A Negative Feedback of the HIF-1a Pathway via Interferon- Stimulated Gene 15 and ISGylation

Purpose: The IFN-stimulated gene 15 (ISG15)and ubiquitin-conjugation pathways play roles in mediating hypoxic and inflammatory responses. To identify interaction(s) between these two tumor microenvironments, we investigated the effect of ISG15 on the activity of the master hypoxic transcription factor HIF-1a. Experimental Design: IFN and desferoxamine treatments were used to induce the expression of ISGs and HIF-1a, respectively. Interactions between HIF-1a and the ISG15 and ISGylation system were studied using knockdown of mRNA expression, immunoblotting, coimmunoprecipitation, and pull-down analyses. Effects of the ISG15 and ISGylation system on the HIF-1a–directed processes were examined using reporter, reverse transcription polymerase chain reaction (RT-PCR), and tumorigenic growth assays. Results: We found that the level of the free form of HIF-1a is differentially regulated by IFN treatment, and that the free ISG15 level is lower under hypoxia. Mechanism-directed studies have shown that HIF-1a not only interacts physically with ISG15, but is also ISGylated in multiple domains. ISG15 expression disrupts the functional dimerization of HIF-1a and -1b. Subsequently, expression of the ISG15 and/or ISGylation system attenuates HIF-1a–mediated gene expression and tumorigenic growth. Conclusion: In summary, our results revealed cross-talk between inflammatory and hypoxic pathways through the ISGylation of HIF-1a. On the basis of these results, we propose a novel negative feedback loop for the HIF-1a–mediated pathway involving the regulation of HIF-1a via IFN-induced ISGylation. Clin Cancer Res; 19(21); 5927–39. 2013 AACR. Introduction Posttranslational modifications (PTM) of proteins contribute significantly to the functional diversity of the proteome (1). For example, the ubiqutin–proteasome pathway (UPP) plays a critical role in hypoxic response and tumorigenesis by regulating protein stability of the master transcription factor HIF-1a (2–5). Both the UPP and IFN-stimulated gene 15 (ISG15) conjugation (ISGylation) pathways, which are regulated in response to IFN, contribute to inflammatory responses and cancer cell-killing activity of drugs (6–9). Notably, hypoxia and inflammation are two microenvironments involved in cancer development (5, 10–12). The initiation andmaintenance of tumor cells rely largely on their ability to interact with and adapt to changes in microenvironments (5, 11). Cells within rapidly growing tissues, whose proliferation rate becomes faster than their angiogenesis rate, such as in embryos and solid tumors, often encounter hypoxic stress. Moreover, hypoxia is a feature of the microenvironment in chronic inflammatory conditions such as arthritis and inflammatory bowel disease, and almost all types of solid tumors encounter hypoxic inflammation (10, 13). These two interdependent microenvironments, thus, impact not only the progression of cancer but also its development (12). However, the potential interaction between cancer-related inflammation and hypoxic environments during tumorigenesis and the underlying cross-talk mechanism remain largely unclear. Cells respond to hypoxia and inflammation through transcriptional programming, allowing them to adjust to the optimal cellular context (4, 5). Hypoxia induces expression of a defined set of genes through hypoxia-inducible factors (HIFs; especially HIF-1a), thereby being associated with erythropoiesis, glycolysis, the epithelial–mesenchymal transition (EMT), angiogenesis, tumor metastasis, therapy resistance, and a poor prognosis (14). Several regulatory mechanisms affecting the activity of HIF-1a have been identified: (i) HIF-1a stability is regulated by changes in oxygen tension. HIF-1a is degraded under normoxia by UPP, which involves prolyl hydroxylase (PHD1/2/3)-mediated hydroxylation and VHL-mediated ubiquitinylation (3, 15); (ii) Factor inhibiting HIF-1 (FIH) hydroxylates Authors' Affiliations: Department and Graduate Institute of Microbiology, College of Medicine; Center for Biotechnology, National Taiwan University, Taipei; and Department of InternalMedicine, KaohsiungArmed Forces General Hospital, Kaohsiung, Taiwan Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). CorrespondingAuthor:Tsai-Kun Li, Department andGraduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 10051. Phone: 886-2231-23456, ext. 88287/88294; Fax: 886-223915293; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-0018 2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 5927 on October 16, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst September 20, 2013; DOI: 10.1158/1078-0432.CCR-13-0018