Expression and content of VHL in kidney cancer tissue, relationship with clinical and morphological parameters of the disease, expression of transcriptional and growth factors

Introduction. The molecular picture of the development of HIF overexpression against VHL deficiency background is a key event associated with the manifestation and progression of clear cell renal cell carcinoma. However, this indicator's significance for the development of the disease and the formation of a response to targeted therapy is not clear. The study aimed to study the relationship between the expression and content of VHL in kidney cancer tissue with clinical, morphological parameters, mRNA level NF-κB p65, NF-κB p50, HIF-1, HIF-2, VEGF, and CAIX. Material and methods. The study included 39 patients with renal cell carcinoma. The disease's localized form (T1-2N0M0) was diagnosed in 20 patients; 19 patients had a disseminated process (T1-3N0-1M1). The study's material was normal, and tumor tissue was obtained during the surgical stage of treatment. VHL expression was determined by real-time PCR. The pVHL protein content was estimated using the Western Blotting method. Results. As a result of the study, it was noted that an increase in the VHL expression, together with a decrease in its protein level, was associated with an increase in tumor size. The prevalence of the disease was accompanied by an increase in both mRNA and VHL content in cancers. There was a negative correlation between the level of VHL expression, the content of the corresponding protein, and a positive association with the expression of NF-κB p65. VHL content in cancers was associated with decreased expression of NF-κB p50, NF-κB p65, and VEGF. Discussion. An increase in VHL expression in tumor tissue promoted the growth of mRNA of transcription factors, in particular NF-κB, which is accompanied by tumor spread. An increase in the VHL protein content led to the suppression of the mRNA level of transcriptional and growth factors, which is probably associated with their inactivation, including through proteasome cleavage, and is an important stage in oncogenesis. Conclusion. The relationship between the expression and content of the VHL protein is of decisive importance in the development of the disease, the formation of distant metastases.

Regulation of Immunity in Clear Cell Renal Carcinoma: Role of PD-1, PD-L1, and PD-L2

Regulation of immunity is a unique oncogenic mechanism that differs in different cancers. VHL deficient clear cell renal cell carcinomas (ccRCC) trigger the immune response resulting in cancer progression. This study aimed to investigate PD-1, PD-L1, and PD-L2 expression in ccRCC primary cancers and metastatic tissues associated with the p-VHL content, transcriptional, and growth factors expression. Methods: A total of 62 patients with RCC were enrolled in the study. Investigation of mRNA level was performed by PCR in real-time. Western blotting analysis was used for detecting the p-VHL protein content in tissues. Results: The PD-L2 prevalence in metastatic cancers is crucial in tumor progression. The VHL expression and p-VHL content determined the aggressive cancer behavior and elevated in disseminated tumors. The cancer dissemination was accompanied by an increase in both mRNA and VHL content. Conclusion: We present a new instrument targeting pathologies with p-VHL/HIF altered function that impact the PD-L2 expression through the change in transcriptional, growth factors, and AKT/mTOR modulation.

The Endothelial Landscape and Its Role in Von Hippel–Lindau Disease

Von Hippel–Lindau disease (VHL) is a rare hereditary disease characterized by the predisposal to develop different types of highly vascularized tumors. VHL patients carry a VHL mutation that causes partial lack of functional VHL protein (pVHL) in all cells, and a total lack thereof in cells harboring a second hit mutation. Absence of pVHL generates a prolonged state of pseudo-hypoxia in the cell due to accumulation of hypoxia inducible factor, an important transcription factor regulating pro-tumorigenic genes. The work here presented focuses on characterizing the endothelium of VHL patients, by means of blood outgrowth endothelial cells (BOECs). Transcriptome analysis of VHL-derived BOECs, further supported by in vitro assays, shows that these cells are at a disadvantage, as evidenced by loss of cell adhesion capacity, angiogenesis defects, and immune response and oxidative metabolic gene downregulation, which induce oxidative stress. These results suggest that the endothelium of VHL patients is functionally compromised and more susceptible to tumor development. These findings contribute to shedding light on the vascular landscape of VHL patients preceding the second hit mutation in the VHL gene. This knowledge could be useful in searching for new therapies for these patients and other vascular diseases.

VHL, BAP1, PBRM1, SETD2 Expression in Clear Cell Renal Carcinoma, Association With PD-1, PD-L1, PD-L2 mRNA Level

Novel mechanism of ccRCC progression is essential, including PBRM1, BAP1, and SETD2 in histone-modifying and chromatin remodeling genes. The study aimed to investigate VHL, PD-1, PD-L1, PD-L2. BAP1, PBRM1, SETD2 expression in ccRCC primary cancers and metastatic tissues associated with the cancer dissemination. A total of 62 patients with RCC were enrolled in the study. Investigation of mRNA level of VHL, PD-1, PD-L1, PD-L2. PCR in real-time performed BAP1, PBRM1, SETD2 with the previous RNA isolation. Western Blotting analysis was used for detecting the p-VHL protein content in tissues. The VHL expression and p-VHL content determined the aggressive cancer behavior and elevated in disseminated tumors. The cancer dissemination was accompanied by an increase in both mRNA and VHL content. The PD-L2 prevalence in metastatic cancers is crucial in tumor progression. ccRCC progression in VHL overexpression is associated with the decrease in BAP1 gene expression. It is revealed the heterogeneity in molecular markers in primary tumors and metastases. The low mRNA level of BAP1, PBRM1, SETD2, PD-1, PD-L1, PD-L2 in metastases compared with primary tumors were found. We show a novel mechanism for VHL tumor progression and present a new instrument and factor targeting tumor-related pathologies with p-VHL/HIF altered function.

VHL inhibitor binding increases intracellular level of VHL

The vonHippel Lindau (VHL) protein is a tumour suppressor protein frequently mutated in the VHL disease, which functions as substrate recognition subunit of a Cul2 E3 ubiquitin ligase (CRL2VHL). CRL2VHL plays an important role in oxygen sensing, by binding and targeting Hypoxia Inducible Factor-alpha subunits (HIF-alpha) for ubiquitination and degradation. VHL is also commonly hijacked by heterobifunctional degrader molecules known as proteolysis-targeting chimeras (PROTACs). In previous work we reported the structure-based design and functional characterisation of VHL inhibitors (VH032 and VH298) that induce the HIF response in cells. Here, we use unbiased quantitative mass spectrometry to identify the proteomic changes elicited by the VHL inhibitor and compare this to hypoxia or broad-spectrum prolyl-hydroxylase domain (PHD) enzyme inhibitor IOX2. Our results demonstrate the VHL inhibitor selectively activates the HIF response that vastly overlaps with hypoxia- and IOX2-induced proteomic changes. Interestingly, VHL inhibitors were found to selectively upregulate a single protein, which is VHL itself. Our analysis revealed that this occurs via protein stabilisation of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn to reduced levels of HIF-1 protein. Our results demonstrate the high specificity of VHL inhibitors and suggest that use of these inhibitors would not produce overtly side effects due to prolonged HIF stabilisation. They also exemplify the concept that small-molecule binding induced protein stabilisation can increase protein levels inside cells.

The Genotype-Phenotype Association of Von Hipple Lindau Disease Based on Mutation Locations: A Retrospective Study of 577 Cases in a Chinese Population

PurposeVon Hippel-Lindau (VHL) disease is a hereditary kidney cancer syndrome, with which patients are more likely to get affected by renal cell carcinoma (RCC), pancreatic cyst or tumor (PCT), central nervous system hemangioblastoma (CHB), retinal angiomas (RA), and pheochromocytoma (PHEO). Mutations of VHL gene located in 3p25 may impair the function of the VHL protein and lead to the disease. It’s unclear why obvious phenotype varieties exist among VHL patients. Here we aimed to ascertain whether the mutation types and locations affect the phenotype.MethodsWe enrolled 577 Chinese VHL patients from 211 families and divided them into three groups and six subgroups according to their mutation types and locations. Cox survival analysis and Kaplan-Meier analysis were used to compare intergroup age-related tumor risks.ResultsPatients with nonsense or frameshift mutations that were located before residues 117 of VHL protein (NoF1 subgroup) hold lower age-related risks of VHL associated tumors (HR = 0.638, 95%CI 0.461–0.883, p = 0.007), CHB (HR = 0.596, 95%CI 0.409–0.868, p = 0.007) or PCT (HR = 0.595, 95%CI 0.368–0.961, p = 0.034) than patients whose mutations were located after residues 117 (NoF2 subgroup). Patients in NoF1 subgroup still had lower age-related risks of CHB (HR = 0.652, 95%CI 0.476–0.893, p = 0.008) and PCT (HR = 0.605, 95%CI 0.398–0.918, p = 0.018) compared with those in combined NoF2 subgroup and other truncating mutation patients. NoF1 subgroup correspondingly had a longer estimated median lifespan (64 vs. 55 year, p = 0.037) than NoF2 subgroup. Among patients with missense mutations of VHL, only a small minority (23 of 286 missense mutations carriers) carried mutations involving neither HIF-α binding region nor elongin C binding region, who were grouped in MO subgroup. MO subgroup seemed to have a higher age-related risk of PHEO. In the whole cohort (n = 577), PHEO was an independent protective factor for CHB (p = 0.001) and survival (p = 0.005). RA and CHB failed to predict the age-related risk of each other.ConclusionThe mutation types and locations of VHL gene are associated with phenotypes. Genetic counselors could predict phenotypes more accurately based on more detailed genotype-phenotype correlations. Further genotype-phenotype studies should focus on the prediction of tumor recurrence, progression, and metastasis. The deep molecular mechanism of genotype-phenotype correlation is worth further exploring.

Spectrum of VHL mutations in clear cell renal cell carcinoma

The VHL gene alterations are the early and characteristic feature of clear cell renal cell carcinoma (ccRCC). We have examined VHL mutations in sporadic 98 ccRCC cases to evaluate their localization in relation to functionally important motifs of the VHL protein. The DNA samples were obtained from snap-frozen carcinoma biopsies and used for Sanger sequencing, while 62 ccRCC DNA cases were studied by next generation sequencing (NGS) analysis in parallel. In 73 (74.4 %) оf 98 ccRCC cases the somatic non-silent VHL mutations were identified. Loss of function VHL mutations (nonsilent, frameshifts or in splicing sites) were detected in 40 (40.8 %) ccRCC, while missense mutations – in 35 (35.7 %) ccRCC. In total 76 mutations important for VHL functioning were detected in 72 (73 %) ccRCC samples, of them 15 mutations (deletion / insertion in-frame or frameshifts) were identified for the first time. Four ccRCC cases contained two mutations each. Most of missense mutations disturb the sites of VHL interactions with HIF, РКС or kinesin. The pathogenicity of p.P154P silent mutation and intronic mutations near mRNA VHL splicing sites was discussed. The obtained results are important for understanding the role of VHL mutations in ccRCC progression and prognosis.

The Genomics and Genetics of Oxygen Homeostasis

Human survival is dependent upon the continuous delivery of O2 to each cell in the body in sufficient amounts to meet metabolic requirements, primarily for ATP generation by oxidative phosphorylation. Hypoxia-inducible factors (HIFs) regulate the transcription of thousands of genes to balance O2 supply and demand. The HIFs are negatively regulated by O2-dependent hydrox-ylation and ubiquitination by prolyl hydroxylase domain (PHD) proteins and the von Hippel–Lindau (VHL) protein. Germline mutations in the genes encoding VHL, HIF-2α, and PHD2 cause hereditary erythrocytosis, which is characterized by polycythemia and pulmonary hypertension and is caused by increased HIF activity. Evolutionary adaptation to life at high altitude is associated with unique genetic variants in the genes encoding HIF-2α and PHD2 that blunt the erythropoietic and pulmonary vascular responses to hypoxia.