VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

The protein tyrosine phosphatase PPH-7 is required for fertility and embryonic development in C. elegans at elevated temperatures.

Post-translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post-translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH-7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH-7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel-Lindau (VHL) E3 ligase, suggesting a potential role for PPH-7 in the regulation of VHL.

VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Belzutifan, HIF-2α Inhibitor, and Clear Cell Renal Cell Carcinoma With Somatic Von-Hippel-Lindau Loss-of-Function Mutation.

The Von-Hippel-Lindau (VHL) gene, acting as a tumor suppressor, plays a crucial role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Approximately 90% of individuals with advanced ccRCC exhibit somatic mutations in the VHL gene. Belzutifan, orally administered small-molecule inhibitor of hypoxia-induced factor-2α, has demonstrated promising efficacy in solid tumors associated with germline loss-of-function mutations in VHL, including ccRCC. However, its impact on cases with somatic or sporadic VHL mutations remains unclear. Here, we present 2 cases where belzutifan monotherapy was employed in patients with advanced ccRCC and somatic loss-of-function mutations in VHL. Both patients exhibited a swift and sustained response, underscoring the potential role of belzutifan as a viable option in second or subsequent lines of therapy for individuals with somatic VHL mutations. Despite both patients experiencing a pulmonary crisis with respiratory compromise, their rapid response to belzutifan further emphasizes its potential utility in cases involving pulmonary or visceral crises. This report contributes valuable insights into the treatment landscape for advanced ccRCC with somatic VHL mutations.

Therapeutic Targeting of Hypoxia-Inducible Factors in Cancer.

In the realm of cancer therapeutics, targeting the hypoxia-inducible factor (HIF) pathway has emerged as a promising strategy. This study delves into the intricate web of HIF-associated mechanisms, exploring avenues for future anticancer therapies. Framing the investigation within the broader context of cancer progression and hypoxia response, this article aims to decipher the pivotal role played by HIF in regulating genes influencing angiogenesis, cell proliferation, and glucose metabolism. Employing diverse approaches such as HIF inhibitors, anti-angiogenic therapies, and hypoxia-activated prodrugs, the research methodologically intervenes at different nodes of the HIF pathway. Findings showcase the efficacy of agents like EZN-2968, Minnelide, and Acriflavine in modulating HIF-1α protein synthesis and destabilizing HIF-1, providing preliminary proof of HIF-1α mRNA modulation and antitumor activity. However, challenges, including toxicity, necessitate continued exploration and development, as exemplified by ongoing clinical trials. This article concludes by emphasizing the potential of targeted HIF therapies in disrupting cancer-related signaling pathways.

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma.

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

Discovery of a novel, highly potent EZH2 PROTAC degrader for targeting non-canonical oncogenic functions of EZH2.

Aberrant expression of EZH2, the main catalytic subunit of PRC2, has been implicated in numerous cancers, including leukemia, breast, and prostate. Recent studies have highlighted non-catalytic oncogenic functions of EZH2, which EZH2 catalytic inhibitors cannot attenuate. Therefore, proteolysis-targeting chimera (PROTAC) degraders have been explored as an alternative therapeutic approach to suppress both canonical and non-canonical oncogenic activity. Here we present MS8847, a novel, highly potent EZH2 PROTAC degrader that recruits the E3 ligase von Hippel-Lindau (VHL). MS8847 degrades EZH2 in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Notably, MS8847 induces superior EZH2 degradation and anti-proliferative effects in MLL-rearranged (MLL-r) acute myeloid leukemia (AML) cells compared to previously published EZH2 PROTAC degraders. Moreover, MS8847 degrades EZH2 and inhibits cell growth in triple-negative breast cancer (TNBC) cell lines, displays efficacy in a 3D TNBC in vitro model, and has a pharmacokinetic (PK) profile suitable for in vivo efficacy studies. Overall, MS8847 is a valuable chemical tool for the biomedical community to investigate canonical and non-canonical oncogenic functions of EZH2.

Design, synthesis, and evaluation of VHL-based EZH2 degraders for breast cancer.

EZH2 (enhancer of zeste homolog 2) is one of the most important histone methyltransferases (HMTs), and overexpression of EZH2 can lead to proliferation, migration and angiogenesis of tumor cells. But most of EZH2 inhibitors are only effective against some hematologic malignancies and have poor efficacy against solid tumors. Here, we report the design, synthesis, and evaluation of highly potent proteolysis targeting chimeric (PROTACs) small molecules targeting EZH2. We developed a potent and effective EZH2 degrader P4, which effectively induced EZH2 protein degradation and inhibited breast cancer cell growth. Further studies showed that P4 can significantly decrease the degree of H3K27me3 in MDA-MB-231 cell line, induce apoptosis and G0/G1 phase arrest in Pfeiffer and MDA-MB-231 cell lines. Therefore, P4 is a potential anticancer molecule for breast cancer treatment.

Journey of Von Hippel-Lindau (VHL) E3 ligase in PROTACs design: From VHL ligands to VHL-based degraders.

The scientific community has shown considerable interest in proteolysis-targeting chimeras (PROTACs) in the last decade, indicating their remarkable potential as a means of achieving targeted protein degradation (TPD). Not only are PROTACs seen as valuable tools in molecular biology but their emergence as a modality for drug discovery has also garnered significant attention. PROTACs bind to E3 ligases and target proteins through respective ligands connected via a linker to induce proteasome-mediated protein degradation. The discovery of small molecule ligands for E3 ligases has led to the prevalent use of various E3 ligases in PROTAC design. Furthermore, the incorporation of different types of linkers has proven beneficial in enhancing the efficacy of PROTACs. By far more than 3300 PROTACs have been reported in the literature. Notably, Von Hippel-Lindau (VHL)-based PROTACs have surfaced as a propitious strategy for targeting proteins, even encompassing those that were previously considered non-druggable. VHL is extensively utilized as an E3 ligase in the advancement of PROTACs owing to its widespread expression in various tissues and well-documented binders. Here, we review the discovery of VHL ligands, the types of linkers employed to develop VHL-based PROTACs, and their subsequent modulation to design advanced non-conventional degraders to target various disease-causing proteins. Furthermore, we provide an overview of other E3 ligases recruited in the field of PROTAC technology.

DNA-encoded library-enabled discovery of proximity-inducing small molecules.

Small molecules that induce protein-protein associations represent powerful tools to modulate cell circuitry. We sought to develop a platform for the direct discovery of compounds able to induce association of any two preselected proteins, using the E3 ligase von Hippel-Lindau (VHL) and bromodomains as test systems. Leveraging the screening power of DNA-encoded libraries (DELs), we synthesized ~1 million DNA-encoded compounds that possess a VHL-targeting ligand, a variety of connectors and a diversity element generated by split-and-pool combinatorial chemistry. By screening our DEL against bromodomains in the presence and absence of VHL, we could identify VHL-bound molecules that simultaneously bind bromodomains. For highly barcode-enriched library members, ternary complex formation leading to bromodomain degradation was confirmed in cells. Furthermore, a ternary complex crystal structure was obtained for our most enriched library member with BRD4BD1 and a VHL complex. Our work provides a foundation for adapting DEL screening to the discovery of proximity-inducing small molecules.

The von Hippel-Lindau protein forms fibrillar amyloid assemblies that are mitigated by the anti-amyloid molecule Purpurin.

The von Hippel-Lindau protein (pVHL) is a tumor suppressor involved in oxygen regulation via dynamic nucleocytoplasmic shuttling. It plays a crucial role in cell survival by degrading hypoxia-inducible factors (HIFs). Mutations in the VHL gene cause angiogenic tumors, characterized as VHL syndrome. However, aggressive tumors involving wild-type pVHL have also been described but the underlying mechanism remains to be revealed. We have previously shown that pVHL possesses several short amyloid-forming motifs, making it aggregation-prone. In this study, using a series of biophysical assays, we demonstrated that a pVHL-derived fragment (pVHL104-140) that harbors the nuclear export motif and HIF binding site, forms amyloid-like fibrillar structures in vitro by following secondary-nucleation-based kinetics. The peptide also formed amyloids at acidic pH that mimics the tumor microenvironment. We, subsequently, validated the amyloid formation by pVHL in vitro. Using the Curli-dependent amyloid generator (C-DAG) expression system, we confirmed the amyloidogenesis of pVHL in bacterial cells. The pVHL amyloids are an attractive target for therapeutics of the VHL syndrome. Accordingly, we demonstrated in vitro that Purpurin is a potent inhibitor of pVHL fibrillation. The amyloidogenic behavior of wild-type pVHL and its inhibition provide novel insights into the molecular underpinning of the VHL syndrome and its possible treatment.

von Hippel-Lindau disease-related neoplasia with an emphasis on renal manifestations.

von Hippel-Lindau (VHL) disease is characterized by biallelic inactivation of the VHL gene leading to abnormal or absent VHL protein function, and constitutive activation of hypoxia-inducible factors (HIF) that leads to pro-tumorigenic signaling. Individuals with VHL disease develop numerous cysts and tumors involving multiple organs including the kidneys, central nervous system, endolymphatic sac, lungs, pancreatobiliary system, adrenal glands, epididymis, and/or broad ligament. On histologic examination, these lesions show morphologic overlap as they are frequently characterized by cells with clear cytoplasm and prominent vascularity. In addition to distinguishing non-renal tumors from metastatic clear cell renal cell carcinoma, understanding site-specific histopathologic and immunophenotypic features of these tumors has several applications. This includes distinguishing VHL-related tumors from those that arise sporadically and lack VHL gene alterations, guiding further genetic workup, and helping distinguish between different genetic predisposition syndromes. In this context, immunohistochemical studies for markers such as paired box 8 (PAX-8), carbonic anhydrase 9 (CA9), and glucose transporter 1 (GLUT-1) have an important role in routine clinical practice and represent cost-effective diagnostic tools. The recent development of targeted therapeutics directed against HIF-mediated signaling represents a significant milestone in the management of VHL disease and highlights the importance of accurately diagnosing and characterizing the wide spectrum of VHL disease-associated lesions.

The kidney imaging surveillance scoring system (KISSS): using qualitative MRI features to predict growth rate of renal tumors in patients with von-Hippel Lindau (VHL) syndrome.

OBJECTIVE: To determine the reliability of an MRI-based qualitative kidney imaging surveillance scoring system (KISSS) and assess which imaging features predict growth rate (GR) of renal tumors in patients with VHL. MATERIALS AND METHODS: We identified 55 patients with VHL with 128 renal tumors who underwent intervention from 2015 to 2020 at the National Cancer Institute. All patients had 2 preoperative MRIs at least 3 months apart. Two fellowship-trained radiologists scored each tumor on location and MR-sequence-specific imaging parameters from the earlier MRI. Weighted kappa was used to determine the degree of agreement between radiologists for each parameter. GR was calculated as the difference in maximum tumor dimension over time (cm/year). Differences in mean growth rate (MGR) within categories of each imaging variable were assessed by ANOVA. RESULTS: Apart from tumor margin and renal sinus, reliability was at least moderate (K > 0.40) for imaging parameters. Median initial tumor size was 2.1 cm, with average follow-up of 1.2 years. Tumor MGR was 0.42 cm/year. T2 hypointense, mixed/predominantly solid, and high restricted diffusion tumors grew faster. When comparing different combinations of these variables, the model with the lowest mean error among both radiologists utilized only solid/cystic and restricted diffusion features. CONCLUSIONS: We demonstrate a novel MR-based scoring system (KISSS) that has good precision with minimal training and can be applied to other qualitative radiology studies. A subset of imaging variables (T2 intensity; restricted diffusion; and solid/cystic) were independently associated with growth rate in VHL renal tumors, with the combination of the latter two most optimal. Additional validation, including in sporadic RCC population, is warranted.

Characterizing and predicting ccRCC-causing missense mutations in Von Hippel-Lindau disease.

BACKGROUND: Mutations within the Von Hippel-Lindau (VHL) tumor suppressor gene are known to cause VHL disease, which is characterized by the formation of cysts and tumors in multiple organs of the body, particularly clear cell renal cell carcinoma (ccRCC). A major challenge in clinical practice is determining tumor risk from a given mutation in the VHL gene. Previous efforts have been hindered by limited available clinical data and technological constraints. METHODS: To overcome this, we initially manually curated the largest set of clinically validated VHL mutations to date, enabling a robust assessment of existing predictive tools on an independent test set. Additionally, we comprehensively characterized the effects of mutations within VHL using in silico biophysical tools describing changes in protein stability, dynamics and affinity to binding partners to provide insights into the structure-phenotype relationship. These descriptive properties were used as molecular features for the construction of a machine learning model, designed to predict the risk of ccRCC development as a result of a VHL missense mutation. RESULTS: Analysis of our model showed an accuracy of 0.81 in the identification of ccRCC-causing missense mutations, and a Matthew's Correlation Coefficient of 0.44 on a non-redundant blind test, a significant improvement in comparison to the previous available approaches. CONCLUSION: This work highlights the power of using protein 3D structure to fully explore the range of molecular and functional consequences of genomic variants. We believe this optimized model will better enable its clinical implementation and assist guiding patient risk stratification and management.

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

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

Development of visible-light-activatable photocaged PROTACs.

Photocaged proteolysis-targeting chimeras (PROTACs), which employ light as a stimulus to control protein degradation, have recently garnered considerable attention as both powerful chemical tools and a promising therapeutic strategy. However, the poor penetration depth of traditionally used ultraviolet light and the deficiency of alternative caging positions have restricted their applications in biological systems. By installing a diverse array of photocaged groups, with excitation wavelengths ranging from 365 nm to 405 nm, onto different positions of cereblon (CRBN) and Von Hippel-Lindau (VHL)-recruiting Brd4 degraders, we conducted the first comprehensive study on visible-light-activatable photocaged PROTACs to the best of our knowledge. We found the A2, A4 and B3 positions to be most effective at regulating the activity of the degraders, and to provide the resulting molecules (9-12 and 17) as potent visible-light-controlled degraders in live cells.

[Activation of HIF-1α/ACLY signaling axis promotes progression of clear cell renal cell carcinoma with VHL inactivation mutation].

Objective: To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1α/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. Methods: Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1α in ccRCC cell lines. HeLa cells were treated with CoCl2 and hypoxia (1%O2) to activate HIF-1α and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. Results: Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1α expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1α in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1α in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1α in Caki-2 cells inhibited the mRNA and protein levels of ACLY (P<0.001 for all). CoCl2 and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α (P<0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1α could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (P<0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1α and ACLY. The depletion of HIF-1α could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1α knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (P<0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (P<0.001). Conclusions: VHL mutation-mediated HIF-1α overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.

Does the VHL polymorphisms rs779805 and rs1642742 affect renal cell carcinoma susceptibility, prognosis and survival in Central European population?

Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system. The von Hippel-Lindau (VHL) tumor suppressor gene play an essential role in the tumorigenic pathway of clear cell RCC (ccRCC). This study was aimed to clarify the influence of VHL polymorphisms on ccRCC susceptibility and survival in Central European population. We genotyped 2 single-nucleotide polymorphisms (SNPs) rs779805 and rs1642742 in VHL gene and assessed their associations with ccRCC risk, clinicopathologic parameters, and prognosis in 171 cases. The selected SNPs were genotyped by ROCHE LifeCycler 96 using tumor tissue-derived DNA. Both SNPs do not directly influence ccRCC susceptibility and overall survival. A significant associations were found between allele G and genotypes AG and GG of rs779805 in the VHL tumor suppressor gene and increased tumor size, as well as high nuclear grade. Furthermore, a statistically significant association was observed between rs1642742 of VHL gene and low pathological tumor stage and between rs779805 of VHL gene and high pathological tumor stage. Both investigated SNPs can be important prognostic indicators of RCC in the Central European population, because statistically significant associations were observed between evaluated VHL polymorphisms and the best known factors with proven prognostic significance in kidney cancer.

Exosomes from Von Hippel-Lindau-Null Cancer Cells Promote Metastasis in Renal Cell Carcinoma.

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.