In vitro and In vivo Models Analyzing von Hippel-Lindau Disease-Specific Mutations

Abstract

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl Ϫ/Ϫ murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1␣ and HIF2␣. All mutant pVHL proteins restored proper hypoxic regulation of HIF1␣, although one VHL mutation (VHL R167Q ) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2␣ regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHL Y112H mutant protein displayed a growth disadvantage, despite restoring HIF␣ regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl Ϫ/Ϫ as well as VHL Y112H , VHL R167Q , and VHL R200W embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.

The VHL tumor suppressor and HIF: insights from genetic studies in mice

“…These findings were surprising, but are in concordance with observations made by Carmeliet et al 129 who showed that HIF-1a-deficient teratocarcinomas had a growth advantage compared to wild type. However, growth suppression in VHLÀ/À teratocarcinomas may not be entirely dependent on HIF-1, as introduction of a mutated pVHL species (Y112H) into the VHLÀ/À background remained growth suppressive despite restoration of HIF-a regulation, 130 suggesting HIF-independent functions of pVHL in this model.…”

Biology of hypoxia-inducible factor-2α in development and disease

“…52 Introduction of pVHL mutations (Y98N, Y112N, W117R, and R167Q) that are associated with a high risk of renal carcinoma (type 2B) into VHL-deficient cell lines leads to increased normoxic HIF-1a and HIF-2a stabilization compared to wildtype VHL. 57,58 Cells harboring these mutations also display increased proliferation in serum-depleted media and growth in orthotopic xenograft assays, which can be reversed by shRNA-mediated knock down of HIF-2a. 57 The R167Q mutation, in particular, uniquely disrupts regulation of HIF-2a in ES cells.…”

“…57 The R167Q mutation, in particular, uniquely disrupts regulation of HIF-2a in ES cells. 58 In contrast, type 2A mutations (Y98H, Y112H, A149T, and T157I), which have a lower risk for renal tumors, display reduced levels of normoxic HIF-a accumulation and growth in xenograft assays compared to type 2B mutants. 57 In contrast to tumor-associated VHL mutations, the R200W mutation in pVHL leads to Chuvash polycythemia, a heritable disease characterized by elevated levels of serum Epo and VEGF.…”

Epstein-Barr Virus Latent Membrane Protein 1 is not Associated with Vessel Density nor with Hypoxia Inducible Factor 1 Alpha Expression in Nasopharyngeal Carcinoma Tissue

“…Control tissues included EBER-positive Hodgkin lymphoma for LMP1, and renal cell carcinoma tissue for HIF-1a and CD31. Additional controls were renal cell carcinoma cell line pellets (RCC4 2-1 over expresses HIF-1a while RCC4 3-14 does not) that were fixed in formalin and paraffin embedded [17]. Specificity was assured in all specimens by parallel stains omitting the primary antibody.…”