Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism

During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the “von Hippel Lindau” (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL⁻/VHL⁺ RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.     

The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.     

Recent advances in ideas on the molecular pathology and clinical aspects of Von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that is associated with various tumors and cysts in the central nervous system (CNS) and visceral organs. Inactivation of the VHL tumor-suppressor protein and subsequent loss of function in the VHL, and Elongin BC (VBC) complex result in dysfunction in the ubiquitination of hypoxia-inducible factor (HIF), which is an important step in the development of angiogenic tumors. The most frequent disorders in VHL disease are hemangioblastoma in the CNS and retina, pheochromocytoma in the adrenal gland, renal cell carcinoma, and pancreatic neuroendocrine tumor. Here, we review recent ideas on the pathogenesis and clinical diagnosis and treatment of VHL disease. Progress in molecular diagnosis and molecular targeting therapy is expected for improvement in the diagnosis and treatment of this disease. The familys support for patients with VHL disease is important, being mutually helpful to overcome various social and psychological problems in the patients.     

Von Hippel-Lindau抑癌基因在散发性肾透明细胞癌中的突变及其意义

背景与目的：von Hippel-Lindau（VHL）基因是一种抑癌基因,其突变在肾透明细胞癌的发生发展中发挥重要的作用.本研究旨在了解VHL抑癌基因在中国人散发性.肾透明细胞癌中的突变特点,并探讨基因突变与肿瘤分期、病理分级等临床病理特征的关系.方法：应用聚合酶链反应（PCR）、双向测序方法检测72例散发性肾透明细胞癌患者肾癌组织及正常肾组织中VHL抑癌基因的突变情况.结果：72例肿瘤组织中检测出VHL抑癌基因突变25例,突变率为35%.其中移码突变13例,错义突变8例,无义突变3例,同义突变l例.第1外显子6例,第2外显子5例,第3外显子12例,内含子区2例.25例突变中,6例突变位于密码子157～166区域.VHL抑癌基因突变与肿瘤分期、分级等临床病理指标无关（P＞0.05）.结论：中国人散发性肾透明细胞癌中VHL抑癌基因突变率较低,突变主要位于第3外显子,该外显子区可能存在一个突变热点区域.VHL抑癌基因突变与肿瘤分期、分级等临床病理指标无关,是肾透明细胞癌发生发展的早期遗传事件.     

53例国人肾透明细胞癌中PBRM 1基因的突变分析

目的 分析Polybromo 1（PBRM 1）基因在国人肾透明细胞癌中的突变特点,探讨基因突变与临床病理特征和预后的关系。方法 选取53例肾透明细胞癌患者,利用PCR结合直接测序的方法检测PBRM 1基因2～5号外显子、15～17号外显子的突变情况。分析PBRM 1基因突变与临床病理特征和预后的相关性。结果 53例样本中检测出PBRM 1基因突变14例,突变率为26％。突变较集中分布在第15、17外显子,第15外显子5例,第17外显子6例。PBRM 1基因突变组和无突变组的中位无进展生存期分别为5个月（95％CI：1.6～8.4个月）和14个月（95％CI：9.7～18.3个月）,中位生存期分别为7个月（95％CI：2.6～11.4个月）和18个月（95％CI：5.6～30.4个月）,差异均有统计学意义（P＜0.05）。 PBRM 1基因突变与性别、年龄、发病部位、分期及疾病控制率等无关（P＞0.05）。结论 中国人肾透明细胞癌中PBRM 1基因突变率较高,突变主要位于第15、17外显子。PBRM 1基因突变可能是肾透明细胞癌患者预后不良的危险因素。     

Tongue cancer patients have a high frequency of allelic loss at the von Hippel-Lindau gene and other loci on 3p

BACKGROUND: Although genetic abnormalities on 3p have been suggested to be linked to the development of squamous cell carcinoma of the head and neck, to the authors' knowledge no study to date has examined such genetic abnormalities in patients with squamous cell carcinoma of the tongue. In the current study, loss of heterozygosity (LOH) was evaluated at several loci within 3p, including the von Hippel-Lindau gene (VHL), in samples of tongue squamous cell carcinoma. In addition, the coding region of the intact VHL allele was screened for sequence mutations. METHODS: DNA was extracted from tumor and nontumor tissues collected from 28 patients with tongue squamous cell carcinoma. LOH was investigated by analysis of single nucleotide polymorphisms within exon 3 of VHL and by microsatellite analysis within another 10 loci. Mutation analysis of the VHL gene was performed by polymerase chain reaction (PCR) amplification and sequencing of the coding region of the gene. RESULTS: LOH within VHL was found at a high frequency (45.5%) within the tumor. However, mutations of the VHL gene were not detected in all tumor samples. LOH of other microsatellite markers on 3p was observed in 27.3% to 50% of tumor samples. Eleven (58%) of 19 samples that were informative at more than 2 loci exhibited LOH of at least 1 locus; 10 of these 11 cases exhibited LOH at multiple loci. CONCLUSIONS: A wide range of deletions in 3p, including at the VHL gene, may play a role in the development of tongue cancer.     

Increased mobilisation of circulating endothelial progenitors in von Hippel-Lindau disease and renal cell carcinoma

Background:

Circulating endothelial cells (CECs) are a candidate biomarker for monitoring angiogenesis in cancer. Circulating endothelial cell subsets are mobilised by angiogenic mediators. Because of the highly angiogenic phenotype of renal cell carcinoma (RCC), we sought to assess the potential of CECs as a marker of RCC in patients with von Hippel-Lindau (VHL) disease and those with sporadic RCC.

Methods:

We performed multicolour flow cytometry to enumerate CECs in patients with RCC, patients with VHL disease with and without RCC, and normal subjects. Two subsets of CECs were evaluated: mature CECs (mCECs) and circulating endothelial progenitors (CEPs).

Results:

In patients with VHL disease and RCC and those with sporadic RCC (N=10), CEPs and the CEP:mCEC ratio were higher than in normal subjects (N=17) (median CEPs: 0.97 vs 0.19 cells μl⁻¹, respectively, P<0.01; median CEP:mCEC: 0.92 vs 0.58, respectively, P=0.04). However, in patients with VHL without RCC, CECs were not increased. In paired pre- and post-nephrectomy RCC patient samples (N=20), CEPs decreased after surgery (median difference 0.02 cells μl⁻¹, −0.06 to 1.2; P=0.05).

Conclusion:

Circulating endothelial progenitors were elevated in RCC, but not in patients with VHL without RCC. Circulating endothelial progenitor enumeration merits further investigation as a monitoring strategy for patients with VHL.     

Detection of von Hippel-Lindau gene mutation in circulating cell-free DNA for clear cell renal cell carcinoma

The therapeutic landscape of metastatic clear cell renal cell carcinoma (ccRCC) has rapidly expanded, and there is an urgent need to develop noninvasive biomarkers that can select an optimal therapy or evaluate the response in real time. To evaluate the clinical utility of circulating tumor DNA (ctDNA) analysis in ccRCC, we established a highly sensitive assay to detect mutations in von Hippel-Lindau gene (VHL) using a combination of digital PCR and multiplex PCR–based targeted sequencing. The unique assay could detect VHL mutations with a variant allele frequency (VAF) <1.0%. Further, we profiled the mutation status of VHL in 76 cell-free DNA (cfDNA) and 50 tumor tissues from 56 patients with ccRCC using the assay. Thirteen VHL mutations were identified in cfDNA from 12 (21.4%) patients with a median VAF of 0.78% (range, 0.13%-4.20%). Of the 28 patients with VHL mutations in matched tumor tissues, eight (28.6%) also had VHL mutation in cfDNA with a median VAF of 0.47% (range, 0.13%-2.88%). In serial ctDNA analysis from one patient, we confirmed that the VAF of VHL mutation changed consistent with tumor size by radiographic imaging during systemic treatment. In conclusion, VHL mutation in cfDNA was detected only in a small number of patients even using the highly sensitive assay; nevertheless, we showed the potential of ctDNA analysis as a novel biomarker in ccRCC.     

Cigarette smoking, von Hippel-Lindau gene mutations and sporadic renal cell carcinoma

We investigated whether smoking is associated with mutations in the Von Hippel-Lindau (VHL) gene in 337 cases of sporadic renal cell carcinoma (RCC) among 120 852 people followed for 11.3 years; the findings suggest that smoking causes RCC independently of VHL gene mutations.     

Low CAIX expression and absence of VHL gene mutation are associated with tumor aggressiveness and poor survival of clear cell renal cell carcinoma

We attempted to describe, in a series of clear cell renal cell carcinoma (RCC), the relationship between CAIX expression, VHL gene mutations, tumor characteristics and outcome. Radical nephrectomy was performed in 100 patients. Genomic DNA was extracted from frozen tumor samples. Four amplimers covering the whole coding sequence of the VHL gene were synthesized by PCR and sequenced. The monoclonal antibody M75 was used to evaluate CAIX protein expression immunohistochemically. VHL mutations were identified in 58 patients (58%) and high CAIX expression (>85%) was observed in 78 (78%). Tumors with VHL mutation showed higher CAIX expression than those without (p = 0.02). Low CAIX expression and absence of VHL mutation were associated with a more advanced tumors e.g., higher T stages and presence of metastases. VHL mutation and high CAIX expression predicted longer progression-free survival (p = 0.037) and disease-specific survival (p = 0.001), respectively. In combination, they defined three prognostic groups (p = 0.002): (i) good prognosis, defined as VHL mutation and high CAIX (2-year survival: 86%), (ii) intermediate prognosis with either VHL mutation or high CAIX (69%), and (iii) poor prognosis with no VHL mutation and low CAIX (45%, median survival 18 months). CAIX expression, but not VHL mutational status, was an independent prognostic factor in multivariate analysis. Taken together, CAIX expression and VHL mutational status are able to stratify patients with clear cell RCC into distinct groups with regards to clinicopathological variables and prognosis, with low CAIX expression and absence of VHL mutation being associated with a poor clinicopathological phenotype and diminished survival.     

A retrospective case study of sunitinib treatment in three patients with Von Hippel-Lindau disease

Von Hippel-Landau (VHL) disease is characterized by malignant and benign tumors in multiple organs. Sunitinib, a tyrosine kinase inhibitor, has been clinically available for treating sporadic patients with recurrent or unresectable and metastatic clear renal cell carcinomas (cRCCs) and metastatic lesions of the lung, but its effect on VHL disease-associated tumors remains poorly understood. This retrospective case series examined the effect of sunitinib on RCC, hemangioblastomas, pheochromocytomas, and pancreatic neuroendocrine tumors in patients with confirmed VHL. Of note, three patients with VHL disease who were treated with sunitinib were identified from a review of their medical records. The efficacy of sunitinib was evaluated by comparing computed tomography (CT) or magnetic resonance imaging (MRI) scans conducted before and after treatment. Adverse side effects associated with sunitinib were assessed and recorded. All three patients with VHL disease exhibited clinical improvement after treatment with sunitinib. Patient 1 exhibited a decrease in the size of both their pheochromocytoma and RCC after 19 months of sunitinib treatment. RCCs in Patients 2 and 3 exhibited stable response to sunitinib for approximately 1 and 6 years, respectively. All the patients reported tolerable side effects. Therefore sunitinib treatment was associated with either partial response or stable control of VHL-related RCCs, pheochromocytomas and pancreatic neuroendocrine tumor (NET) with acceptable side effects. Further evaluation of sunitinib in patients with VHL disease in larger prospective studies is warranted.     

肾透明细胞癌VHL基因突变与Cyclin D1过度表达的相关性

背景与目的:VHL基因突变与肾透明细胞癌发生关系密切,而CyclinD1基因通过促进细胞过度增殖,对肾透明细胞癌发生和发展起重要作用。本研究拟初步探讨肾透明细胞癌中VHL基因突变的状况及其与CyclinD1表达上升的相关性。方法:取50例肾透明细胞癌手术标本,抽提病变组织和癌周正常组织中的基因组DNA和总RNA。通过PCR扩增VHL基因各外显子序列并测序以及内切酶鉴定的方法,确定肿瘤组织中VHL基因突变的比率和具体位点。通过RT-PCR和Westernblot方法,检测相应肿瘤组织中CyclinD1的表达水平是否发生改变。结果:VHL基因序列检测结果表明,有42例(84.0%)肾透明细胞癌标本的VHL基因发生各种形式的突变,有12(24.0%)例甚至发生2种以上的基因突变。在57例VHL基因外显子突变中,1号外显子突变有17例(29.8%);2号外显子突变有26例(45.6%);3号外显子突变有14例(24.6%)。CyclinD1的表达水平在42例VHL基因发生突变的组织中均有不同程度的上升,达到正常对照组的2～10倍(3.91±1.54倍)(P<0.01),另8例表现为正常。结论:在肾透明细胞癌中存在各种VHL基因突变,导致CyclinD1过度表达。     

Downregulation of Cap43 gene by von Hippel-Lindau tumor suppressor protein in human renal cancer cells

We previously identified 9 genes (i.e., thymosin beta4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Igamma) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel-Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL-positive cell lines, 786/VHL-1 and 786/VHL-2, after the introduction of wild-type VHL into VHL-negative renal cancer 786-O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL-positive renal cancer cell lines than in VHL-negative 786-O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from -286 base pairs (bp) to -62 bp was partly responsible for VHL-induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL-positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer.     

Chromosome 3p Deletion in a Renal Cell Carcinoma Cell Line Established from a Patient with von Hippel-Lindau Disease

von Hippel-Lindau disease (VHL) is an autosomal dominant inheriteddisease, frequently accompanied by occurrences of renal cellcarcinoma (RCC). Both the VHL gene and tumor suppressor genesfor RCC have been mapped to the short arm of chromosome 3, althoughthe genes have not yet been identified. An RCC cell line, KC12,was established from a VHL patient. Molecular genetic analysesin conjunction with cytogenetic studies revealed that the shortarm of chromosome 3 distal to the D3S4 locus at 3p11 was lostin the RCC cell line as a result of an unbalanced translocationbetween chromosomes 3p and 5q. Structural and numerical aberrations,including those on chromosome 3p, were not detected in T-lymphocytesfrom the patient, suggesting that the inherited mutation ofthe VHL gene at 3p25-26 in this patient was too subtle to bedetected by either Southern blot or karyotype analysis. Sinceno permanent RCC cell line has been established from a VHL patient,this cell line will be a useful source for analyzing the VHLgene at 3p25-26 and tumor suppressor gene(s) at 3p13-21.     

Involvement of PBRM1 in VHL disease-associated clear cell renal cell carcinoma and its putative relationship with the HIF pathway

Von Hippel-Lindau (VHL) disease is the main cause of inherited clear-cell renal cell carcinoma (ccRCC) and is caused by germline mutations in the VHL tumor suppressor gene. Bi-allelic VHL alterations lead to inactivation of pVHL, which plays a major role by downstream activation of the hypoxia inducible factor (HIF) pathway. Somatic VHL mutations occur in 80% of sporadic ccRCC cases and the second most frequently mutated gene is polybromo 1 (PBRM1). As there is currently no data regarding PBRM1 involvement in VHL disease-associated ccRCC, the aim of the present study was to assess the PBRM1 mutational status, and PBRM1 and HIF expression in VHL disease-associated ccRCC series compared with a sporadic series. PBRM1 gene was screened by Sanger sequencing for 23 VHL-disease-associated ccRCC and 22 sporadic ccRCC cases. Immunohistochemical studies were performed to detect the expression of PBRM1, HIF1 and HIF2 for all cases. In VHL-associated tumors, 13.0% (n=3/23) had PBRM1 somatic mutations and 17.4% (n=4/23) had a loss of PBRM1 nuclear expression. In sporadic cases, 27.3% (n=6/22) showed PBRM1 somatic mutations and 45.5% (n=10/22) had a loss of PBRM1 nuclear expression. Loss of PBRM1 was associated with an advanced tumor stage. HIF1-positive tumors were observed more frequently in the VHL-associated ccRCC than in the sporadic series. Furthermore, in the VHL cohort, PBRM1 expression appeared to be associated more with HIF1 than with HIF2. Given that hereditary tumors tend to be less aggressive, these results would suggest that co-expression of PBRM1 and HIF1 may have a less oncogenic role in VHL-associated ccRCC.     

Molecular genetic analysis of the von Hippel-Lindau disease (VHl) tumour suppressor gene in gonadal tumours

Chromosome 3p allele loss is frequent in ovarian and testicular tumours. The von Hippel-Lindau (VHL) disease tumour suppressor gene maps to chromosome 3p25. Gonadal tumours may occur in patients with VHL disease, so somatic VHL gene mutations might be involved in the pathogenesis of sporadic gonadal tumours. To investigate this hypothesis, we screened 60 gonadal tumours (36 ovarian and 24 testicular) for VHL gene mutations and chromosome 3p allele loss. Although 38% (10/26) of informative ovarian and 54% (7/13) of testicular tumours demonstrated 3p allele loss, no somatic VHL gene mutations were detected in the 60 gonadal tumours analysed. This suggested that chromosome 3p tumour suppressor gene(s) other than VHL are involved in gonadal tumorigenesis.