Mammalian Tumor Suppressor Int6 Specifically Targets Hypoxia Inducible Factor 2α for Degradation by Hypoxia- and pVHL-independent Regulation

Abstract

The hypoxia-inducible factors HIF-1␣ and HIF-2␣ are structurally similar as regards their DNA-binding and dimerization domains, but differ in their transactivation domains and, as is shown by experiments using hif-1␣؊/؊ and hif-2␣ ؊/؊ mice, in their functions. This implies that HIF-1␣ and HIF-2␣ may have unique target genes. To address this discrepancy and identify HIF-2␣-specific target genes, we performed yeast two-hybrid analysis and identified the tumor suppressor Int6/eIF3e/p48 as a novel target gene product involved in HIF-2␣ regulation. The int6 gene was first identified from a screen in which the mouse mammary tumor virus was employed as an insertional mutagen to identify genes whose functions are critical for breast tumor formation. Here, by using two-hybrid analysis, immunoprecipitation in mammalian cells, and HRE-reporter assays, we report the specific interaction of HIF-2␣ (but not HIF-1␣ or HIF-3␣) with Int6. The results indicate that the direct interaction of Int6 induces proteasome inhibitor-sensitive HIF-2␣ degradation. This degradation was clearly observed in renal cell carcinoma 786-O cells, and was found to be both hypoxia-and pVHL-independent. Furthermore, Int6 protein knockdown by int6-siRNA vectors or the dominant-negative mutant Int6-⌬C increased endogenous HIF-2␣ expression, even under normoxia, and induced sets of critical angiogenic factors comprising vascular endoplasmic growth factor, angiopoietin, and basic fibroblast growth factor mRNA. These results indicate that Int6 is a novel and critical determinant of HIF-2␣-dependent angiogenesis as well as cancer formation, and that int6-siRNA transfer may be an effective therapeutic strategy in pathological conditions such as heart and brain ischemia, hepatic cirrhosis, and obstructive vessel diseases.Hypoxia-responsive genes are involved in glucose transport, glycolysis, erythropoiesis, angiogenesis, vasodilation, and respiratory rate. However, they are also involved in the pathogenesis of many cardiovascular diseases and cancer (1, 2). Central to many molecular and physiological responses to hypoxia in most mammals cells are the hypoxia-inducible factors HIF-1␣ and HIF-2␣ (HIFs), 2 heterodimers with HIF-1␤ (also referred to as aryl hydrocarbon receptor nuclear translocator, ARNT) (3-5). Both HIF-1␣ and ARNT belong to the basic helix loop helix Per-Arnt-Sim (PAS) family of transcription factors, which share several conserved structural domains (6).Under normoxia, HIF-1␣ is ubiquitinated via interaction with the von Hippel-Lindau tumor suppressor protein (pVHL) and is subsequently degraded by the 26 S proteasome (7-12). pVHL, the recognition component of an E3 ubiquitin ligase complex, binds HIF-1␣ when it is hydroxylated at proline residues 402 and 577 (13-15). The proline hydroxylation of HIF-1␣ is catalyzed by prolyl hydroxylase domain-containing proteins, which are members of the 2-oxoglutarate-dependent dioxygenase superfamily whose activity requires O 2 as a cofactor (16,17). During hypoxia, the activity of prolyl hydroxylase domainco...

An oncogenic role of eIF3e/INT6 in human breast cancer

“…In Drosophila eIF3e was shown to be a positive regulator of cullin neddylation, thus regulating degradation through the ubiquitin-proteasome pathway of substrates of cullin-containing ubiquitin ligases (RencusLazar et al, 2008). Other studies reported specific interaction of eIF3e with HIF-2a (Chen et al, 2007) or with MCM7 (Buchsbaum et al, 2007), leading to proteasomal degradation or stabilization of the eIF3e partner protein, respectively. In our experiments, eIF3e knockdown induced a slight accumulation of polyubiquitinylated proteins (Figure 2c), including MCM7 (Supplementary Figure 1), leaving open the possibility that some of the biological consequences observed might reflect an involvement of eIF3e in ubiquitin-dependent proteolysis.…”

“…Individual overexpression of any of five subunits of human eIF3 (eIF3a, b, c, h or i) promoted malignant transformation of immortal fibroblasts (Ahlemann et al, 2006;Savinainen et al, 2006;Zhang et al, 2007). By contrast, studies of eIF3e/INT6 have suggested various roles for this accessory eIF3 subunit either as an oncoprotein or a tumor suppressor (Marchetti et al, 2001;Rasmussen et al, 2001;Buttitta et al, 2005;Chen et al, 2007).…”

Translation Regulation as a Therapeutic Target in Cancer

“…The murine Int-6 gene, encoding eIF3e, was first identified as a mouse mammary tumor virus integration site (34,35) that results in the production of a truncated oncoprotein with transforming activity (36) capable of inducing cap-independent translation, as reported recently (37). Several subsequent studies of full-length eIF3e showed its tumor suppressor activity (38)(39)(40). However, further reports indicated its oncogenic activity (41,42), and a recent screen identified eIF3e as a potential biomarker for the early detection of breast cancer (43).…”

Int6 /eIF3e Silencing Promotes Functional Blood Vessel Outgrowth and Enhances Wound Healing by Upregulating Hypoxia-Induced Factor 2α Expression

“…We previously reported that the tumor suppressor INT6/ eIF3e binds in a subtype-specific manner to HIF2␣ and is involved in HIF2␣ regulation. 26 The Int6 gene was first identified as a frequent integration site of the mouse mammary tumor virus in preneoplastic and neoplastic mammary lesions. 27 It was later characterized as the translation initiation factor subunit e (eIF3e) in rabbits, 28 Schizosaccharomyces pombe, 29 -31 and Arabidopsis thaliana 32,33 ; however, little is known about the functional role of eIF3e.…”