Inhibition of Hypoxia-Inducible Factor Is Sufficient for Growth Suppression of VHL–/– Tumors Michael Zimmer, Darrell Doucette, Naila Siddiqui, and Othon Iliopoulos Hematology-Oncology Unit, Department of Medicine, Massachusetts General Hospital and the Massachusetts General Hospital Cancer Center, Boston, MA Abstract The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate receptor for the mammalian SCF-2 E3 ubiquitin ligase complex that targets several substrates for ubiquitination and proteasomal degradation. Among these targets are the A-regulatory subunits of the hypoxia-inducible factor (HIF). VHL – / – cells constitutively overexpress hypoxia-inducible genes through both transcriptional and posttranscriptional mechanisms and form tumors when injected into nude mice. Reintroduction of pVHL into VHL – / – cell lines restores normal oxygen-dependent regulation of these genes and suppresses tumor formation in the mouse xenograft assay. We report here that short hairpin RNA-mediated inactivation of HIF phenocopies the effects of pVHL reintroduction with respect to decreased expression of hypoxia-inducible genes, decreased ability to promote vascular endothelial cell proliferation in vitro , and tumor growth suppression in vivo . In addition, HIF inactivation abrogated the cellular response to hypoxia, indicating that HIF is the only pVHL target required for this response. These data suggest that deregulation of hypoxia-inducible genes in VHL – / – cells can be attributed mainly to deregulation of HIF and validate HIF as a therapeutic anticancer drug target. Introduction The von Hippel-Lindau tumor suppressor protein (pVHL) is the substrate receptor of a mammalian E3 ubiquitin ligase complex consisting of Cullin-2, Elongins B/C, and the RING finger protein Rbx-1 (1, 2). Germ line mutations in the VHL gene predispose affected individuals to a variety of hyper- vascular tumors including renal cell carcinomas (RCC), hemangioblastomas, and pheochromocytomas (3). The VHL gene is also somatically inactivated in the majority of sporadic RCC and hemangioblastoma cases (4). Several substrates of pVHL have been reported thus far, including the a-regulatory subunits of hypoxia-inducible factor (HIF), the subunits of the RNA polymerase II complex, and the activated form of protein kinase C-y (5 – 7). The relative importance of each substrate for tumorigenesis as mediated by loss of VHL function is currently under investigation. A hallmark of the VHL–/– cells is the constitutive overexpression of hypoxia-inducible genes under normoxic conditions (8, 9). This up-regulation has been attributed to both transcriptional and posttranscriptional events (8 – 10). Reintro- duction of pVHL into VHL–/– cells reconstitutes normal regulation of these genes and suppresses the ability of VHL – / – cell lines to grow as tumors in the mouse xenograft assay without altering their in vitro growth characteristics (9, 11). Regulation of HIF activity by pVHL provides a link between VHL being a tumor suppressor gene and the function of pVHL as the substrate recognition component of an ubiquitin ligase complex. Under normoxic conditions, pVHL binds to the a-regulatory subunits of HIF leading to their polyubiquitination and proteasomal degradation (5, 12). This interaction depends on hydroxylation of HIF on conserved proline residues by a family of HIF prolyl hydroxylases (HPHs; 13, 14). Hypoxia inactivates the HPHs and thereby disrupts the pVHL-HIF interaction. Once stabilized, HIF can be detected in the nucleus within seconds. Tumor-associated VHL mutations inhibit the ability of the SCF-2 VHL complex to ubiquitinate HIF, resulting in HIF stabilization and constitutive overexpression of hypoxia- inducible genes (5, 12). Many of the growth and angiogenic factors not regulated in the absence of pVHL are HIF target genes (e.g. , VEGF, PDGF, GLUT-1 , and MMP ) and up- regulation of these genes is generally considered part of the malignant phenotype (15 – 18). However, these genes are regulated by factors other than HIF because basal expression is observed even in pVHL-expressing cell lines. Moreover, pVHL targets multiple proteins for proteasomal degradation and hypoxia-inducible genes are also subject to posttranscrip- tional regulation. It is therefore important to evaluate the role of HIF with regard to the ability of pVHL to mediate regulation of hypoxia-inducible genes and tumor suppression. It is currently not clear whether HIF activity is itself critical for tumor growth. Overexpression of HIF1a in the epidermis of transgenic mice leads to the formation of mature vessels only, without evidence of tumorigenesis (19). Carmeliet et al. (20) reported that HIF1a –/– embryonic stem cells generate aggressive vascularized tumors, albeit in a latent fashion, when injected in syngeneic animals. Moreover, normoxic stabilization of HIF1a was not sufficient to reproduce tumorigenesis in pVHL-reconstituted RCC cells (21). Despite these observa- tions, there is mounting evidence that HIF is critical for tumor growth. HIF1a is overexpressed in a variety of human tumors Received 11/5/03; revised 12/30/03; accepted 1/5/04. Grant support: NIH grant R29CA78358-06 (O. I.), Bertucci Fund for Urologic Malignancies (O. I.), David P. Foss Fund (O. I.), and VHL Family Alliance 2003 award (M. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Othon Iliopoulos, Massachusetts General Hospital, 55 Fruit Street, GRJ904, Boston, MA 02114. Phone: (617) 724-3404; Fax: (617) 726-8623. E-mail: oiliopoulos@partners.org Copyright D 2004 American Association for Cancer Research. Vol. 2, 89 – 95, February 2004 Molecular Cancer Research 89 Research. on November 24, 2021. © 2004 American Association for Cancer mcr.aacrjournals.org Downloaded from