A novel mode of action of YC-1 in HIF inhibition: stimulation of FIH-dependent p300 dissociation from HIF-1A

Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing f60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which downregulates HIF-1A and HIF-2A at the post-translational level, is being developed as a novel anticancer drug. We here found that YC-1 acts in a novel manner to inhibit HIF-1. In the Gal4 reporter system, which is not degraded by YC-1, YC-1 was found to significantly inactivate the COOH-terminal transactivation domain (CAD) of HIF-1A, whereas it failed to inactivate CAD(N803A) mutant. In coimmunoprecipitation assays, YC-1 stimulated factor inhibiting HIF (FIH) binding to CAD even in hypoxia, whereas it failed to increase the cellular levels of hydroxylated Asn of CAD. It was also found that YC-1 prevented p300 recruitment by CAD in mammalian two-hybrid and coimmunoprecipitation assays. The involvement of FIH in YC-1-induced CAD inactivation was confirmed in EPO-enhancer and Gal4 reporter systems using FIH small interfering RNA and dimethyloxalylglycine FIH inhibitor. Indeed, FIH inhibition rescued HIF target gene expressions repressed by YC-1. In cancer cell lines other than Hep3B, YC-1 inhibits HIF-1A via the FIHdependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF-A contributes to the YC-1induced deregulation of hypoxia-induced genes. [Mol Cancer Ther 2008;7(12):3729–38] Introduction Hypoxia is a common feature occurring in growing solid tumors and plays a pivotal role in tumor promotion, as hypoxia-driven genome changes promote tumor progression to more clinically aggressive phenotypes. This increased aggression is the result of the ‘‘turning-on’’ of the many genes required to overcome oxygen and nutrient deficiency and cell death. Hypoxic genome changes are mainly induced by hypoxia-inducible factors (HIF), which function to control z60 hypoxia-induced genes (1). HIFs are heterodimeric transcription factors composed of HIF-a and HIF-h (also known as ARNT) subunits; HIF-1 is composed of HIF-1a/ARNT, and HIF-2 is composed of HIF-2a/ARNT. HIF-as are prime transcription factors that transactivate genes, and ARNT assists HIF-a/DNA binding (2–4). Under normoxic conditions, HIF-as are prolylhydroxylated by PHD enzymes, then ubiquitinated by von Hippel-Lindau protein, and consequently degraded by 26S proteasomes (5). HIF-as are also regulated functionally by factor inhibiting HIF (FIH), a HIF asparagine hydroxylase. The transactivation domains of HIF-as are hydroxylated by FIH and then cannot recruit p300/CBP coactivator, which leads to the repression of HIF-mediated transcriptional activity (6). In contrast, under hypoxic conditions, both proline and asparagine hydroxylation are inhibited; thus, HIF-as are protected from von Hippel-Lindau protein binding and ubiquitination and activated by recruiting p300/CBP. YC-1 [3-(5¶-hydroxymethyl-2¶-furyl)-1-benzyl indazole] is a synthetic compound with a variety of pharmacologic actions (7). Because we showed the HIF-a-inhibitory activity of YC-1, YC-1 has been widely used as a pharmacologic tool for investigating the physiologic and pathologic roles of HIF (8). We also showed that YC-1 inhibits the growths of several human tumors xenografted in nude mice due to anti-HIF action (9). Subsequently, many researches have confirmed the antiHIF and anticancer activities of YC-1 in vitro and in vivo (10). Moreover, the potential use of YC-1 has expanded to radiosensitization (11), tumor metastasis prevention (12), and choroidal neovascularization inhibition (13). Mechanistically, it has been found that YC-1 accelerates HIF-1a degradation by targeting its amino acids 720 to 780 region (14) or by inhibiting Mdm2 (15) and that it inhibits the de novo synthesis of HIF-1a by inactivating the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway (16). However, the precise mechanism that underlies HIF-a down-regulation by YC-1 remains uncertain. In addition to HIF-a down-regulation, its functional inhibition might also offer a good cancer therapy strategy. For example, Kung et al. have found a functional inhibitor of HIF-a (chetomin) through a high-throughput screen. Received 1/23/08; revised 8/15/08; accepted 8/31/08. Grant support: National R&D Program for Cancer Control, Korean Ministry of Health & Welfare Research Fund grant 0520260-2. 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: Jong-Wan Park, Department of Pharmacology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Phone: 82-2-740-8289; Fax: 82-2-745-7996. E-mail: [email protected] Copyright C 2008 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0074 3729 Mol Cancer Ther 2008;7(12). December 2008 on July 6, 2017. © 2008 American Association for Cancer Research. mct.aacrjournals.org Downloaded from This compound was identified to repress the transcriptional activity of HIF-a by disrupting p300 binding to HIF-a. Moreover, its anticancer activity was shown in xenografted human tumors (17). Bortezomib proteasome inhibitor is also known to have anticancer effect by inhibiting HIF-a. Bortezomib has been investigated clinically for the treatment of multiple myeloma and several solid tumors (18). In addition to its direct proapoptotic activity, bortezomib indirectly inhibits tumor angiogenesis by inactivating the COOH-terminal transactivation domain (CAD) of HIF-1a and subsequently repressing vascular endothelial growth factor (VEGF) expression (19, 20). Another proteasome inhibitor, MG132, also inactivates HIF-1a by stimulating the CITED2-mediated interference of CAD-p300 binding (21). Moreover, amphotericin B (an antifungal agent) inactivates HIF-1a by stimulating the CAD-FIH interaction, which may cause erythropoietin-deficient anemia associated with amphotericin B therapy (22). Given these reports, it appears that CAD inactivation could contribute to the pharmacologic actions of anti-HIF agents. In terms of the anti-HIF mechanism of YC-1, no mechanisms, other than HIF-a down-regulation, have been investigated thus far, because HIF-a down-regulation is sufficient for HIF inhibition. However, we questioned this mechanism having conducted many experiments using YC-1, because the efficacy of functional HIF inhibition by YC-1 is always superior to that of HIF-a down-regulation by YC-1. In the present study, therefore, we addressed the possibility that the anti-HIF action of YC-1 is due in part to some mechanism other than HIF-a