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In Vitro and in Vivo Models Analyzing Von Hippel-Lindau Disease-Specific Mutations

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In Vitro and in Vivo Models Analyzing Von Hippel-Lindau Disease-Specific Mutations [CANCER RESEARCH 64, 8595–8603, December 1, 2004] In vitro and In vivo Models Analyzing von Hippel-Lindau Disease-Specific Mutations W. Kimryn Rathmell,1,3 Michele M. Hickey,1,2 Natalie A. Bezman,1 Christie A. Chmielecki,3 Natalie C. Carraway,3 and M. Celeste Simon1,2 1Abramson Family Cancer Research Institute and the Department of Cell and Molecular Biology, and 2Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and 3Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina ABSTRACT transport and droplet formation (8, 9). Unregulated expression of the full complement of hypoxia response genes is surmised to contribute Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene much of the clinical and pathological phenotype of renal cell carci- cause tissue-specific tumors, with a striking genotype-phenotype correla- noma, which is characterized as a highly vascular, glycolytic, lipid- tion. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to rich tumor that can be associated with polycythemia (10, 11). Detailed pheochromocytoma, polycythemia, or combinations of hemangioblas- studies of the effects of VHL loss have identified the regulation of the toma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein hypoxia response pathway via the proteasomal degradation of HIF1␣ (pVHL) has been implicated in many cellular activities including the and HIF2␣ as a major activity of the VHL protein (pVHL; refs. hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix 12–15). The pVHL acts as the substrate receptor for an E3 ubiquitin Ϫ Ϫ remodeling. We have expressed missense pVHL mutations in Vhl / ligase complex and targets HIF1␣ and HIF2␣ for degradation under murine embryonic stem cells to test genotype-phenotype correlations in normal oxygen (O2) tension (16). The O2-dependent regulation of euploid cells. We first examined the ability of mutant pVHL to direct HIF␣ (referring to either HIF1␣ or HIF2␣) is imparted by posttrans- degradation of the hypoxia inducible factor (HIF) subunits HIF1␣ and lational hydroxylation of specific prolines located in the HIF␣ O - HIF2␣. All mutant pVHL proteins restored proper hypoxic regulation of 2 ␣ HIF1␣, although one VHL mutation (VHLR167Q) displayed impaired bind- dependent degradation domain by HIF prolyl hydroxylases (17–19). ␣ ␣ ing to Elongin C. This mutation also failed to restore HIF2␣ regulation. In When O2 levels drop, HIF1 and HIF2 accumulate (5). In the separate assays, these embryonic stem cells were used to generate terato- absence of pVHL, HIF1␣ and HIF2␣ are maximally stabilized in mas in immunocompromised mice, allowing independent assessment of the presence of atmospheric O2 levels and show no additional O2- the effects of specific VHL mutations on tumor growth. Surprisingly, dependent accumulation (13). Y112H teratomas expressing the VHL mutant protein displayed a growth We previously reported that VhlϪ/Ϫ murine embryonic stem cells ␣ Ϫ Ϫ disadvantage, despite restoring HIF regulation. Finally, we observed predictably disrupt the hypoxia response pathway (20). In Vhl / increased microvessel density in teratomas derived from VhlϪ/Ϫ as well as embryonic stem cells, HIF1␣ and HIF2␣ are highly expressed under VHLY112H, VHLR167Q, and VHLR200W embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the normoxic conditions, and protein levels are not additionally stabilized cell, and that these activities can be separated via discrete VHL point in response to O2 deprivation. Furthermore, Vhl deletion gives rise to mutations. The ability to dissect specific VHL functions with missense highly glycolytic cells, as would be expected in cells with elevated Ϫ Ϫ mutations in a euploid model offers a novel opportunity to elucidate the levels of glycolytic enzymes. When Vhl / embryonic stem cells are activities of VHL as a tumor suppressor. grown in a teratoma model system the tumors display a highly vascular phenotype, although with a surprising reduction in tumor volume (20). INTRODUCTION The spectrum of tumors arising in individual VHL patients strongly Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene correlates with specific VHL mutations (21, 22), and the syndrome has give rise to a variety of tumors and other abnormalities in affected been divided into type 1 and type 2 disease based on predisposition for individuals including renal clear cell carcinoma, pheochromocytoma, pheochromocytoma (Fig. 1A). Type 1 disease is characterized by hemangioblastoma, and polycythemia (1). Some of these disease mutations, which delete, silence, or destabilize the VHL gene, and this manifestations derive from the unregulated expression of the hypoxia patient group has a low frequency of pheochromocytoma (23). Type inducible factor (HIF) transcription complex. HIF is a heterodimer 2 disease is characterized by a strong predisposition for pheochromo- composed of a highly regulated ␣ subunit (either HIF1␣ or HIF2␣/ cytoma, and it is additionally subdivided into 2A (pheochromocy- EPAS1) and a ubiquitously expressed ␤ subunit (HIF1␤/ARNT; refs. toma, hemangioblastoma, and low risk for renal cell carcinoma), 2B 2–4). This complex, in cooperation with the coactivators cAMP- (pheochromocytoma, hemangioblastoma, and high risk for renal cell responsive element binding protein and p300, activates transcription carcinoma), and 2C (pheochromocytoma only). Molecular analysis of via a hypoxia response element in the promoter or enhancer regions of these mutations shows that type 2A mutants and type 2C mutants many hypoxia-responsive genes. Genes transcriptionally activated by maintain interaction with the ubiquitin ligase complex, whereas 2B HIF include factors involved in angiogenesis, glucose transport, gly- mutants are deficient in this interaction (23). In addition, homozygos- colysis, and erythropoiesis as recently reviewed (5–7), and lipid ity for a VHL mutation in the extreme COOH-terminal domain has been associated with a highly penetrant congenital form of polycy- Received 4/22/04; revised 9/8/04; accepted 10/1/04. themia termed Chuvash Polycythemia (24, 25). No tumor predispo- Grant support: NIH Grant K08-CA098410 and a V Foundation Scholar Award sition has yet been identified with this mutation. (W. Rathmell), Howard Hughes Medical Institute Predoctoral Fellowships and the Ruth L. Kirschstein National Research Service Award (M. Hickey and N. Bezman), and NIH In addition to VHL disease, mutations in VHL have been identified Grant HL66130 and the Howard Hughes Medical Institute (M. Simon). in the majority of patients with sporadic clear cell renal cell carcinoma The costs of publication of this article were defrayed in part by the payment of page (26–28), which constitutes the majority of carcinomas of the kidney. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This form of renal cell carcinoma is characterized by a highly vascular Requests for reprints: M. Celeste Simon, University of Pennsylvania, Abramson cystic or solid mass comprised of nests of tumor cells containing Family Cancer Research Institute, BRB II/III, Room 450, Philadelphia, PA 19104. Phone: (215) 746-5526; Fax: (215) 746-5511; E-mail: [email protected]. abundant clear cytoplasm consisting of glycogen and neutral lipid. ©2004 American Association for Cancer Research. Although loss of VHL is not a transforming event in such cells, VHL 8595 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. ANALYSIS OF VHL MUTATIONS Fig. 1. Embryonic stem cell expression of representative VHL missense mutations. A. VHL disease subtypes, characteristic patterns of disease, and mutations used in this analysis to represent each subtype of VHL disease. B, schematic of VHL gene. The hemagglutinin tag is located at the NH2 terminus as indicated. Important residues are identified, as are the known interaction domains. C, human pVHL immunoblot. Protein extracts from embryonic stem cells expressing the indicated version of pVHL, pVHL null, and endogenous mouse pVHL were compared with in vitro translated human pVHL protein. Note the lack of signal in the endogenous pVHL extract from Vhlϩ/Ϫ extracts. D, murine pVHL immunoblot. Protein extracts from pVHL wild-type, heterozygous, and null embryonic stem cells. (HA, hemagglutinin) mutations have been identified in renal cysts and dysplastic lesions, ally, the mutation associated with VHL type 2B disease (VHLR167Q) strongly suggesting they represent an early event in renal tumorigen- uniquely conferred impaired interaction with Elongin C as well as a ␣ ␣ esis (29). Furthermore, renal cell carcinoma cells regain O2-regulated disruption of HIF2 hypoxia responsive regulation, whereas HIF1 gene expression (15, 30) and lose their transformed phenotype on protein regulation remained intact. Additionally, a mutant associated reintroduction of wild-type pVHL activities (31, 32). with VHL type 2A disease (VHLY112H) showed a suppressive growth Whereas VHL clearly regulates the hypoxia response pathway, the effect similar to that observed with Vhl loss in teratomas. Consistent cohort of mutations giving rise to type 2C disease has not been found with previous observations, we detected no impact on the hypoxia to affect
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