to properly investigate research questions. This review addresses common model systems for renal cell carcinoma and critically evaluates their performance and ability to measure tumor characteristics.
... Hypoxia-inducible gene expression is regulated by HIF2a in the VHL -/ -RCC cell line 786-O while HIF1a is not expressed (5,27). It has recently been shown that overexpression of a HIF2a, but not HIF1a, mutant that escapes pVHL-mediated degradation in this cell line overrode the ability of reintroduced pVHL to suppress tumor formation, indicating that ubiquitin-dependent degradation of HIF is necessary for pVHL-mediated tumor suppression (28). Notwithstanding the limitations of overexpression gain-of-function experiments, we wanted to investigate whether inactivation of HIF2a is sufficient for tumor suppression in VHL -/ -786-O cells. ...
... It is currently not known to what extent the functions of HIF1a and HIF2a overlap if expressed in the same cellular context. Overexpression of a transcriptionally active HIF2a mutant, which escapes regulation by pVHL, overrides the tumor suppression effect of pVHL (28). Interestingly, the equivalent HIF1a mutant isoform failed to do so in the same cell line (21). ...
The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate receptor for the mammalian SCF-2 E3 ubiquitin ligase complex that targets several substrates for ubiquitination and proteasomal degradation. Among these targets are the α-regulatory subunits of the hypoxia-inducible factor (HIF). VHL−/− cells constitutively overexpress hypoxia-inducible genes through both transcriptional and posttranscriptional mechanisms and form tumors when injected into nude mice. Reintroduction of pVHL into VHL−/− cell lines restores normal oxygen-dependent regulation of these genes and suppresses tumor formation in the mouse xenograft assay. We report here that short hairpin RNA-mediated inactivation of HIF phenocopies the effects of pVHL reintroduction with respect to decreased expression of hypoxia-inducible genes, decreased ability to promote vascular endothelial cell proliferation in vitro, and tumor growth suppression in vivo. In addition, HIF inactivation abrogated the cellular response to hypoxia, indicating that HIF is the only pVHL target required for this response. These data suggest that deregulation of hypoxia-inducible genes in VHL−/− cells can be attributed mainly to deregulation of HIF and validate HIF as a therapeutic anticancer drug target.
... Therefore, hypoxia or VHL inactivation results in the stabilization and accumulation of HIFα isoforms, mainly HIF-1α and/or HIF-2α 10 . HIF-1/2α then translocates into the nucleus and forms a functional heterodimer with the constitutively expressed β subunit (HIF-β), thus leading to the expression of multiple target genes involved in angiogenesis, cell proliferation, and other biological processes [11][12][13] . HIF-1α and HIF-2α were previously thought to promote tumor progression through largely overlapping functions 10 . ...
... Whether MIIP regulates HIF-1α and HIF-2α through these 2 mechanisms simultaneously, differentially, or in a cancer-type dependent manner remains to be investigated. Although HIF-1α and HIF-2α share significant homology, a tripartite structure, and a degradation pathway, HIF-2α, but not HIF-1α, has been clearly established as the dominant isoform that plays a crucial role in ccRCC initiation and progression 8,10,12,47,48 . Importantly, eliminating HIF-2α is sufficient to suppress VHL-defective tumor growth 49,50 . ...
Objective:
In various cancers, migration and invasion inhibitory protein (MIIP) is expressed at low level and is involved in cancer pathogenesis. Herein, we sought to explore the function of MIIP in clear cell renal cell carcinoma (ccRCC).
Methods:
CCK-8, colony formation, cell cycle, and endothelial cell tube formation assays were performed to evaluate the roles of MIIP in ccRCC proliferation and angiogenesis. To explore the underlying mechanism, we conducted RNA-sequencing, GSEA, qRT-PCR, Western blot, ELISA, cell transfection, coimmunoprecipitation, and ubiquitination assays in ccRCC cell lines. Furthermore, xenograft tumor growth in nude mice, and Ki-67 and CD31 staining in xenograft tissues were examined. Finally, the association of MIIP expression with clinical pathology and the expression status of HIF-2α and cysteine-rich 61 (CYR61) were further analyzed in human RCC tissues through Western blot and immunohistochemistry.
Results:
Both in vitro and in vivo functional experiments indicated that forced expression of MIIP inhibited ccRCC proliferation and angiogenesis, whereas silencing MIIP either in normal HK-2 cells or in ccRCC cells had the opposite effect (P < 0.05). Mechanistically, CYR61 was identified as a gene significantly downregulated by MIIP overexpression, and was required for the suppressive role of MIIP in ccRCC. MIIP was found to promote HSP90 acetylation and thus impair its chaperone function toward HIF-2α. Consequently, RACK1 binds HIF-2α and causes its ubiquitination and proteasomal degradation, thus decreasing the transcription of its target, CYR61. Finally, analyses of clinical samples demonstrated that MIIP is significantly downregulated in cancer vs. normal tissues in RCC cases, and its expression is negatively associated with histological grade, metastasis, the prognosis of patients with RCC, and the expression of HIF-2α and CYR61 (P < 0.05).
Conclusions:
MIIP is a novel tumor suppressor in ccRCC via negative regulation of HIF-2α-CYR61 axis.
... The VHL protein, pVHL, plays an essential role in the mammalian oxygen-sensing pathway and regulates the expression of hypoxia-inducible factors (HIF1α and HIF2α) via ubiquitinmediated degradation [7][8][9]. In the absence of pVHL, HIF1α and HIF2α are stabilized and induce the hypoxia response program, such as angiogenesis [10,11]. Consequently, ccRCC tumors are highly vascularized, with high metastatic potential frequently disseminating to the lungs [12][13][14]. ...
Exosomes are extracellular vesicles that modulate essential physiological and pathological signals. Communication between cancer cells that express the von Hippel-Lindau (VHL) tumor suppressor gene and those that do not is instrumental to distant metastasis in renal cell carcinoma (RCC). In a novel metastasis model, VHL(−) cancer cells are the metastatic driver, while VHL(+) cells receive metastatic signals from VHL(−) cells and undergo aggressive transformation. This study investigates whether exosomes could be mediating metastatic crosstalk. Exosomes isolated from paired VHL(+) and VHL(−) cancer cell lines were assessed for physical, biochemical, and biological characteristics. Compared to the VHL(+) cells, VHL(−) cells produce significantly more exosomes that augment epithelial-to-mesenchymal transition (EMT) and migration of VHL(+) cells. Using a Cre-loxP exosome reporter system, the fluorescent color conversion and migration were correlated with dose-dependent delivery of VHL(−) exosomes. VHL(−) exosomes even induced a complete cascade of distant metastasis when added to VHL(+) tumor xenografts in a duck chorioallantoic membrane (dCAM) model, while VHL(+) exosomes did not. Therefore, this study supports that exosomes from VHL(−) cells could mediate critical cell-to-cell crosstalk to promote metastasis in RCC.
... The disease is caused by mutations of the tumor suppressor gene VHL [310,311]. The VHL gene product (pVHL) is best known to target the transcription factor hypoxia-induced factor (HIF) for polyubiquitination and thus destruction [312][313][314]. In 2006, a study by Nakamura et al. showed the importance of CLU in VHL tumorigenesis and how CLU induction led to an HIF-independent pVHL function that could prove crucial for tumor suppression. ...
Clusterin (CLU) is a glycoprotein originally discovered in 1983 in ram testis fluid. Rapidly observed in other tissues, it was initially given various names based on its function in different tissues. In 1992, it was finally named CLU by consensus. Nearly omnipresent in human tissues, CLU is strongly expressed at fluid–tissue interfaces, including in the eye and in particular the cornea. Recent research has identified different forms of CLU, with the most prominent being a 75–80 kDa heterodimeric protein that is secreted. Another truncated version of CLU (55 kDa) is localized to the nucleus and exerts pro-apoptotic activities. CLU has been reported to be involved in various physiological processes such as sperm maturation, lipid transportation, complement inhibition and chaperone activity. CLU was also reported to exert important functions in tissue remodeling, cell–cell adhesion, cell–substratum interaction, cytoprotection, apoptotic cell death, cell proliferation and migration. Hence, this protein is sparking interest in tissue wound healing. Moreover, CLU gene expression is finely regulated by cytokines, growth factors and stress-inducing agents, leading to abnormally elevated levels of CLU in many states of cellular disturbance, including cancer and neurodegenerative conditions. In the eye, CLU expression has been reported as being severely increased in several pathologies, such as age-related macular degeneration and Fuch’s corneal dystrophy, while it is depleted in others, such as pathologic keratinization. Nevertheless, the precise role of CLU in the development of ocular pathologies has yet to be deciphered. The question of whether CLU expression is influenced by these disorders or contributes to them remains open. In this article, we review the actual knowledge about CLU at both the protein and gene expression level in wound healing, and explore the possibility that CLU is a key factor in cancer and eye diseases. Understanding the expression and regulation of CLU could lead to the development of novel therapeutics for promoting wound healing.
... Unsurprisingly, given the accumulation of HIF under normoxic conditions in ccRCC, HIF driven transcriptional activation plays a prominent role in tumor growth. Evidence from studies using HIFα knockdown, VHL reintroduction, or stabilized HIFα variants implicate the HIF-2α isoform as the tumorigenic driver in ccRCC [13][14][15][16][17][18]. Selective small molecule antagonism of HIF-2α could therefore have important applications in the treatment of ccRCC. ...
Pharmacokinetic properties of our first-generation HIF-2α antagonist PT2385, including modest solubility, resulted in a high recommended phase 2 dose (RP2D) of 800 mg BID and motivated the pursuit of novel scaffolds which could improve solubility and formulation parameters with the goal of improved pharmacokinetics. Herein we disclose our successful efforts to identify such HIF-2α antagonists through an optimization strategy characterized by: (1) increasing the fraction of sp³ hybridized carbons (Fsp³), (2) replacing the aromatic portion of the indane core with pyridine heterocycles, and (3) improving a putative Olp→π*Ar interaction, an underutilized electrostatic contact in medicinal chemistry. These efforts emphasize the importance of employing multiple strategies in parameter optimization. In isolation, modifications to areas (1) and (2) improved solubility, but with the compromise of reduced potency. In area (3), understanding the importance of an Olp→π*Ar interaction, as documented through a wealth of crystal structures and retrospective calculations, proved essential in guiding SAR and identifying the trifluoromethyl group as a suitable replacement of the sulfone. Only by combining these three strategies could inhibitors with substantially improved solubility and comparable potency be discovered. Finally, the overall improvement in pharmacokinetic properties of the newly identified inhibitors is highlighted through a battery of ADME and in vivo data, including use of pharmacodynamic biomarkers indicative of HIF-2α antagonism.
... Mutations in EPAS1 are considered to be drivers in pheochromocytoma and paraganglioma [7][8][9]. In renal cell carcinoma (RCC), the HIF-2α regulation is linked to the cancer development in vivo and in vitro [10][11][12]. And preclinical studies and clinical data validate that HIF-2α inhibitors have antitumor activity in RCC cell lines and heavily pretreated patients [13][14][15][16]. ...
EPAS1 plays an important role in the development and progression of multiple tumor types by interacting with a series of other molecules. However, the prognostic and diagnostic values of EPAS1 in acute myeloid leukemia (AML) remain unknown. Here, we systematically explored and clarified the potential functions of EPAS1 in AML using data from Xena Browser and TCGA database. The expression of EPAS1 was significantly lower in AML patients than that in healthy people. The GO, KEGG, GSEA, and GSVA were performed to explore the potential functions and signaling pathways. The survival analysis was conducted using Cox regression analysis and the Kaplan-Meier method. Immune cell infiltration was evaluated via single-sample GSEA (ssGSEA). The results of enrichment analyses suggested that low-EPAS1 expression was related to the initiation, development, and prognosis of AML. The immune microenvironment landscape in AML was described by ssGSEA. ROC analysis of EPAS1 showed high discrimination ability between AML patients and healthy people. Kaplan-Meier method indicated that low-EPAS1 expression correlated significantly with a poor overall survival. Multivariate Cox regression analysis revealed that both age and EPAS1 expression were independent prognostic factors in AML patients. Furthermore, the nomogram based on these two variables performed well in discrimination and calibration. In summary, our study may provide new insights into the molecular mechanisms underlying AML and demonstrate the diagnostic and prognostic value of EPAS1 in AML for the first time.
... These oncometabolites competitively inhibit the HIF-PHD interaction, which requires 2-oxoglutarate. This pseudohypoxic phenotype results in HIFα-stabilization and upregulation of HIF-inducible genes [125][126][127][128]. In addition, they block a group of α-KG dependent dioxygenases leading to epigenetic dysregulation. ...
Renal cancer management is challenging from diagnosis to treatment and follow-up. In cases of small renal masses and cystic lesions the differential diagnosis of benign or malignant tissues has potential pitfalls when imaging or even renal biopsy is applied. The recent artificial intelligence, imaging techniques, and genomics advancements have the ability to help clinicians set the stratification risk, treatment selection, follow-up strategy, and prognosis of the disease. The combination of radiomics features and genomics data has achieved good results but is currently limited by the retrospective design and the small number of patients included in clinical trials. The road ahead for radiogenomics is open to new, well-designed prospective studies, with large cohorts of patients required to validate previously obtained results and enter clinical practice.
... . WT8 + HIF2αP531A cells fully restored their ability to form tumors in nude mice irrespective of their positive status of pVHL.58 Because tumor suppression by pVHL could be overridden by a HIF-2α variant that escapes pVHL control, our results strongly suggested that HIF-2α might be a driver in the tumor progression of ccRCC.In a subsequent study in 2005, Dr. Raval et al. infected 786-O VHL −/− RCC cells with retroviruses expressing HIF-1α, HIF-2α, or GFP alone and implanted cells subcutaneously into nude mice. ...
Intra-tumoral hypoxia is associated with tumor progression and therapeutic resistance. The VHL tumor suppressor gene was identified in 1993, and later studies revealed that the gene product pVHL interacts with other proteins to form the VBC complex. The VBC complex functions as an E3 ubiquitin ligase and regulates the abundance of the α-subunit of the transcription factor HIF. HIF regulates thousands of genes required for cells to adapt and survive in hypoxic conditions, and thus pVHL play a major role in oxygen-sensing pathways. Patients with VHL disease, harboring a germline mutation of the VHL gene, develop renal cell carcinomas and a series of tumors exhibiting hypervascular phenotypes. The extensive findings that have clarified the function of VHL have contributed to the development of novel first-in-human drugs, including belzutifan, a HIF-2α inhibitor. The 2019 Nobel Prize in Physiology or Medicine was awarded to Dr. William G. Kaelin Jr., Dr. Peter J. Ratcliffe, and Dr. Gregg L. Semenza as researchers contributing to clarifying the mechanism of the oxygen-sensing pathway of cells. The first report of VHL disease was in 1894, meaning the development of a specific drug for this disease took almost 125 years. In this article, we describe how researchers and clinician scientists successfully clarified the function of VHL and achieved a preclinical proof of concept to apply for clinical trials, key requirements for drug development.
... Expression of HIF2α can induce xenograft renal cancer cell line formation and overcome VHL suppressive effects. In contrast, HIF2α suppression inhibits renal cancer cell in mice (22,23), while HIF1α has the opposite effect in these models (24). While it is largely accepted that overexpression of HIF2α promotes angiogenesis and tumor invasion, its complete loss may contribute to oncogenesis in some models, suggesting that therapeutic benefit of HIF2α inhibition may be context dependent (12,25). ...
Tumors driven by deficiency of the VHL gene product, which is involved in degradation of the hypoxia inducible factor subunit 2 alpha (HIF2α), are natural candidates for targeted inhibition of this pathway. Belzutifan, a highly specific and well-tolerated HIF2α inhibitor, recently received FDA approval for the treatment of nonmetastatic renal cell carcinomas, pancreatic neuroendocrine tumors, and central nervous system hemangioblastomas from patients with von Hippel Lindau disease, who carry VHL germline mutations. Such approval is a milestone in oncology; however, the full potential, and limitations, of HIF2α inhibition in the clinic are just starting to be explored. Here we briefly recapitulate the molecular rationale for HIF2α blockade in tumors and review available preclinical and clinical data, elaborating on mutations that might be particularly sensitive to this approach. We also outline some emerging mechanisms of intrinsic and acquired resistance to HIF2α inhibitors, including acquired mutations of the gatekeeper pocket of HIF2α and its interacting partner, ARNT. Lastly, we propose that the high efficacy of Belzutifan observed in tumors with genetically driven hypoxia caused by VHL mutations suggests that a focus on other mutations that similarly lead to HIF2α stabilization, such as those occurring in neuroendocrine tumors with disruptions in tricarboxylic acid cycle (TCA) cycle (SDHA/B/C/D, FH, MDH2, IDH2), HIF hydroxylases (EGLN/PHDs) and the HIF2α-encoding gene, EPAS1, are warranted.
... 31,32 HIF-2α has been established as the major tumor-promoting isoform of HIFα in pVHL-defective ccRCC. [33][34][35] While HIF-1α has been shown to have a tumor-promoting role in several other malignancies, 36,37 its cumulative role in pVHLdefective ccRCC is extensively debated, and continues to be an area of active investigation. 35,[38][39][40][41] Both isoforms have overlapping as well as distinct functions/ target genes mediating the hypoxic response, which varies with cellular contexts. ...
Von Hippel Lindau (VHL) disease is a tumor predisposition syndrome caused by mutations in the VHL gene that presents with visceral neoplasms and growths, including clear cell renal cell carcinoma, and central nervous system manifestations, such as hemangioblastomas of the brain and spine. The pathophysiology involves a dysregulation of oxygen sensing caused by inability to degrade HIFα, leading to overactivation of hypoxic pathways. Hemangioblastomas are the most common tumors in patients with VHL and cause significant morbidity. Until recently, there were no systemic therapies available for patients that could effectively reduce the size of these lesions. Belzutifan, the first approved HIF-2α inhibitor, has demonstrated benefit in VHL-associated tumors, with a 30% response rate in hemangioblastomas and ~30-50% reduction in their sizes over the course of treatment. Anemia is the most prominent adverse effect, affecting 76-90% of participants and sometimes requiring dose reduction or transfusion. Other significant adverse events include hypoxia and fatigue. Overall, belzutifan is well tolerated; however, long term data on dosing regimens, safety, and fertility are not yet available. Belzutifan holds promise for treatment of neurological manifestations of VHL and its utility may influence the clinical management paradigms for this patient population.
... Clear cell renal cell carcinoma (ccRCC) makes up~75% of renal malignancies and accounts for most of the renal cancerassociated death. ccRCC (> 90%) is mainly caused by loss-of-function mutations or deletion of the Von Hippel-Lindau (VHL) tumor suppressor gene, which results in stabilization and constitutive activation of Hypoxia-inducible factor 2α (HIF-2α) and ectopic expression of its target genes including those encoding glucose transporter type 1 (GLUT1) and vascular endothelial growth factor (VEGF) that regulate glycolysis and angiogenesis, respectively 1 . ...
... Very few respondents (2%) preferred to start belzutifan prophylactically to prevent the development of tumors. In patients with renal tumors <3 cm and low anticipated morbidity with intervention, 36% preferred to recommend active surveillance and local therapy only if the lesion surpassed the size threshold, 36% would initiate belzutifan to prevent growth, and 28% preferred to recommend active surveillance with consideration of belzutifan oncogenic driver of both malignant and benign tumors (14,15). Although prior attempts at drugging this key transcription factor have been largely unsuccessful (16), belzutifan recently demonstrated high efficacy in treating VHL-associated tumors with low toxicity. ...
Belzutifan was recently approved for the management of Von Hippel–Lindau disease (VHL). Given the morbidity of recurrent treatment, systemic therapy to reduce or eliminate the need for surgery has been long-awaited. Herein, we sought to gain insight about future utilization by surveying VHL experts in the United States. A survey developed by members of the VHL Alliance (VHLA) Clinical Advisory Council was distributed to kidney cancer providers at VHLA and National Comprehensive Cancer Network (NCCN) centers. Surveys were administered on a secure web-based platform. A total of 60 respondents from 29 institutions participated. Urologists (50%) and medical oncologists (43%) represented the majority of participants. The majority (98%) of respondents anticipated that belzutifan’s approval would signifi-cantly change the current treatment landscape. Most reported that therapy should be continuous (76%). There was a difference in willingness to prescribe belzutifan by specialty (38% of urologists vs 91% of medical oncologists (P = 0.02). In individuals with renal tumors <3 cm, 36% would still recommend surveillance, while 36% would initiate belzutifan to prevent growth. In those with multifocal renal lesions and growth of a solitary tumor on belzutifan, 50% would proceed with only treatment of that site. In conclusion, VHL kidney cancer specialists anticipate a paradigm shift with the approval of belzutifan. Provider roles may change with movement away from surgical management. Opinions on treatment indications, such as when to initiate therapy and how to best salvage, vary widely and collaborative efforts among experts may assist in the development of clinical guidelines.
... The earliest recognizable pVHL-defective lesions arising in the kidneys of VHL patients demonstrate an increase in HIF and HIF target genes, with the appearance of HIF2 in conjunction with HIF1 in such lesions correlating with worsening cellular atypia and more advanced disease (13,27). In mouse VHL -/-ccRCC xenograft experiments, silencing HIF2α using shRNAs or CRISPR/Cas9 suppresses tumor growth whereas forced production of HIF2α, such as through the expression of a non-hydroxylatable HIF2α mutant, bypasses pVHL's tumor suppressor activity (28)(29)(30)(31)(32). In stark contrast, HIF1α constrains VHL -/-ccRCC growth in such assays (31)(32)(33)(34). ...
Germline loss-of-function mutations of the VHL tumor suppressor gene cause von Hippel-Lindau disease, which is associated with an increased risk of hemangioblastomas, clear cell renal cell carcinomas (ccRCCs), and paragangliomas. This Review describes mechanisms involving the VHL gene product in oxygen sensing, protein degradation, and tumor development and current therapeutic strategies targeting these mechanisms. The VHL gene product is the substrate recognition subunit of a ubiquitin ligase that targets the α subunit of the heterodimeric hypoxia-inducible factor (HIF) transcription factor for proteasomal degradation when oxygen is present. This oxygen dependence stems from the requirement that HIFα be prolyl-hydroxylated on one (or both) of two conserved prolyl residues by members of the EglN (also called PHD) prolyl hydroxylase family. Deregulation of HIF, and particularly HIF2, drives the growth of VHL-defective ccRCCs. Drugs that inhibit the HIF-responsive gene product VEGF are now mainstays of ccRCC treatment. An allosteric HIF2 inhibitor was recently approved for the treatment of ccRCCs arising in the setting of VHL disease and has advanced to phase III testing for sporadic ccRCCs based on promising phase I/II data. Orally available EglN inhibitors are being tested for the treatment of anemia and ischemia. Five of these agents have been approved for the treatment of anemia in the setting of chronic kidney disease in various countries around the world.
... 786-O cells transfected with HIF2α P405A/P531A double mutants (unable to be hydroxylated by PHDs) can induce tumors in nude mice when implanted. Growth of these tumors can be reduced via siRNA-mediated knockdown of HIF2α, but this defect cannot be rescued by expression of a basic helix-loop-helix (bHLH) HIF2α mutant incapable of DNA binding Kondo et al., 2002;Zimmer et al., 2004). This suggests HIF2α suppression is necessary for pVHL tumor suppression and that HIF2α is, therefore, a driver of RCC. ...
von Hippel-Lindau (VHL) disease is a rare hereditary cancer syndrome that causes a predisposition to renal clear-cell carcinoma, hemangioblastoma, pheochromocytoma, and autosomal-recessive familial polycythemia. pVHL is the substrate conferring subunit of an E3 ubiquitin ligase complex that binds to the three hypoxia-inducible factor alpha subunits (HIF1-3α) for polyubiquitylation under conditions of normoxia, targeting them for immediate degradation by the proteasome. Certain mutations in pVHL have been determined to be causative of VHL disease through the disruption of HIFα degradation. However, it remains a focus of investigation and debate whether the disruption of HIFα degradation alone is sufficient to explain the complex genotype-phenotype relationship of VHL disease or whether the other lesser or yet characterized substrates and functions of pVHL impact the development of the VHL disease stigmata; the elucidation of which would have a significant ramification to the direction of research efforts and future management and care of VHL patients and for those manifesting sporadic counterparts of VHL disease. Here, we examine the current literature including the other emergent pseudohypoxic diseases and propose that the VHL disease-phenotypic spectrum could be explained solely by the varied disruption of HIFα signaling upon the loss or mutation in pVHL.
... The unique cancer genetics that underlie ccRCC patient tumors further support the importance of intracellular iron concentration in this cancer. Specifically, the von Hippel Lindau (VHL)/hypoxia-inducible factor-a (HIF-a) axis, whose genetic or epigenetic inactivation underlies the vast majority of ccRCC tumors (69,70), serves as a master axis for sensing and responding to intracellular iron levels (71)(72)(73). We recently described that VHL inactivation introduces a novel iron dependency in ccRCC cells to escape apoptosis and cell cycle arrest and that ccRCC cell lines maintain significantly higher reactive iron levels than benign renal cell lines (74). ...
Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.
... VHL is a part of E3 ubiquitin ligase complex, mediating the degradation of hypoxia-inducible transcription factors (HIFs) 13 . Inactivation of VHL induces the stabilization and accumulation of HIFs. ...
The molecular heterogeneity of primary clear cell renal cell carcinoma (ccRCC) has been reported. However, the classifications of Von Hippel–Lindau (VHL) mutant ccRCC are unclear. Here, VHL mutant ccRCC from The Cancer Genome Atlas and E-MTAB-1980 datasets were divided into two sub-clusters through non-negative matrix factorization algorithm. Most VHL mutant ccRCC patients in sub-cluster2 were with pathological T1 stage and VHL mutant ccRCC patients in sub-cluster1 were with decreased overall survival. DNA replication and homologous recombination scores were higher, while, WNT signaling pathway and regulation of autophagy scores were lower in sub-cluster1 VHL mutant ccRCC. Moreover, PBX1 transcriptional scores and mRNA expressions were lower in sub-cluster1 VHL mutant ccRCC patients and were associated with the overall survival of VHL mutant ccRCC. Furthermore, PBX1 associated genes EMCN and ERG were down-regulated in sub-cluster1 VHL mutant ccRCC and overall survival was decreased in EMCN or ERG lowly expressed VHL mutant ccRCC patients. Also, PBX1 and EMCN were down-regulated in ccRCC tissues, compared with normal kidney tissues. At last, we constructed risk models based on PBX1, EMCN and EGR expression features. With the increase of the risk score, the number of death of VHL mutant ccRCC patients was increased.
... RIP140-expressing vectors (pEFcmyc-RIP140 [47], pEGFP-RIP140 [48]) and control vectors, pEGFP (Clontech), are described elsewhere. HA-HIF1alpha-pcDNA3 (Addgene plasmid # 18949) and HA-HIF2alpha-pcDNA3 (Addgene plasmid # 18950) plasmids were gifts from Dr Kaelin [49]. The GLUT3-Luc reporter gene was a gifts from Dr. Yuan [50]. ...
Glycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mam-mary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation.
... The activity disabling C187S mutation in USP25 was introduced using QuickChange site-directed mutagenesis kit (Agilent). V5/Histagged expression plasmids encoding human HIF-1α were kindly provided by Ya-Min Tian (University of Oxford), and HA-tagged human HIF-1α was a gift from William Kaelin (Addgene #18949) 64 . All plasmids used in this study were isolated using ZymoPURE II Plasmid Maxiprep kit (Zymo) and correctness verified by sequencing. ...
Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited. The biological roles of deubiquitinating enzymes (DUBs) in pancreatic ductal adenocarcinoma (PDAC) are not fully explored. Here the authors perform activity based proteomics with a loss of function genetic screen and identify that USP25 promotes PDAC growth and survival through HIF-1 protein stability and transcriptional activity.
... VHL is not only the most frequently studied gene but also has the highest mutation prevalence, accounting for 64% (51). Mutant VHL lacks the ability to target hypoxia-inducible factor (HIF) involved in angiogenesis and mitogenesis for destruction by the pVHL-E3 ligase complex ubiquitin-proteasome pathway (52). Interestingly, stabilization and increased transcription and expression of HIF-1 are clearly affected by human oncogenic viruses by disrupting degradation of HIF-1 (53). ...
Renal cell carcinoma (RCC), one of the most common genitourinary tumors, is induced by many factors, primarily smoking, obesity, and hypertension. As a non-acquired immunodeficiency syndrome (AIDS)-defining cancer, human immunodeficiency virus (HIV) may also play a critical role in the incidence and progression of RCC. It is evident that individuals who are infected with HIV are more likely than the general population to develop RCC. The age of RCC diagnosis among HIV-positive patients is younger than among HIV-negative individuals. However, many other characteristics remain unknown. With the increase in RCC incidence among HIV-infected patients, more research is being conducted to discover the relationship between RCC and HIV, especially with regard to HIV-induced immunodeficiency, diagnosis, and treatment. Unexpectedly, the majority of the literature suggests that there is no relationship between RCC and HIV-induced immunodeficiency. Nonetheless, differences in pathology, symptoms, or treatment in HIV-positive patients diagnosed with RCC are a focus. In this review, we summarize the association of RCC with HIV in terms of epidemiology, risk factors, diagnosis, and treatment.
... pVHL is the substrate recognition component of an E3 ubiquitin ligase that targets the alpha subunits of the heterodimeric HIF (hypoxiainducible factor) transcription factor for destruction when oxygen is plentiful. In the absence of pVHL, deregulation of HIF, in particular HIF2, drives the growth of ccRCC (8)(9)(10)(11)(12)(13). ...
Significance
VHL tumor suppressor gene inactivation is a hallmark of clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and promotes tumor growth by stabilizing the hypoxia-inducible factor 2 (HIF2) transcription factor. HIF2 inhibitors appear to be helpful for some, but not all, ccRCC patients in clinical trials. Previous preclinical and clinical data suggested that only ccRCCs that can activate the p53 tumor suppressor in response to DNA damage would respond to HIF2 inhibitors. Here, we show that an intact p53 pathway is neither necessary nor sufficient for the sensitivity of ccRCCs to HIF2 inhibitors, suggesting that it would be premature to use p53 status to determine which ccRCC patients should be treated with a HIF2 inhibitor.
... Recently, the loss of VHL has been estimated to be as high as 91% of ccRCC cases, rooting the VHL the genetic basis in the ccRCC (16). Besides, VHL gene-dependent tumor suppression in ccRCC responded to the VHL-mediated ubiquitination process, enabling the possibility of ccRCC treatment through VHL ubiquitination inhibition (17). Considering the ambiguous relationship between ubiquitination and prognosis of ccRCC, developing a lncRNA signature-based model has an important clinical value in the prognosis of ccRCC. ...
Background: The purpose of this study was to identify the ubiquitination-associated genes and co-expressed lncRNAs on the prognosis of clear cell renal cell carcinoma (ccRCC) patients.
Methods and patients: We downloaded 530 cases and the corresponding transcriptome profiling from The Cancer Genome Atlas (TCGA) database. We distinguished mRNA and lncRNA expression data from the transcriptome profiling, then extracted the expression of mRNAs that regulate protein ubiquitination. We obtained lncRNAs associated with protein ubiquitination regulation from the lncRNA data by Gene Co-expression Analysis. Cox Regression Analysis of survival time, survival status, and lncRNA expression level was carried out and a Prognostic Index (PI) was constructed.
Results: The PI was established based on 8 prognostic lncRNAs that regulate protein ubiquitination, and distinguish the high-risk group patients from all patients. Multivariate analysis indicated that this PI was an individualized clinical prognostic factor for patients with ccRCC. Regarding clinical characteristics, a Ubiquitination-associated Clinical-Prognostic Index (UCPI), containing 8 ubiquitination-related lncRNAs and age, was established and validated with high prognostic efficacy.
Conclusion: We established a UCPI containing 8 lncRNAs related to protein ubiquitination. This UCPI may become an appropriate model to predict the prognosis in ccRCC patients and guide clinicians to adjust the follow-up regimen.
... The VHL gene product regulates the hypoxic gene response by stabilizing alpha subunits of hypoxia-inducible factor (HIF)-1 and HIF-2 transcription factors [4][5][6]. Mutated VHL protein does not bind to HIF-1α and 2α, resulting in gene transcription and upregulation of growth factors, including erythropoietin, vascular endothelial growth factor (VEGF), platelet-derived growth factor β, and other genes involved in metabolism. Thus, VHL mutation drives angiogenesis and proliferation. ...
Hereditary renal cancer syndromes represent approximately 5% of renal malignancies and have distinctive clinical, histopathologic, and genetic features. Next-generation sequencing and other molecular testing methods have uncovered several hereditary renal cancer syndromes. Several autosomal dominant hereditary renal cell carcinoma (RCC) syndromes, including those related to germline pathogenic variants in VHL, BAP1, MITF, MET, FH, TSC1/TSC2, FLCN, SDH, and CDC73 have been confirmed. FH- and BAP1-related RCCs are associated with more aggressive disease. Identifying the clinical and pathological features in these hereditary RCC syndromes is important as, relative to familial cohorts, these patients require early screening and intervention and regular surveillance to improve their clinical prognosis and long-term outcomes. More importantly, identification of these syndromes plays a vital role in personalized management and systemic treatment selection in this modern era of precision medicine. Ongoing studies have demonstrated that treatment based on genetic pathway targeting is a promising approach for hereditary renal cancer management. This review describes updates in the diagnostic criteria for and management of familial kidney cancer syndromes.
N6-Methyladenosine (m6A), a prevalent posttranscriptional modification, plays an important role in cancer progression. Clear cell renal cell carcinoma (ccRCC) is chiefly associated with the loss of the von Hippel-Lindau (VHL) gene, encoding a component of the E3 ubiquitin ligase complex. In this issue of the JCI, Zhang and colleagues unveiled a function of VHL beyond its canonical role as an E3 ubiquitin ligase in regulating hypoxia-inducible factors (HIFs). It also governed m6A modification by orchestrating the assembly of m6A writer proteins METTL3 and METTL14, thereby stabilizing PIK3R3 mRNA. Mechanistically, PIK3R3 contributed to p85 ubiquitination, which restrained PI3K/AKT signaling and consequently impeded ccRCC growth in cell and mouse models. This discovery provides potential treatment targets in VHL-deficient ccRCCs.
Chondrosarcomas represent the second most common primary bone malignancy. Despite the vulnerability of chondrosarcoma cells to nicotinamide adenine dinucleotide (NAD⁺) depletion, targeting the NAD⁺ synthesis pathway remains challenging due to broad implications in biological processes. Here, we establish SIRT1 as a central mediator reinforcing the dependency of chondrosarcoma cells on NAD⁺ metabolism via HIF-2α-mediated transcriptional reprogramming. SIRT1 knockdown abolishes aggressive phenotypes of chondrosarcomas in orthotopically transplanted tumors in mice. Chondrosarcoma cells thrive under glucose starvation by accumulating NAD⁺ and subsequently activating the SIRT1-HIF-2α axis. Decoupling this link via SIRT1 inhibition unleashes apoptosis and suppresses tumor progression in conjunction with chemotherapy. Unsupervised clustering analysis identifies a high-risk chondrosarcoma patient subgroup characterized by the upregulation of NAD⁺ biosynthesis genes. Finally, SIRT1 inhibition abolishes HIF-2α transcriptional activity and sensitizes chondrosarcoma cells to doxorubicin-induced cytotoxicity, irrespective of underlying pathways to accumulate intracellular NAD⁺. We provide system-level guidelines to develop therapeutic strategies for chondrosarcomas.
Background:
The eukaryotic elongation factor, EEF1A2, has been identified as an oncogene in various solid tumors. Here, we have identified a novel function of EEF1A2 in angiogenesis.
Methods:
Chick chorioallantoic membrane, tubulogenesis, aortic ring, Matrigel plug, and skin wound healing assays established EEF1A2's role in angiogenesis.
Result:
Higher EEF1A2 levels in breast cancer cells enhanced cell growth, movement, blood vessel function, and tubule formation in HUVECs, as confirmed by ex-ovo and in-vivo tests. The overexpression of EEF1A2 could be counteracted by Plitidepsin. Under normoxic conditions, EEF1A2 triggered HIF1A expression via ERK-Myc and mTOR signaling in TNBC and ER/PR positive cells. Hypoxia induced the expression of EEF1A2, leading to a positive feedback loop between EEF1A2 and HIF1A. Luciferase assay and EMSA confirmed HIF1A binding on the EEF1A2 promoter, which induced its transcription. RT-PCR and polysome profiling validated that EEF1A2 affected VEGF transcription and translation positively. This led to increased VEGF release from breast cancer cells, activating ERK and PI3K-AKT signaling in endothelial cells. Breast cancer tissues with elevated EEF1A2 showed higher microvessel density.
Conclusion:
EEF1A2 exhibits angiogenic potential in both normoxic and hypoxic conditions, underscoring its dual role in promoting EMT and angiogenesis, rendering it a promising target for cancer therapy.
Oxygen (O 2 ) is essential for cellular metabolism and biochemical reactions. When the demand for O 2 exceeds the supply, hypoxia occurs. Hypoxia-inducible factors (HIFs) are essential to activate adaptive and survival responses following hypoxic stress. In the gut (intestines) and liver, oxygen gradients or physiologic hypoxia are necessary to maintain normal homeostasis. While physiologic hypoxia is beneficial and aids in normal functions, pathological hypoxia is harmful as it exacerbates inflammatory responses and tissue dysfunction and is a hallmark of many cancers. In this review, we discuss the role of gut and liver hypoxia-induced signaling, primarily focusing on HIFs, in the physiology and pathobiology of gut and liver diseases. Additionally, we examine the function of HIFs in various cell types during gut and liver diseases, beyond intestinal epithelial and hepatocyte HIFs. This review highlights the importance of understanding hypoxia-induced signaling in the pathogenesis of gut and liver diseases and emphasizes the potential of HIFs as therapeutic targets.
Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 19 is January 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Despite the initiation of tumor arises from tumorigenic transformation signaling in cancer cells, cancer cell survival, invasion, and metastasis also require a dynamic and reciprocal association with extracellular signaling from tumor microenvironment (TME). Primary cilia are the antenna‐like structure that mediate signaling sensation and transduction in different tissues and cells. Recent studies have started to uncover that the heterogeneous ciliation in cancer cells and cells from the TME in tumor growth impels asymmetric paracellular signaling in the TME, indicating the essential functions of primary cilia in homeostasis maintenance of both cancer cells and the TME. In this review, we discussed recent advances in the structure and assembly of primary cilia, and the role of primary cilia in tumor and TME formation, as well as the therapeutic potentials that target ciliary dynamics and signaling from the cells in different tumors and the TME.
The ERM (ezrin, radixin, and moesin) family of proteins and the related protein merlin participate in scaffolding and signaling events at the cell cortex. The proteins share an N-terminal FERM [band four-point-one (4.1) ERM] domain composed of three subdomains (F1, F2, and F3) with binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that displays peptides representing the intrinsically disordered regions of the human proteome, we identified a large number of novel ligands. We determined the affinities for the ERM and merlin FERM domains interacting with 18 peptides and validated interactions with full-length proteins through pull-down experiments. The majority of the peptides contained an apparent Yx[FILV] motif; others show alternative motifs. We defined distinct binding sites for two types of similar but distinct binding motifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computational peptide docking protocols and mutational analysis. We provide a detailed molecular understanding of how the two types of peptides with distinct motifs bind to different sites on the moesin FERM phosphotyrosine binding-like subdomain and uncover interdependencies between the different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin and suggests that the FERM domain acts as a switchable interaction hub.
Not all aspects of the disruption of iron homeostasis in cancer have been fully elucidated. Iron accumulation in cancer cells is frequent for many solid tumours, and this is often accompanied by the contemporary rise of two key iron regulators, HIF2α and Hepcidin. This scenario is different from what happens under physiological conditions, where Hepcidin parallels systemic iron concentrations while HIF2α levels are inversely associated to Hepcidin. The present review highlights the increasing body of evidence for the pro-tumoral effect of HIF2α and Hepcidin, discusses the possible imbalance in HIF2α, Hepcidin and iron homeostasis during cancer, and explores therapeutic options relying on these pathways as anticancer strategies.
Myocardial ischemia–reperfusion injury (MIRI) induces life-threatening damages to the cardiac tissue, and pharmacological means to achieve cardioprotection are sorely needed. MIRI severity varies along the day–night cycle and is molecularly linked to components of the cellular clock, including the nuclear receptor REV-ERBα, a transcriptional repressor. Here we show that digoxin administration in mice is cardioprotective when timed to trigger REV-ERBα protein degradation. In cardiomyocytes, digoxin increases REV-ERBα ubiquitinylation and proteasomal degradation, which depend on REV-ERBα’s ability to bind its natural ligand, heme. Inhibition of the membrane-bound Src tyrosine-kinase partially alleviated digoxin-induced REV-ERBα degradation. In untreated cardiomyocytes, REV-ERBα proteolysis is controlled by several E3 ubiquitin ligases and the proteasome subunit PSMB5. Among these, only SIAH2 and PSMB5 contributed to digoxin-induced degradation of REV-ERBα. Thus, controlling REV-ERBα proteostasis through the ubiquitin–proteasome system is an appealing cardioprotective strategy. Our data support the timed use of clinically approved cardiotonic steroids in prophylactic cardioprotection. The heart’s tolerance to ischemia–reperfusion injury varies according to a day–night cycle. Vinod et al. show that a timed low dose of cardiac glycosides, such as digoxin, is protective against heart ischemia–reperfusion injury by promoting the proteasomal degradation of the molecular-clock component and transrepressive nuclear receptor REV-ERBα.
In clear cell renal cell carcinoma, the VHL/HIF axis lays the groundwork for tumorigenesis and is the target of many therapeutic agents. However, HIF activation alone is largely insufficient for kidney tumor development, and the secondary mutations in PBRM1, BAP1, SETD2, KDM5C or other tumor suppressor genes are strong enablers of tumorigenesis. Interestingly, it has been discovered that VHL loss, and subsequent HIF activation, result in ISGF3 upregulation a negative feedback loop mediated by ISGF3, a transcription factor activated by type I interferon, and the secondary mutations in multiple tumor suppressor genes all partially disable this negative feedback loop to facilitate tumor growth. The convergence of so many cancer genes on this pathway suggests that it plays an important role in ccRCC development and maintenance. The tumors with secondary mutations that dampen the negative feedback loop might be exquisitely sensitive to its reactivation, and pharmacological activation of ISGF3 could be an effective method to treat ccRCC patients either alone or in combination with other effective therapies. In this review, we examine the relevance of the type I IFN pathway to ccRCC, synthesize our current knowledge of the ccRCC tumor suppressors in its regulation, and explore how this may impact the future treatment of ccRCC patients.
Hintergrund und Ziele: Die Kinase mechanistic Target of Rapamycin (mTOR) und der Transkriptionsfaktor Hypoxia-inducible Factor (HIF) spielen beide eine übergeordnete Rolle bei der Tumorentstehung und dem Tumorwachstum. Diese beiden Systeme können das Tumorwachstum eigenständig fördern, zeigen möglicherweise aber auch eine gegenseitige Potenzierung [1]. Das Ziel der Arbeit war es, ein detaillierteres Verständnis über die Interaktion von mTOR und HIF zu erhalten und welchen Einfluss HIF auf die Tumorentstehung und dem Tumorwachstum bei Aktivierung von mTOR, durch Inaktivierung des Tuberous Sclerosis Complex (TSC), hat. Methoden: In vitro wurden die Auswirkungen von HIF-Stabilisatoren sowie von mTOR-Inhibition in Immortalized human kidney epithelial cells (IHKE Zellen) und human urinary primary tubular cells (hUPT Zellen) von Tuberösen Sklerose (TS) Patienten untersucht. In vivo wurden, um spezifisch in die beiden Signalwege einzugreifen, 9 Monate alte Mäuse mit einer TSC2 Deletion (TSC2+/- Mäuse) 12 Wochen intraperitoneal (i. p.) entweder mit Lösungsmittel, einem HIF-Stabilisator oder mTOR-Inhibitor behandelt. Nach der Tötung wurden die Nieren der Mäuse entnommen und anschließend die makroskopisch und mikroskopisch sichtbaren Tumore gezählt. Anschließend wurden Proteine mittels Immunhistochemie (IHC), Immunoblots und quantitativer Real-Time Polymerase-Kettenreaktion (PCR) untersucht, welche eine Relevanz in dem Signalweg der mTOR-HIF Interaktion haben könnten. Da die phänotypischen Auswirkungen einer TSC-Inaktivierung zwischen Maus und Mensch differieren können, wurden Nieren von TS Patienten zusätzlich immunhistochemisch untersucht. Ergebnisse und Beobachtungen: Die TSC2+/- Mäuse wiesen in den Vorversuchen in unseren Händen, im Gegensatz zu publizierten Daten [2], erst nach 9 bis 12 Monaten eine 100%ige Tumorentwicklung auf. Im Vergleich zu den Wildtyp-Mäusen zeigte sich eine deutliche Aktivierung des mTOR-Signalweges, welches durch die Phosphorylierung von dem ribosomalen Protein S6 (rpS6) nachgewiesen werden konnte. Ebenso konnte gezeigt werden, dass in den TSC2+/- Mäusen im Vergleich zu Wildtyp-Mäusen vermehrt HIF-1α positiven Zellen vorhanden waren. In den TSC2+/- Mäusen, welchen 3 Monate der mTOR Inhibitor Temsirolimus (Tem) injiziert wurde, konnten signifikant weniger Tumore gefunden werden. Die erfolgreiche mTOR Inhibition konnte nachgewiesen werden und es konnte gezeigt werden, dass Tem-Gabe ebenfalls die HIF-Zielgene senkt. Eine pharmakologische Stabilisierung von HIF durch 2-(1-Chloro-4-Hydroxyisochinolin-3Carboxamido) Acetat (ICA) in den TSC2+/- Mäuse wiederum führte zu einer signifikant erhöhten Anzahl von Tumoren. Dass ICA funktionell in der Niere wirkte, konnte durch eine Erhöhung des Hämatokrit(Hk)-Wertes und der HIF-Zielgene gezeigt werden. In Färbungen von Folgeschnitten konnten in gesunden Nierenabschnitten und in den entstandenen Tumoren gemeinsame räumliche Lokalisationen der Signale von rpS6-P, Carboanhydrase 9 (CA9) und des Fibroblastenspezifischen Proteins 1 (FSP-1) gefunden werden. Dies bestätigt den Verdacht, dass eine mTOR Aktivierung die Translation von HIF und dessen Zielgene erhöht und daraus eine epitheliale-mesenchymale Transition (EMT) resultieren kann. Durch die Untersuchung von Gewebeproben aus Nephrektomiepräparaten von einem TS Patienten und gesunden Nieren konnte gezeigt werden, dass es auch hier zu einer verstärkten Aktivierung von mTOR und Steigerung von HIF und seiner Zielgene kam. Schlussfolgerungen: Die Doktorarbeit stärkt die Hypothese, dass die Translation von HIF über den Phosphatidyl¬inositol-3-Kinase/Akt/mTOR Signalweg beeinflusst werden kann. Durch die TSC2 Deletion kam es zu einer verstärkten mTOR Aktivierung und einer Steigerung von HIF-Zielgenen. Durch die Gabe von Tem konnten diese Zielgene wiederum verringert werden. Um detaillierte Aussagen über die mTOR-HIF Interaktion tätigen zu können, werden weitere spezifische Arbeiten in diesem Bereich nötig sein. Der Nutzen der mTOR Inhibitoren bei TS als Anti-Tumor-Therapie kann durch die Ergebnisse nur unterstützet werden, da eine signifikante Reduktion der Nierentumore bei den TSC2+/- Mäusen, welche mit einem mTOR Inhibitor behandelt wurden, nachgewiesen werden konnte. Therapien mit HIF-Stabilisatoren sollten hingegen kritisch hinterfragt werden. HIF wird in vielen Tumoren überexprimiert und trägt, wie in dieser Arbeit gezeigt werden konnte, zum Tumorwachstum bei. Es könnte daher kritisch sein, Patienten mit einer Prädisposition, wie z.B. terminalen Niereninsuffizienz, mit HIF-Stabilisatoren zu behandeln.
Targeting the tumor microenvironment is a promising strategy to prevent metastasis, overcome acquired drug resistance, and improve the therapeutic effect. Hypoxia is one of the characteristics of the tumor microenvironment, which is mainly regulated by hypoxia-inducible factors. Hypoxia-inducible factors (HIFs) including HIF-1α, HIF-2α, and HIF-3α, of which HIF-2α has assumed a more important role in tumor hypoxia environment. It has been demonstrated that HIF-2α plays an important role in tumor diseases, including renal cell carcinoma, breast cancer, non-small cell lung cancer, and gastric cancer, among others. Therefore, targeting HIF-2α has become one of the important strategies for treating cancers. HIF-2α inhibitors can be divided into two categories: specific inhibitors and non-specific inhibitors. The former includes synthetic monomer compounds and traditional Chinese medicine extracts. In this review, we summarized, classified, and discussed current research on the structure, structure-activity relationship (SAR), and pharmacology of HIF-2α inhibitors, which is helpful to the rational design of effective drugs for various types of malignant tumors.
The hypoxia‐inducible factors (HIF)‐1α and HIF‐2α are central regulators of transcriptional programs in settings such as development and tumor expansion. HIF‐2α moonlights as a cap‐dependent translation factor. We provide new insights into how the interferon‐stimulated gene 15 (ISG15), a ubiquitin‐like modifier, and the HIFs regulate one another in hypoxia and interferon‐induced cells. We show that upon ISGylation induction and HIF‐α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post‐transcriptional pathways. We show the first evidence of HIF‐2α modification by ISG15. ISGylation induces system‐level alterations to the HIF transcriptional program and increases the cytoplasmic/nuclear fraction and translation activity of HIF‐2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression. We show reciprocal regulation between the hypoxia‐inducible factors (HIFs) and interferon‐stimulated gene 15 (ISG15), a ubiquitin‐like modifier. ISG15 participates in negative‐ and positive‐feedback systems with HIF‐1α and HIF‐2α, respectively, and induction of ISG15 conjugation in hypoxia is associated with transcriptional alterations of HIF‐dependent pathways. Furthermore, whereas HIF‐1α turnover is increased, ISGylation of HIF‐2α promotes its cytoplasmic localization and association with polysomes and eIF4FH‐dependent translation.
BRCA1-associated protein 1 (BAP1) is a deubiquitinase that is mutated in 10–15% of clear cell renal cell carcinomas (ccRCC). Despite the association between BAP1 loss and poor clinical outcome, the critical tumor suppressor function(s) of BAP1 in ccRCC remains unclear. Previously, we found that hypoxia-inducible factor 2α (HIF2α) and BAP1 activate interferon-stimulated gene factor 3 (ISGF3), a transcription factor activated by type I interferons and a tumor suppressor in ccRCC xenograft models. Here, we aimed to determine the mechanism(s) through which HIF and BAP1 regulate ISGF3. We found that in ccRCC cells, loss of the von Hippel-Lindau tumor suppressor (VHL) activated interferon beta (IFN-β) expression in a HIF2α-dependent manner. IFN-β was required for ISGF3 activation and suppressed the growth of Ren-02 tumors in xenografts. BAP1 enhanced the expression of IFN-β and stimulator of interferon genes (STING), both of which activate ISGF3. Both ISGF3 overexpression and STING agonist treatment increased ISGF3 activity and suppressed BAP1-deficient tumor growth in Ren-02 xenografts. Our results indicate that BAP1 loss reduces type I interferon signaling, and reactivating this pathway may be a novel therapeutic strategy for treating ccRCC.
Urologic diseases are commonly diagnosed health problems affecting people around the world. More than 26 million people suffer from urologic diseases and the annual expenditure was more than 11 billion. The urologic cancers, like bladder cancer, prostate cancer and kidney cancer are always the leading causes of death worldwide, which account for approximately 22% and 10% of the new cancer cases and death, respectively. Organ transplantation is one of the major clinical treatments for urological diseases like end-stage renal disease and urethral stricture, albeit strongly limited by the availability of matching donor organs. Tissue engineering has been recognized as a highly promising strategy to solve the problems of organ donor shortage by the fabrication of artificial organs/tissue. This includes the prospective technology of three-dimensional (3D) bioprinting, which has been adapted to various cell types and biomaterials to replicate the heterogeneity of urological organs for the investigation of organ transplantation and disease progression. This review discusses various types of 3D bioprinting methodologies and commonly used biomaterials for urological diseases. The literature shows that advances in this field toward the development of functional urological organs or disease models have progressively increased. Although numerous challenges still need to be tackled, like the technical difficulties of replicating the heterogeneity of urologic organs and the limited biomaterial choices to recapitulate the complicated extracellular matrix components, it has been proved by numerous studies that 3D bioprinting has the potential to fabricate functional urological organs for clinical transplantation and in vitro disease models.
Oxygen plays a fundamental role in respiration and metabolism, and quantifying oxygen levels is essential in many environmental, industrial, and research settings. Microdevices facilitate the study of dynamic, oxygen-dependent effects in real time. This review is organized around the key needs for oxygen measurement in microdevices, including integrability into microfabricated systems; sensor dynamic range and sensitivity; spatially resolved measurements to map oxygen over two- or three-dimensional regions of interest; and compatibility with multimodal and multianalyte measurements. After a brief overview of biological readouts of oxygen, followed by oxygen sensor types that have been implemented in microscale devices and sensing mechanisms, this review presents select recent applications in organs-on-chip in vitro models and new sensor capabilities enabling oxygen microscopy, bioprocess manufacturing, and pharmaceutical industries. With the advancement of multiplexed, interconnected sensors and instruments and integration with industry workflows, intelligent microdevice-sensor systems including oxygen sensors will have further impact in environmental science, manufacturing, and medicine.
Background
ccRCC is considered as the main subtype of RCC, which accounted for sixth deadliest cancer worldwide. Recently, ubiquitination has been reported to be closely involved in the progression of tumore. The purpose of this study was to identify the ubiquitination-associated genes and co-expressed lncRNAs on the prognosis of clear cell renal cell carcinoma (ccRCC) patients.
Methods and patients
We downloaded 530 cases and the corresponding transcriptome profiling from The Cancer Genome Atlas (TCGA) database. We distinguished mRNA and lncRNA expression data from the transcriptome profiling and then extracted the expression of mRNAs that regulate protein ubiquitination. We obtained lncRNAs associated with protein ubiquitination regulation from the lncRNA data by gene co-expression analysis. Cox regression analysis of survival time, survival status, and lncRNA expression level was carried out, and a prognostic index (PI) was constructed.
Results
The PI was established based on 8 prognostic lncRNAs that regulate protein ubiquitination and distinguish the high-risk group patients from all patients. Multivariate analysis indicated that this PI was an individualized clinical prognostic factor for patients with ccRCC. Regarding clinical characteristics, a ubiquitination-associated clinical-prognostic index (UCPI), containing 8 ubiquitination-related lncRNAs and age, was established and tested with AUC of 0.80.
Conclusion
We established a UCPI containing 8 lncRNAs related to protein ubiquitination. This UCPI may become an appropriate model to predict the prognosis in ccRCC patients and guide clinicians to adjust the follow-up regimen.
Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) encodes the 4EBP1 protein, a negative regulator of mRNA translation and a substrate of the mechanistic target of rapamycin (mTOR), whose function and relevance in cancer is still under debate. Here, we analyzed EIF4EBP1 expression in different glioma patient cohorts and investigated its mode of transcriptional regulation in glioblastoma cells. We verified that EIF4EBP1 mRNA is overexpressed in malignant gliomas, including isocitrate dehydrogenase (IDH)-wildtype glioblastomas, relative to non-neoplastic brain tissue in multiple publically available datasets. Our analyses revealed that EIF4EBP1 overexpression in malignant gliomas is neither due to gene amplification nor to altered DNA methylation, but rather results from aberrant transcriptional activation by distinct transcription factors. We found seven transcription factor candidates co-expressed with EIF4EBP1 in gliomas and bound to the EIF4EBP1 promoter, as revealed by chromatin immunoprecipitation (ChIP)-sequencing data. We investigated the ability of these candidates to activate the EIF4EBP1 promoter using luciferase reporter assays, which supported four transcription factors as candidate EIF4EBP1 regulators, namely MYBL2, ETS1, HIF-1A, and E2F6. Finally, by employing transient knock-down experiments to repress either of these transcription factors, we identified MYBL2 and ETS1 as the relevant transcriptional drivers of enhanced EIF4EBP1 expression in malignant glioma cells. Taken together, our findings confirm enhanced expression of EIF4EBP1 in malignant gliomas relative to non-neoplastic brain tissue and characterize the underlying molecular pathomechanisms.
Hypoxia regulates tumor angiogenesis, metabolism, and therapeutic response in malignant cancers including glioblastoma, the most lethal primary brain tumor. The regulation of HIF transcriptional factors by the ubiquitin-proteasome system is critical in the hypoxia response, but hypoxia-inducible deubiquitinases that counteract the ubiquitination remain poorly defined. While the activation of ERK1/2 also plays an important role in hypoxia response, the relationship between ERK1/2 activation and HIF regulation remains elusive. Here, we identified USP33 as essential deubiquitinase that stabilizes HIF-2alpha protein in an ERK1/2-dependent manner to promote hypoxia response in cancer cells. USP33 is preferentially induced in glioma stem cells by hypoxia and interacts with HIF-2alpha, leading to its stabilization through deubiquitination. The activation of ERK1/2 upon hypoxia promoted HIF-2alpha phosphorylation, enhancing its interaction with USP33. Silencing of USP33 disrupted glioma stem cells maintenance, reduced tumor vascularization, and inhibited glioblastoma growth. Our findings highlight USP33 as an essential regulator of hypoxia response in cancer stem cells, indicating a novel potential therapeutic target for brain tumor treatment.
Latency is a hallmark of herpesviruses, allowing them to persist into their host without virions production. Acute exposure to hypoxia (below 3% O 2 ) was identified as a trigger of latent-to-lytic switch (reactivation) for human oncogenic gamma-herpesviruses (KSHV and EBV). Therefore, we hypothesized that hypoxia could also induce reactivation of Marek’s disease virus (MDV), sharing biological properties with EBV and KSHV (notably oncogenic properties), into lymphocytes. Acute exposure to hypoxia (1% O 2 ) of two MDV-latently infected cell lines derived from MD tumors (3867K and MSB-1) induced MDV reactivation. A bioinformatic analysis of the RB-1B MDV genome revealed 214 putative hypoxia-response element consensus sequences on 119 open reading frames. RT-qPCR analysis showed five MDV genes strongly upregulated early after hypoxia. In 3867K cells under normoxia, pharmacological agents mimicking hypoxia (MLN4924 and CoCl 2 ) increased MDV reactivation, but to a lower level than real hypoxia. Overexpression of wild-type or stabilized human hypoxia inducible factor-1α (HIF-1α) in MSB-1 cells in normoxia also promoted MDV reactivation. In such conditions, lytic cycle was detected in cells with a sustainable HIF-1α expression, but also in HIF-1α negative cells, indicating that MDV reactivation is mediated by HIF-1, in a direct and/or indirect manner. Lastly, we demonstrated by a reporter assay that HIF-1α overexpression induced the transactivation of two viral promoters, shown upregulated in hypoxia. These results suggest that hypoxia may play a crucial role in the late lytic replication phase observed in vivo in MDV-infected chickens exhibiting tumors, since a hypoxic microenvironment is a hallmark of most solid tumors.
IMPORTANCE
Latent-to-lytic switch of herpesviruses (aka reactivation) is responsible for pathology recurrences and/or viral shedding. Studying physiological triggers of reactivation is therefore important for health to limit lesions and viral transmission. Marek's disease virus (MDV) is a potent oncogenic alpha-herpesvirus establishing latency in T-lymphocytes and causing lethal T-lymphomas in chickens. In vivo , a second lytic phase is observed during tumoral stage. Hypoxia being a hallmark of tumors, we wondered whether hypoxia induces MDV reactivation in latently-infected T-lymphocytes, like previously shown for EBV and KSHV in B-lymphocytes. In this study, we demonstrated that acute hypoxia (1% O2) triggers MDV reactivation in two MDV transformed T-cell lines. We provide some molecular basis of this reactivation by showing that hypoxia inducible factor (HIF-1) overexpression induces MDV reactivation to a similar extend than hypoxia after 24 hours. Hypoxia is therefore a reactivation stimulus shared by mammalian and avian oncogenic herpesviruses of different genus.
Hypoxia is a hallmark of all solid tumors and their metastases. This leads to activation of the hypoxia-inducible factor (HIF) family of transcription factors, which modulate gene expression within both tumor cells and immune cells within the tumor microenvironment, influencing tumor progression and treatment response. The best characterized HIF isoforms, HIF-1α and HIF-2α, show nonoverlapping and often antagonistic roles. With the recent availability of inhibitors that target one or both HIFs, including the first-in-class selective HIF-2α inhibitor belzutifan, the prospect of HIF-α isoform-selective targeting is now a reality. Here, we summarize current knowledge on the unique contributions of the two HIF-α isoforms to tumor progression in the context of the complex tumor immune microenvironment, highlighting important considerations for therapy.
A prevalent characteristic of solid tumors is intra-tumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) predominantly mediates the adaptive response to O2 oscillation and is linked to multiple malignant hallmarks. Here we describe a strategy to robustly target HIF1α by dual inhibition of CDK(s) and heat shock protein 90 (HSP90). We show that CDK1 may contribute to HSP90-mediated HIF1α stabilization. CDK1 knockdown enhances the decrease of HIF1α by HSP90 inhibition. Dual inhibition of CDK1 and HSP90 significantly increases apoptosis and synergistically inhibits cancer cell viability. Similarly, targeting CDK4/6 using FDA-approved inhibitors in combination with HSP90 inhibition shows a class effect on HIF1α inhibition and cancer cell viability suppression not only in colorectal but also in various other cancer types, including Rb-deficient cancer cells. Dual inhibition of CDK4/6 and HSP90 suppresses tumor growth in vivo. In summary, combined targeting of CDK(s) (CDK1 or CDK4/6) and HSP90 remarkably inhibits the expression level of HIF1α and shows promising anti-cancer efficacy with therapeutic potential.
Objectives
Retinal angioma frequently occurs in von Hippel-Lindau (VHL) disease. However, VHL gene alterations have not been documented in retinal angiomas.Methods
Using tissue microdissection and polymerase chain reaction amplification, we have analyzed 7 retinal angiomas associated with VHL disease for loss of heterozygosity of the VHL gene. In addition, vascular endothelial growth factor expression was evaluated in these tumors by immunohistochemistry and in situ hybridization.Results
All 6 informative retinal angiomas showed loss of heterozygosity of the VHL gene. Loss of heterozygosity was detected in vacuolated "stromal" cells, but not in vascular cells or reactive glial tissue. Vascular endothelial growth factor protein and messenger RNA were also present in vacuolated "stromal" cells.Conclusions
These findings suggest that vacuolated "stromal" cells represent the true neoplastic component in retinal angioma. These cells express vascular endothelial growth factor and therefore may be responsible for abundant neovascularization of retinal angioma.
The transcription factor hypoxia-inducible factor (HIF)-1 is an important mediator of hypoxic adaptation of tumor cells and controls several genes that have been implicated in tumor growth. Oxygen-dependent degradation of HIF-1alpha, the regulatory subunit, requires binding to the von Hippel Lindau (VHL) protein. Because functional inactivation of the VHL tumor suppressor gene occurs in up to 70% of clear cell renal carcinomas, we investigated whether this results in overexpression of HIF-1alpha and its target genes. Immunoblotting revealed increased expression of HIF-1alpha in 24 of 32 (75%) clear cell renal carcinomas but only 3 of 8 non-clear cell renal tumors. Somatic mutations of the VHL gene were detected only in clear cell renal carcinomas that overexpressed HIF-1alpha. None of the HIF-1alpha-negative tumors displayed a VHL mutation. The level of HIF-1alpha mRNA was not different between tumors and adjacent kidney tissue. Immunohistochemistry revealed distinct patterns of nuclear staining for HIF-1alpha, depending on histological type and overall abundance of HIF-1alpha. In those clear cell renal carcinomas that showed increased expression on immunoblots, HIF-1alpha was expressed in almost all cells. In the remaining clear cell and in non-clear cell tumors, staining was focal; these different patterns thus were compatible with genetic stabilization in contrast to microenvironmental stimulation of HIF-1alpha as the primary mechanism. The mRNA expression of two known target genes of HIF-1alpha, vascular endothelial growth factor and glucose transporter 1, increased progressively with increasing amounts of HIF-1alpha in tumor extracts. In addition, glucose transporter 1 protein levels correlated with HIF-1alpha abundance. In conclusion, the data provide in vivo evidence for a constitutive up-regulation of HIF-1alpha in the majority of clear cell renal carcinomas, which leads to more widespread accumulation of this transcription factor than hypoxic stimulation. These observations are most likely linked to functional inactivation of the VHL gene product. Increased expression of HIF-1alpha is associated with alterations in gene expression patterns that are likely to contribute to tumor phenotype and progression
Multiple, bilateral renal carcinomas are a frequent occurrence in von Hippel-Lindau (VHL) disease. To elucidate the aetiological role of the VHL gene in human kidney tumorigenesis, localized and advanced tumours from 110 patients with sporadic renal carcinoma were analysed for VHL mutations and loss of heterozygosity (LOH). VHL mutations were identified in 57% of clear cell renal carcinomas analysed and LOH was observed in 98% of those samples. Moreover, VHL was mutated and lost in a renal tumour from a patient with familial renal carcinoma carrying the constitutional translocation, t(3;8)(p14;q24). The identification of VHL mutations in a majority of localized and advanced sporadic renal carcinomas and in a second form of hereditary renal carcinoma indicates that the VHL gene plays a critical part in the origin of this malignancy.
Background:
The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors-with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene for clear-cell RCC specifically and with long-term exposure to high doses of trichloroethylene (TRI), an industrially important solvent, for RCC generally. We investigated whether TRI exposure produces RCC through a specific mutational effect on the VHL gene by analyzing VHL sequences in the RCCs of patients exposed to high, cumulative doses of TRI.
Methods:
The level of exposure for each of 44 patients with RCC who had known industrial exposure to TRI was classified according to the duration, frequency, and mode of exposure. Samples of normal and cancerous tissues were microdissected from paraffin-embedded tissue. DNA was isolated from these samples, and somatic VHL mutations were identified by polymerase chain reaction analysis, single-strand conformation polymorphism analysis, DNA sequencing, and restriction enzyme digestion. Control samples included RCC DNA from 107 patients without known TRI exposure and lymphocyte DNA from 97 healthy subjects.
Results:
RCCs of TRI-exposed patients showed somatic VHL mutations in 33 (75%) of 44 cases. The mutations were frequently multiple and accompanied by loss of heterozygosity, and there was an association between the number of mutations and the severity of TRI exposure. We observed a specific mutational hot spot at VHL nucleotide 454 in the RCCs of 13 (39%) of the patients, and this mutation was present in adjacent non-neoplastic kidney parenchyma in four of these patients. The nucleotide 454 mutation was neither detected in any of the RCCs from patients without TRI exposure nor in any of the healthy subjects.
Conclusion:
Our results suggest that RCC in patients with high, cumulative TRI exposure is associated with a unique mutation pattern in the VHL gene.
Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.
Capillary hemangioblastoma is the most frequent manifestation of the autosomal dominantly inherited von Hippel-Lindau (VHL) disease but also presents as a nonfamilial, sporadic vascular tumor. Hemangioblastomas are characterized by a dense network of capillaries in association with cysts. To investigate the mechanisms underlying neovascularization and cyst formation, we analyzed eight VHL disease-associated and five sporadic hemangioblastomas. Histologically, both tumor types showed a similar phenotype. The capillaries expressed the endothelial cell markers von Willebrand factor and CD31 antigen. We investigated the expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen which is also known to induce vascular permeability in vivo, and its high affinity tyrosine kinase receptors flt-1 and KDR. Northern blot and in situ hybridization analysis revealed significant up-regulation of VEGF and VEGF receptor expression in VHL disease-associated and sporadic hemangioblastomas compared to normal brain and tumor stromal cells as sites of abundant VEGF transcription. Endothelial cells did not express detectable amounts of VEGF mRNA but coexpressed flt-1 and KDR. By immunohistochemistry, VEGF protein was detectable in the tumor interstitium and was found to be concentrated around capillaries. Performing reverse transcription-PCR, we demonstrated that VEGF121 and VEGF165 were the splice variants predominantly expressed, whereas mRNA encoding VEGF189 was present at smaller amounts. Our findings suggest that, in VHL disease-associated and sporadic hemangioblastomas, VEGF121 and VEGF165 are secreted by stromal cells and interact with the corresponding VEGF receptors expressed on tumor endothelial cells. This paracrine mechanism may mediate neovascularization and cyst formation in capillary hemangioblastomas.
The expression of epidermal growth factor receptor (EGFR) and the pre-pro form of one of its ligands, transforming growth factor-alpha (TGF-alpha), was studied by Northern blotting in a series of 14 capillary hemangioblastomas of the central nervous system. A constant coexpression of EGFR and pre-pro-TGF-alpha mRNAs was found. Immunocytochemical investigation of an extended series of 51 capillary hemangioblastomas revealed that the stromal cells in these tumors showed immunoreactivity with monoclonal antibodies to EGFR and TGF-alpha. Analysis of gene dosage by Southern blotting in 20 tumors indicated a normal gene copy number of EGFR and TGF alpha in all cases. Our findings suggest that autocrine and/or juxtacrine growth stimulation via the EGFR may contribute to tumor growth in capillary hemangioblastomas.
The clinical importance of microvessel quantitation as a prognostic indicator in invasive breast cancer was examined. This study included 155 patients with invasive breast cancer, with a median follow-up of 82 months. Microvessels were identified by immunohistochemical staining for factor VIII-related antigen in formalin-fixed, paraffin-embedded primary tumours. For each tumour, microvessels were counted within a 200 x magnification field in the area of highest microvessel density. Microvessel counts (MVCs) had no correlation with tumour size, lymph node status or histological grade. When patients were classified by MVC, higher counts were associated with shorter disease-free survival and overall survival (P < 0.025 and P < 0.01 respectively). Multivariate analysis showed that MCV is an independent prognostic factor. Microvessel quantitation may be a useful predictor for identifying breast cancer patients at high risk for relapse and death.
Images
Figure 1
We have developed a microdissection technique that allows for procurement and analysis of specific, minute cell populations from routine, 5-mu, formalin-fixed, paraffin-embedded histological tissue sections. Lesions < 1 mm in size can be specifically examined. Cells of interest are procured under direct microscopic visualization followed by a single-step DNA extraction and subsequent polymerase chain reaction. Amplification of DNA from selected cell populations was demonstrated by detecting a loss of heterozygosity (LOH) at the von Hippel-Lindau disease (VHL) gene in an atypical renal lesion and a renal cell carcinoma in a kidney of a VHL patient. Moreover, previously unrecognized LOH on the short arm of chromosome 3 (3p25-26) was detected in microdissected colorectal carcinoma cells in a non-VHL patient with sporadic colon carcinoma. This technique should prove useful in DNA studies of small lesions and cell populations. Furthermore, microscopic premalignant, in situ, and invasive lesions can be selectively examined.
Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a secreted protein implicated in tumor-associated microvascular hyperpermeability and angiogenesis. Tumor cells in 11 of 12 renal cell carcinomas expressed high levels of VPF messenger RNA (mRNA) by in situ hybridization, the only exception being a case of the relatively avascular papillary variant. Expression was further accentuated adjacent to areas of necrosis. Both tumor cells and endothelial cells in small vessels adjacent to tumor stained strongly for VPF protein by immunohistochemistry. Endothelial cells did not express detectable VPF mRNA, but did express high levels of mRNA for the VPF receptors flt-1 and KDR indicating that the endothelial cell staining likely reflects binding of VPF secreted by adjacent tumor cells. Three transitional cell carcinomas also labeled strongly for VPF mRNA. These data suggest an important role for VPF in the vascular biology of these two common human malignancies.
Mutations or loss of both alleles of the von Hippel-Lindau (VHL) tumor suppressor gene has been documented in sporadic renal cell carcinomas and neoplasms that arise in individuals having the VHL syndrome. The well-vascularized phenotype of tumors that form in VHL disease let us consider vascular endothelial growth factor (VEGF) as a mediator of tumor growth in VHL disease. Human renal carcinoma cells that either lacked endogenous wild-type VHL or were transfected with an inactive mutant VHL showed deregulated expression of VEGF on the mRNA and protein level that was reverted by introduction of wild-type VHL. Stimulation of proliferation of endothelial cells by conditioned medium of cells expressing mutant VHL was almost abolished by neutralizing the VEGF. In contrast, expression of basic fibroblast growth factor and of c-myc proto-oncogene was not affected by VHL. Our data suggest VEGF as the key tumor angiogenesis factor in VHL disease.
Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both normoxic and hypoxic conditions. Reintroduction of wild-type, but not mutant, pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in the transduction of signals generated by changes in ambient oxygen tension.
The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.
Patients with von Hippel-Lindau (VHL) disease develop a spectrum of bilateral clear-cell renal lesions including cysts and renal cell carcinomas (RCCs). VHL gene deletions have been previously reported in VHL-associated macroscopic RCC. Although histological analysis suggests that microscopic cystic lesions in the VHL patients may represent precursors of the RCC, there is at present no direct molecular evidence of their relationship. To investigate the relationship between cystic lesions and RCC, 26 microdissected archival renal lesions from two VHL disease patients were studied for loss of heterozygosity at the VHL gene locus using polymerase chain reaction single-strand conformation polymorphism analysis. The renal lesions included 2 benign cysts, 5 atypical cysts, 5 microscopic RCCs in situ, 5 cysts lined by a single layer of cells, in which RCCs in situ were developing, and 2 microscopic and 7 macroscopic RCCs. Except for a single benign cyst, 25 of 26 renal lesions showed nonrandom allelic loss of the VHL gene. In either of the 2 patients, the same VHL allele was deleted in all of the lesions tested, indicating loss of the wild-type allele and retention of the inherited, mutated VHL allele. The results suggest that all clear-cell lesions in the VHL kidney represent neoplasms and that the loss of the VHL gene occurs early in their development. Atypical and benign cysts most likely represent the initial phenotype in malignant transformation to the RCC.
The inactivation of the von Hippel-Lindau (VHL) gene predisposes affected individuals to VHL syndrome and is an early genetic event associated with sporadic renal cell carcinoma and CNS hemangioblastomas. The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A. Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex in vitro and in vivo. Nearly 70% of naturally occurring cancer-predisposing mutations of VHL disrupt this interaction. The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex. Immunofluorescence studies show Hs-CUL-2 to be a cytosolic protein that can be translocated to the nucleus by pVHL. Recently it has been shown that a yeast Hs-CUL-2 homolog, Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway. In Caenorhabditis elegans, a null mutation of another Hs-cul-2 homolog, Ce-cul-1, results in hyperplasia in all tissues and is required for cell cycle exit. Hence, Hs-cul-2 may be required for VHL function and, therefore, may be a candidate human tumor-suppressor gene.
Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1beta. Because HIF-1beta is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major influence on gene expression, tumor angiogenesis, and growth.
We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously "on," tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.
The von Hippel-Lindau (VHL) tumor suppressor gene has a critical role in the pathogenesis of clear cell renal cell carcinoma (RCC), because VHL mutations have been found in both VHL disease-associated and sporadic RCC. Overexpression of transforming growth factor (TGF)-alpha has been observed in numerous RCC tumors and cell lines, and TGF-alpha has been demonstrated to support RCC cell growth through an autocrine loop. We demonstrate here that VHL substantially decreases TGF-alpha message and protein by shortening TGF-alpha mRNA half-life. By Northern analysis TGF-alpha mRNA steady-state levels were suppressed 5-fold in permanent 786-0 RCC cell lines expressing wild-type VHL compared with 786-0 cells expressing an empty vector or a mutant VHL protein lacking COOH-terminal residues 116-213 (deltaVHL). By Western analysis, VHL also substantially down-regulated the unprocessed, cell-associated Mr 20,000 TGF-alpha protein. Moreover, secreted TGF-alpha was undetectable in VHL-expressing cells. In contrast, VHL did not down-regulate the TGF-alpha receptor, epidermal growth factor receptor, either at the mRNA or protein level. Nuclear run-on in vitro transcription experiments in 786-0 cells showed that VHL did not affect transcriptional control of the endogenous TGF-alpha gene. However, actinomycin D experiments revealed a long TGF-alpha mRNA half-life in 786-0 cells that was significantly decreased by wild-type VHL but not by deltaVHL. We have, therefore, identified TGF-alpha, an important growth factor for RCC, as a new target gene for VHL and demonstrated that VHL acts by decreasing TGF-alpha mRNA stability.
The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O
2
) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the
Caenorhabditis elegans
Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.
von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germ line mutation of the von Hippel-Lindau tumor suppressor gene (VHL). Tumors observed in this disorder include retinal and central nervous system hemangioblastomas, clear cell renal carcinomas and pheochromocytomas. The VHL gene product, pVHL, is a component of a ubiquitin ligase which targets the transcription factor known as hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. pVHL also plays roles in the control of extracellular matrix formation and cellcycle exit. Different VHL mutations confer different site-specific risks of cancer. Type 2C VHL mutations confer an increased risk of pheochromocytoma without the other stigmata of VHL disease. Here we report that the products of such type 2C VHL alleles retain the ability to down regulate HIF but are defective for promotion of fibronectin matrix assembly. Furthermore, pVHL L188V, a well studied type 2C mutant, retained the ability to suppress renal carcinoma growth in vivo. These studies strengthen the notion that HIF deregulation plays a causal role in hemangioblastoma and renal carcinoma, and raises the possibility that abnormal fibronectin matrix assembly contributes to pheochromocytoma pathogenesis in the setting of VHL disease.
The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC), To date, the only VCBC substrates identified are the hypoxia-inducible factor alpha subunits (HIF-1 alpha and HIF-2 alpha), However, pVHL is thought to have multiple functions and the significance of HIF-1 alpha and HIF-2 alpha regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes, Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF-alpha subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF-alpha ubiquitylation in vitro and demonstrated wildtype binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 28) demonstrated variable effects on HIF-a subunit and elongin binding, but all resulted in defective HIF-alpha( regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF-alpha dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF-alpha subunit being required for HAB development and RCC susceptibility.
Background: The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors—with mutations in the von Hippel‐Lindau (VHL) tumor suppressor gene for clear-cell RCC specifically and with long-term exposure to high doses of trichloroethylene (TRI), an industrially important solvent, for RCC generally. We investigated whether TRI exposure produces RCC through a specific mutational effect on the VHL gene by analyzing VHL sequences in the RCCs of patients exposed to high, cumulative doses of TRI. Methods: The level of exposure for each of 44 patients with RCC who had known industrial exposure to TRI was classified according to the duration, frequency, and mode of exposure. Samples of normal and cancerous tissues were microdissected from paraffin-embedded tissue. DNA was isolated from these samples, and somatic VHL mutations were identified by polymerase chain reaction analysis, singlestrand conformation polymorphism analysis, DNA sequencing, and restriction enzyme digestion. Control samples included RCC DNA from 107 patients without known TRI exposure and lymphocyte DNA from 97 healthy subjects. Results: RCCs of TRI-exposed patients showed somatic VHL mutations in 33 (75%) of 44 cases. The mutations were frequently multiple and accompanied by loss of heterozygosity, and there was an association between the number of mutations and the severity of TRI exposure. We observed a specific mutational hot spot at VHL nucleotide 454 in the RCCs of 13 (39%) of the patients, and this mutation was present in adjacent non-neoplastic kidney parenchyma in four of these patients. The nucleotide 454 mutation was neither detected in any of the RCCs from patients without TRI exposure nor in any of the healthy subjects. Conclusion: Our results suggest that RCC in patients with high, cumulative TRI exposure is associated with a unique mutation pattern in the VHL gene. [J Natl Cancer Inst 1999; 91:854‐61]
Richard S, Campello C, Taillandier L, Parker F, Resche F (Hôpital Necker, Paris, CHU Hôpital Caremeau, Nîmes, CHU Hôpital Saint-Julien, Nancy, CHU Hôpital de Bic tre, Le Kremlin-Bic tre, Hôpital Laennec, Nantes, France). Haemangioblastoma of the central nervous system in von Hippel–Lindau disease (Minisymposium: MEN & VHL). J Intern Med 1998; 243: 547–53.
Haemangioblastoma of the central nervous system (CNS) is the most characteristic lesion and the most common presenting manifestation of von Hippel–Lindau (VHL) disease and has a striking tendency to multiple occurrence. Its sites of predilection are the posterior fossa (cerebellum++), and the spinal cord. Haemangioblastoma may cause increased intracranial pressure and/or neurological deficits and remains the main cause of morbidity and mortality in VHL. Treatment of symptomatic haemangioblastoma remains neurosurgical and is often in emergency. Haemangioblastoma appears to be more commonly associated with VHL than previously reported and suggests that all patients with ‘sporadic’ haemangioblastoma should be investigated for evidence of VHL disease. From a fundamental point of view, haemangioblastoma is a benign neoplastic entity with a double, vascular and cellular differentiation. Mutational inactivation of both copies of the VHL gene plays a major role in the pathogenesis of haemangioblastoma. Over-expression of vascular endothelial growth factor (VEGF) and VEGF-receptors has been recently demonstrated in these tumours, raising the possibility of angioblastic origin, and is of very great interest in view of the direct implication of the VHL gene in negative regulation of VEGF.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PAS transcription factor consisting of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha expression and HIF-1 transcriptional activity increase exponentially as cellular O2 concentration is decreased. Several dozen target genes that are transactivated by HIF-1 have been identified, including those encoding erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The products of these genes either increase O2 delivery or allow metabolic adaptation to reduced O2 availability. HIF-1 is required for cardiac and vascular development and embryonic survival. In fetal and postnatal life, HIF-1 is required for a variety of physiological responses to chronic hypoxia. HIF-1 expression is increased in tumor cells by multiple mechanisms and may mediate adaptation to hypoxia that is critical for tumor progression. HIF-1 thus appears to function as a master regulator of O2 homeostasis that plays essential roles in cellular and systemic physiology, development, and pathophysiology.
Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/− RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL−/− RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-α (TGF-α), another VHL-regulated growth factor. We show that TGF-α mRNA and protein are hypoxia-inducible in VHL−/− RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL−/− RCC cells, TGF-α can also act as a specific growth-stimulatory factor for VHL−/− RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL−/− RCC cells, such as vascular endothelial growth factor and TGF-β1, which do not stimulate RCC cell proliferation. A TGF-α-specific antisense oligodeoxynucleotide blocked TGF-α production in VHL−/− RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL−/− RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-α stimulates proliferation. These results suggest that the generation of a TGF-α autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.
Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on chromosome 3p are a critical event In the pathogenesls of non-familial renal cell carcinoma (RCC). Germllne mutations of the von Hippel — Lindau (VHL) disease gene predispose to early onset and multifocal clear cell renal cell carcinoma, and the mechanism of tumorigenesls In VHL disease is consistent with a one-hit mutation model. To Investigate the role of somatic VHL gene mutations in non-familial RCC, we analysed 99 primary RCC for VHL gene mutations by SSCP and heteroduplex analysis. Somatic VHL gene mutations were Identified In 30 of 65 (46%) sporadic RCC with chromosome 3p allele loss and one of 34 (3%) tumours with no LOH for chromosome 3p. The VHL gene mutations were heterogeneous (17 frameshift deletions, eight missense mutations, four frameshift insertions, one nonsense and one splice site mutation), but no mutations were detected in the first 120 codons of cloned coding sequence. Most RCCs with somatic VHL mutations (23 of 27 (85%) informative cases) had chromosome 3p25 allele loss in the region of the VHL gene so that both alleles of the VHL gene had been inactivated as expected from a two–hit model of tumorigenesis. Detailed histopathology was available for 59 of the tumours investigated: 18 of 43 (42%) RCC with a clear cell appearance had a somatic VHL gene mutation but none of 16 non–clear cell RCC (eight chromophilic, three chromophobe and five oncocytoma) (x2= 7.77, P<0.025). These results suggest that somatic mutations of the VHL gene are a critical event in the pathogenesis of non-familial clear cell renal carcinoma, but do not exclude a role for other chromosome 3p tumour suppressor genes.
Findings of increased numbers of epidermal growth factor receptors (EGF-R) and increased expression of transforming growth factor alpha (TGF-alpha) in surgical specimens of human renal cell carcinoma have led to the proposal that growth of these tumors may be regulated by TGF-alpha in an autocrine manner. In the studies presented here, we have examined this hypothesis using two human renal carcinoma cell lines, SKRC-4 and SKRC-29. We demonstrated that both SKRC-4 and SKRC-29 cells were growth stimulated by greater than 35% when cultured in the presence of TGF-alpha or EGF and were inhibited by 29% to 46% if cultured in the presence of anti-EGF-R monoclonal antibody 225. Treatment of cells with TGF-alpha enhanced the levels of expression of EGF-R mRNA and TGF-alpha mRNA. In addition, incubation of cells with monoclonal antibody 225 significantly elevated the levels of excreted TGF-alpha species in the culture medium. Our findings suggest that proliferation of human renal carcinoma cells may be regulated by endogenously produced TGF-alpha and that this regulatory pathway can be interrupted using antibody to its receptor, EGF-R.
Experimental evidence suggests that the growth of a tumor beyond a certain size requires angiogenesis, which may also permit metastasis. To investigate how tumor angiogenesis correlates with metastases in breast carcinoma, we counted microvessels (capillaries and venules) and graded the density of microvessels within the initial invasive carcinomas of 49 patients (30 with metastases and 19 without).
Using light microscopy, we highlighted the vessels by staining their endothelial cells immunocytochemically for factor VIII. The microvessels were carefully counted (per 200x field), and their density was graded (1 to 4+), in the most active areas of neovascularization, without knowledge of the outcome in the patient, the presence or absence of metastases, or any other pertinent variable.
Both microvessel counts and density grades correlated with metastatic disease. The mean (+/- SD) count and grade in the patients with metastases were 101 +/- 49.3 and 2.95 +/- 1.00 vessels, respectively. The corresponding values in the patients without metastases were significantly lower--45 +/- 21.1 and 1.38 +/- 0.82 (P = 0.003 and P less than or equal to 0.001, respectively). For each 10-microvessel increase in the count per 200x field, there was a 1.59-fold increase in the risk of metastasis (95 percent confidence interval, 1.19 to 2.12; P = 0.003). The microvessel count and density grade also correlated with distant metastases. For each 10-microvessel increase in the vessel count per 200x field, there was a 1.17-fold increase in the risk of distant metastasis (95 percent confidence interval, 1.02 to 1.34; P = 0.029).
The number of microvessels per 200x field in the areas of most intensive neovascularization in an invasive breast carcinoma may be an independent predictor of metastatic disease either in axillary lymph nodes or at distant sites (or both). Assessment of tumor angiogenesis may therefore prove valuable in selecting patients with early breast carcinoma for aggressive therapy.
Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.
A hereditary form of renal cell carcinoma exists in rats that results from a single gene mutation and is histologically similar to that described in humans. Cell lines derived from these rat tumors were shown to express abundant transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF)-receptor RNA transcripts, but no EGF mRNA. In contrast, normal kidney expressed EGF and EGF-receptor transcripts, but TGF-alpha transcripts were barely detectable. Other kidney epithelial cell lines examined (NRK 52E, MDCK, and LLCPK) were negative for expression of both TGF-alpha and EGF transcripts, but expressed EGF receptors. In addition, the renal cell carcinoma-derived lines secreted TGF-alpha into the media. Immunohistochemistry of normal kidney with a TGF-alpha specific antibody revealed a characteristic pattern of staining of collecting ducts and, to a lesser degree, proximal tubules. In the neoplastic kidney tissue, both the cystic and solid portions of the tumors displayed intense immunoreactivity, indicating that altered expression of this growth factor by the transformed cells occurred in situ. These results suggest that altered TGF-alpha expression is an important aspect of the neoplastic phenotype in rodent as well as human renal cell carcinoma, and support the use of this hereditary rodent tumor model for studying the pathogenesis of this disease.
We have analyzed the expression of the genes for the precursors of epidermal growth factor (pro-EGF) and transforming growth factor alpha (proTGF-alpha) as well as for the EGF receptor in tissue specimens of a large number of adult patients with renal cell carcinoma. Since normal kidney tissue was available from the same patients we could directly compare the expression of these genes in tumors with that in adjacent normal renal tissue. Our experiments reveal underexpression of the proEGF gene in all tumors analyzed (21 of 21) and overexpression of the genes for proTGF-alpha (33 of 33 analyzed) and EGF receptor (22 of 23 analyzed) in tumor samples, when compared with normal kidney tissue. The expression of the proTGF-alpha gene appeared to depend on grade and differentiation of the tumor, since well differentiated tumors (grade 1) expressed more proTGF-alpha mRNA than the adjacent normal tissue but significantly less than poorly differentiated tumors (grade 2 or 3), which are the most aggressive ones. In none of these tissue specimens did we find, by Southern analysis, amplification of the proTGF-alpha or EGF receptor gene. Therefore, overexpression of these genes must be due to another effect, perhaps an alteration of their mRNA turnover. Although the EGF receptor gene (c-erbB1) is overexpressed in nearly all carcinomas analyzed, there was no linear coexpression with the proTGF-alpha gene. In contrast, transcription of the proEGF gene was completely turned off in tumor tissue. Although we have found by restriction fragment length polymorphism analysis, in one of three tumor samples, evidence for a somatic mutation within the proEGF gene, we do not know yet, due to the limited number of Southern analyses, whether this somatic mutation is causally involved in the decrease of proEGF mRNA expression and, hence, is representative of renal cell carcinoma. To our knowledge, this is the first observation on primary tumor tissue in humans that upon malignant transformation the gene for a polypeptide growth factor gene is underexpressed.
Hypoxic states are associated with abnormal proliferation and constriction of the smooth muscle cells surrounding the distal vessels of the lung. In hypoxic as well as in normal states, the endothelial cell layer may play a key role in controlling smooth muscle tone by secreting a number of vasoactive agents. Platelet-derived growth factor (PDGF), produced by endothelial cells, is a major growth factor for vascular smooth muscle cells and a powerful vasoconstrictor. It consists of a disulfide-linked dimer of two related peptides, A and B, that are products of two different genes. We found that hypoxic conditions (0-3% oxygen environments) significantly increased PDGF-B mRNA in cultured human umbilical vein endothelial cells by enhancing the transcriptional rate of this gene. This increase was inversely proportional to oxygen tension and was reversible upon reexposure of cells to a 21% oxygen atmosphere. mRNA levels of PDGF-A were not affected nor was the overall rate of cellular gene transcription increased in response to hypoxia. These studies indicate that endothelial cells are not only capable of sensing oxygen tension, but are also able to discriminate and respond to even small differences in oxygen tension resulting in dramatic upregulation of the PDGF-B chain gene.
Epidermal growth factor (EGF) is a cell-regulating polypeptide that appears important to the maintenance and function of some benign tissues and to the transformation and proliferation of certain malignancies. In humans the highest concentrations of EGF are found in the urine. We investigated possible interactions between EGF and normal and neoplastic tissues of the urinary system with indirect immunohistochemical staining of paraffin-embedded tissue sections. A polyclonal antibody directed against mouse EGF but shown to react with human EGF was used in the assays. Positive staining was granular in nature and confined to the cytoplasm. Staining of the renal parenchyma (N = 5) was observed in the epithelium of the proximal and distal tubules and the collecting ducts. There was staining of clear cell (N = 6) and papillary (N = 3) carcinomas of the kidney. Staining of the normal urothelium (N = 5) was limited to superficial cells. All transitional cell (N = 21) and squamous (N = 2) carcinomas of the bladder stained. Subjectively, the staining intensity of the transitional cell carcinomas correlated inversely with tumor differentiation. In light of evidence that internalized, receptor-bound EGF is rapidly degraded, the striking immunohistochemical demonstration of cytoplasmic EGF suggests active synthesis. EGF synthesized by urothelial and renal carcinomas may be involved in an autocrine mechanism of malignant proliferation.
A partial cDNA sequence for the gene linked to the von Hippel-Lindau (VHL) syndrome was reported in 1993. Mutation or loss of both VHL alleles has been documented in sporadic renal cell carcinomas and in the neoplasms that arise in von Hippel-Lindau kindreds. We have determined that the protein product of the VHL gene is an approximately 30 kilodalton cytoplasmic protein. The renal carcinoma cell line 786-O is known to harbour a VHL mutation and, as shown here, fails to produce a wild-type VHL protein. Reintroduction of wild-type, but not mutant, VHL into these cells had no demonstrable effect on their growth in vitro but inhibited their ability to form tumours in nude mice.
Germ-line mutations of the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of human tumors,
and somatic mutations of this gene have been identified in sporadic renal cell carcinomas and cerebellar hemangioblastomas.
Two transcriptional elongation factors, Elongin B and C, were shown to bind in vitro and in vivo to a short, colinear region
of the VHL protein (pVHL) that is frequently mutated in human tumors. A peptide replica of this region inhibited binding of
pVHL to Elongin B and C whereas a point-mutant derivative, corresponding to a naturally occurring VHL missense mutation, had
no effect. These results suggest that the tumor suppression function of pVHL may be linked to its ability to bind to Elongin
B and C.
Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioblastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, Elongin (SIII), is identified as a functional target of the VHL protein. Elongin (SIII) is a heterotrimer consisting of a transcriptionally active subunit (A) and two regulatory subunits (B and C) that activate transcription elongation by RNA polymerase II. The VHL protein was shown to bind tightly and specifically to the Elongin B and C subunits and to inhibit Elongin (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.
Immunohistochemical stains using antibody to epidermal growth factor receptor (EGFR) and transforming growth factor alpha (TGF-alpha) were applied to 67 cases of renal cell carcinoma retrieved from the files of the Division of Surgical Pathology. The 64 patients (33 females, 31 males) ranged in age from 35 to 87 years (mean, 61 years). Two patients had more than one renal carcinoma included in this study. Fifty-seven cases (85%) expressed EGFR, with staining largely confined to the cell membrane. Staining intensity was directly correlated with tumor grade (P = 0.02, T test), size (P = 0.04), and stage (P = 0.01). Those cases with more intense EGFR staining also appear to have shorter patient survival than those showing less intense staining (43 mo versus 63 mo, P = 0.05). Forty-nine cases (73%) expressed TGF-alpha in a distribution similar to that of EGFR. There was no significant correlation between TGF-alpha staining intensity and tumor size, stage, or grade. When the tumor expressed either EGFR or TGF-alpha but not both proteins, average patient survival was 38 months, while the average survival of those patients whose tumors expressed both EGFR and TGF-alpha was 61 months (P = 0.04). Three of eleven cases, all of which expressed EGFR, were felt to show EGFR gene amplification using a modification of the differential polymerase chain reaction on archival, formalin-fixed, paraffin-embedded tissue. EGFR and TGF-alpha likely play a role in the progression of renal cell carcinoma, and their coexpression may have favorable prognostic implications.
Hemangioblastoma is one of the benign tumors in the central nervous system. It is often associated with the von Hippel-Lindau (VHL) disease, a well known hereditary tumor syndrome. It is believed that inactivation of both alleles of VHL tumor suppressor gene is essential in the tumorigenic processes in hemangioblastomas associated with VHL disease. The molecular basis for the development of sporadic hemangioblastomas is not known. Here, we analyzed 13 cases of primary sporadic hemangioblastomas for somatic mutations of VHL gene with single strand conformational polymorphism analyses of the tumor DNAs. We detected abnormal single strand conformational polymorphism pattern in 7 tumors (54%). Of these 7 possibly mutated tumors, we successfully characterized 3 tumors by direct sequencing. We were unable to sequence 4 tumors because of the poor quality of DNA obtained from paraffin blocks. Somatic mutations in the 3 tumors were 2 missense mutations and 1 microdeletion. These mutations were observed in 1 tumor in exon 1 and 2 tumors in exon 2. Our results suggest that mutations of VHL tumor suppressor gene are involved in the development of at least 20% of sporadic central nervous system hemangioblastomas.
Transforming growth factor-alpha (TGF-alpha) is a member of the epidermal growth factor (EGF) family of proteins and, like EGF, elicits its cellular function by binding to the EGF receptor. EGF stimulation may have a role in several normal and pathologic processes in the kidney, and EGF has been implicated in the development of renal cysts in vitro and in human autosomal dominant polycystic kidney disease. We sought to determine whether renal expression of an EGF-like protein (TGF-alpha) could lead to the formation of renal cysts in vivo. We examined morphologic alterations to the normal kidney caused by renal expression of a TGF-alpha transgene linked to a mouse metallothionein promoter stably integrated into the genome of the CD1 mouse. TGF-alpha transgene expression was induced with exogenous zinc treatment starting at 4 weeks of age, and mice were killed at 8 weeks of age. The transgene was expressed at higher levels in female transgenic mice than in male transgenic mice. The augmented expression of the TGF-alpha transgene in females was associated with increased renal size and the development of renal epithelial cysts. Both male and female mice exhibited increases in glomerular size and mesangial volume density. These results provide evidence that stimulation by an endogenous EGF-like protein can lead to renal enlargement, glomerular mesangial expansion, and renal cyst formation.
We analyzed 47 primary sporadic human renal cell carcinomas (39 clear cell and 8 non-clear cell) for mutations of the von Hippel-Lindau (VHL) tumor suppressor gene using the polymerase chain reaction and single strand conformational polymorphism analysis of DNA. All of the positive cases in single strand conformational polymorphism analyses were further characterized by direct sequencing. Somatic mutations were detected in 22 (56%) of 39 clear cell renal carcinomas including 15 deletions, 3 insertions, 3 missense mutations, and 1 nonsense mutation. Nineteen of these mutations predicted to produce truncation of the VHL protein. These mutations mainly occurred in the last one-third region of exons 1, 2, and 3. In addition, loss of heterozygosity of the VHL gene was observed in 16 (84%) of 19 informative clear cell renal carcinomas. No somatic mutations were detected in 8 non-clear cell carcinomas. These results show that the VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinomas, especially in the clear cell subtype renal cell carcinoma. Clear cell carcinoma might be distinguished from other pathological types of renal cell carcinomas by molecular genetic techniques.
To elucidate the mechanisms underlying the regulation of growth and differentiation of capillary hemangioblastoma we studied the expression of selected growth factors and growth factor receptors by immunocytochemistry. As stromal cells of capillary hemangioblastoma express high levels of vascular endothelial growth factor (VEGF) and placental growth factor (P1GF) mRNA, we studied the distribution of the corresponding VEGF and P1GF proteins. We also studied the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptors (PDGFR) because their ligands have been reported to promote angiogenesis. The stromal cells expressed abundant EGFR and, in addition, some stromal cells expressed PDGFR-alpha but not PDGFR-beta. In contrast, the endothelial cells co-expressed PDGFR-alpha and PDGFR-beta. VEGF and P1GF were expressed by scattered stromal cells; however, more intense staining was observed in the endothelial cells of the intratumoral blood vessels, possibly indicating the secreted growth factors bound to their target receptors. We conclude that capillary hemangioblastomas express a variety of growth factor receptors and ligands, potentially involved in both autocrine and paracrine loops. The uniformly high EGFR expression is unique among brain tumors and may be associated with the typical morphology of capillary hemangioblastoma. The expression of highly angiogenic growth factors and their receptors may contribute to the rich vascularity of this enigmatic tumor.
The von Hippel-Lindau (VHL) disease gene on chromosome 3p25.5 has been cloned and shown to be mutated in the germline DNA of patients with VHL disease. In addition, approximately 60% of sporadic renal cell carcinomas (RCCs) have been shown to have a VHL gene mutation in fresh frozen tumor tissue and tumor-derived cell lines. The objective of this study was to test whether VHL gene mutations could be detected in archival sporadic RCC cases. We studied three sporadic RCCs, two oncocytomas, and the corresponding adjacent normal renal parenchyma by polymerase chain reaction and single-strand conformation polymorphism analysis using paraffin-embedded, formalin-fixed material. Tumor and normal tissue were microdissected from eosin-stained 5-microns-thick histologic sections. Mutations in exon 2 of the VHL gene were detected in all three of the sporadic RCCs but were not observed in the matched normal renal tissues or in the two oncocytomas tested. The mutations were identical to those detected in tumor cell lines from the same patients. This report represents the first detection of VHL gene mutations in sporadic RCCs in archival, paraffin-embedded tissue. A high percentage of sporadic RCCs show VHL gene mutations in fresh frozen tissue but the availability of frozen material is limited, so the evaluation of archival tumors for similar mutations should prove useful in future RCC studies. Furthermore, the results suggest that the genetic events leading to the development of clinically benign renal oncocytoma may differ from those leading to malignant RCC.
To compare the expression of VEGF in renal cell carcinoma (RCC) and normal kidney.
RT-PCR and Western blot analysis were performed on tumour and normal adjacent kidney collected from 31 patients (29 RCC and 2 oncocytomas) as well as proliferating vascular endothelial cells (VEC) in culture.
Expression of 3 VEGF isoforms was detected in normal renal parenchyma and all ROC by RT-PCR, but was not apparent in proliferating VEC. In 27 RCC, Western blot analysis demonstrated 3-37 fold increases in VEGF expression when compared to normal parenchyma. Immunohistochemistry demonstrated VEGF staining of both tumour cells and adjacent vascular endothelium. Normal kidney showed no staining for VEGF. In the 2 remaining RCC and both oncocytomas VEGF was not increased.
VEGF expression is increased in RCC and may have a paracrine effect in these tumours in stimulating angiogenesis.
Central nervous system hemangioblastoma is a neoplasm with characteristic and well-described histopathological features, including proliferation of vascular and stromal cells. yet, the histogenesis of the stromal cell component and its neoplastic capacity as compared with the vascular component are still controversial. Stromal cells were selectively procured from formalin-fixed, paraffin-embedded archival tissue from a von Hippel-Lindau (VHL) disease patient with a cerebellar hemangioblastoma and studied for loss of heterozygosity (LOH) of the VHL gene locus and associated microsatellite regions. The stromal cells consistently showed LOH. Analysis of mixed stromal anti vascular areas of this tumor and four other hemangioblastomas of VHL patients showed that loss of heterozygosity was partially obscured. These preliminary results suggest that the stromal component of hemangioblastomas contains genetic alterations consistent with a neoplastic nature. Additional samples of pure stromal cells need to be analyzed to establish the prevalence of VHL gene deletion in stromal cells of capillary hemangioblastoma and, hence, its pathogenetic significance.
Transforming growth factor-alpha (TGF-alpha) is a multifunctional cell regulatory protein with a wide range of effects on cell growth and differentiation and has been implicated in the neoplastic transformation of a variety of cell types. Altered expression of TGF-alpha and its cognate receptor (epidermal growth factor receptor) is enhanced in human and rat renal cell carcinomas. The objective of the study reported here was to determine whether altered TGF-alpha expression is an early or late event in renal tubular oncogenesis. The immunohistochemical expression of TGF-alpha was studied in preneoplastic renal tubular lesions in a rat model of hereditary renal cell carcinoma. Strong TGF-alpha immunoreactivity was present at all stages of renal cell tumor development, including the earliest detectable dysplasias. In contrast, the non-neoplastic regenerating tubular epithelium of rat degenerative nephropathy did not stain for TGF-alpha, although this tissue exhibited a proliferative capacity similar to that observed in the dysplastic and neoplastic lesions. This study indicated that altered TGF-alpha expression was detectable early in the development of renal cell tumors and may be an important feature of the transformed phenotype.
The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.
von Hippel-Lindau disease is a hereditary cancer syndrome characterized by the development of vascular tumors of the central nervous system and retina, clear cell renal carcinomas, pheochromocytomas, pancreatic islet cell tumors, endolymphatic sac tumors, and benign cysts affecting a variety of organs. VHL disease is caused by germline mutations of the von Hippel-Lindau tumor suppressor gene located on chromosome 3p25. Tumor development in this setting is due to inactivation or loss of the remaining wild-type allele in a susceptible cell. The highly vascular nature of VHL-associated neoplasms can be understood in light of the recent finding that the VHL gene product (pVHL) inhibits the accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This property of pVHL appears to be linked to its ability to bind to complexes containing elongin B, elongin C, and cullin 2 (Cul2). Elongin C and Cul2, based on their homology with Skp1 and Cdc53, respectively, are suspected of targeting certain proteins for covalent modification with ubiquitin and hence for degradation. One model, which remains to be tested, is that the binding of pVHL to elongins B/C and Cul2 affects the ubiquitination of RNA-binding proteins that regulate the stability of hypoxia-inducible mRNAs.
Cerebellar hemangioblastoma is a benign central nervous system neoplasm with characteristic proliferation of vascular and stromal cells. There is increasing evidence that the stromal cell population may represent the neoplastic component of hemangioblastoma, whereas the vascular component may be composed of reactive, nonneoplastic cells. Therefore, successful genetic testing for loss of heterozygosity requires selective analysis of target cell populations. Here, tissue microdissection was used to selectively analyze the stromal cell component of 20 archival sporadic cerebellar hemangioblastomas for loss of heterozygosity at the Von-Hippel Lindau (VHL) gene and somatic VHL gene mutations. Allelic deletions at the VHL gene locus were detected in the stromal cell component with one or more markers (D3S1038, D3S1110, and/or 104/105) in 10 of 19 (52.6%) informative cases. In all cases, heterozygosity at the VHL gene locus was retained in the vascular component. In two cases, aberrant bands in exon 2 of the VHL gene were demonstrated in the stromal cells by PCR-based single-strand conformation polymorphism analysis, and somatic missense mutations were successfully characterized in two of the sporadic hemangioblastomas by direct sequencing. The results suggest that allelic losses and mutations of the VHL tumor suppressor gene play a role in sporadic cerebellar hemangioblastoma tumorigenesis. Furthermore, because the genetic changes were detected in selectively procured stromal cell areas, the data provide strong evidence that the stromal cell represents a neoplastic component of hemangioblastoma.