Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis.

The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.

Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia.

BACKGROUND: Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma. DESIGN AND METHODS: Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively. RESULTS: This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category. CONCLUSIONS: As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.

PHD2 mutation and congenital erythrocytosis with paraganglioma.

Prolyl hydroxylase domain (PHD) proteins play a major role in regulating the hypoxia-inducible factor (HIF) that induces expression of genes involved in angiogenesis, erythropoiesis, and cell metabolism, proliferation, and survival. Germ-line mutations in the prolyl hydroxylase domain 2 gene (PHD2) have been reported in patients with familial erythrocytosis but not in association with tumors. We describe a patient with erythrocytosis and recurrent paraganglioma who carries a newly discovered PHD2 mutation. This mutation affects PHD2 function and stabilizes HIF-alpha proteins. In addition, we demonstrate loss of heterozygosity of PHD2 in the tumor, suggesting that PHD2 could be a tumor-suppressor gene.

[Genetics and angiogenesis: the example of von Hippel-Lindau disease]. / Génétique et angiogenèse: l'exemple de la maladie de von Hippel-Lindau.

Von Hippel-Lindau (VHL) disease is the main cause of inherited kidney cancer and the model of tumoral angiogenesis. This rare syndrome is caused by germline mutations of the VHL tumor-suppressor gene that predispose to the development of a panel of highly vascularized tumors. Main manifestations include CNS and retinal haemangioblastomas, endolymphatic sac tumors, clear-cell renal cell carcinomas (RCC), phaeochromocytomas and pancreatic neuroendocrine tumors. The VHL gene plays a major role in regulation of the oxygen-sensing pathway by targeting the hypoxia-inducible factor HIF for degradation in proteasome. Somatic inactivation of the VHL gene occurs also in most sporadic RCC. Recent progress are pawing the way for the development of antiangiogenic targeted therapies that have already shown promising results in metastatic sporadic RCC.

Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo.

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is limited by the associated risk of insertional mutagenesis. We have overcome this problem by developing a nonintegrative lentiviral vector derived from HIV type 1 with a class 1 integrase (IN) mutation (replacement of the 262RRK motif by AAH). We generated and characterized HIV type 1 vectors carrying this deficient enzyme and expressing the GFP or neomycin phosphotransferase transgene (NEO) under control of the immediate early promoter of human CMV. These mutant vectors efficiently transduced dividing cell lines and nondividing neural primary cultures in vitro. After transduction, transient GFP fluorescence was observed in dividing cells, whereas long-term GFP fluorescence was observed in nondividing cells, consistent with the viral genome remaining episomal. Moreover, G418 selection of cells transduced with vectors expressing the NEO gene showed that residual integration activity was lower than that of the intact IN by a factor of 500-1,250. These nonintegrative vectors were also efficient in vivo, allowing GFP expression in mouse brain cells after the stereotactic injection of IN-deficient vector particles. Thus, we have developed a generation of lentiviral vectors with a nonintegrative phenotype of great potential value for secure viral gene transfer in clinical applications.