Contrasting Properties of Hypoxia-Inducible Factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-Associated Renal Cell Carcinoma

Abstract

Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor ␣ subunits (HIF-1␣ and HIF-2␣), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1␣ and HIF-2␣ in RCC and non-RCC cells. We demonstrate common patterns of HIF-␣ isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-␣ isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2␣ suppressing HIF-1␣ and vice-versa. In VHL-defective RCC cells, we demonstrate that the protumorigenic genes encoding cyclin D1, transforming growth factor alpha, and vascular endothelial growth factor respond specifically to HIF-2␣ and that the proapoptotic gene encoding BNip3 responds positively to HIF-1␣ and negatively to HIF-2␣, indicating that HIF-1␣ and HIF-2␣ have contrasting properties in the biology of RCC. In keeping with this, HIF-␣ isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1␣ retarding and HIF-2␣ enhancing tumor growth. These findings indicate that therapeutic approaches to targeting of the HIF system, at least in this setting, will need to take account of HIF isoform-specific functions.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

“…Over expression of both HIF-1a and HIF-2a is seen in many human cancers (Semenza, 2003). Although the HIF-1a and HIF-2a proteins are very similar and are both induced upon hypoxia, they elicit different transcriptional responses and have different roles during embryonic development (Ravi et al, 2000;Wiesener et al, 2003;Raval et al, 2005;Wang et al, 2005).…”

Target gene selectivity of hypoxia-inducible factor-α in renal cancer cells is conveyed by post-DNA-binding mechanisms

“…Taken together, these findings strongly suggest that HIF-2a has pro-tumorigenic actions in RCC that are isoform specific, and not shared by HIF-1a. In keeping with this, HIF-1a and HIF-2a show clear transcriptional selectivity, with studies to date defining at least two types of isoform-specific responses; certain genes appear exclusively responsive to HIF-1a in both RCC and non-RCC cells, whereas others respond to both HIF-1a and HIF-2a in non-RCC cells, and are dominantly regulated by HIF-2a in RCC cells (Hu et al, 2003;Sowter et al, 2003;Raval et al, 2005). Hence, RCC cells display marked HIF-a chain selectivity in target gene responses that likely underlie differences in the role of these molecules in promoting tumour growth.…”

“…These chimaeric HIF-a sequences were transferred into the retroviral vector pLZRS-IRES-GFP (Jacobs et al, 1999). Retroviral production and infection of target cells were conducted as previously described (Raval et al, 2005). A 1.1 kb fragment of the PHD3 first intron (14 328 421 to 14 327 303, antisense strand, NT_026437.10) and a 1 kb fragment of the PHD3 promoter upstream to the translation start site (33 490 609 to 33 489 711, antisense strand, NC_000014.7) were amplified by PCR from human genomic DNA using primers containing the appropriate restriction sites (Sigma Genosys, UK).…”

“…Whole cell extracts from six-well plates were prepared in ureasodium dodecyl sulphate (SDS) buffer, normalised for total protein content, resolved and transferred as previously described (Raval et al, 2005). Primary antibodies were mouse monoclonal antibodies against HIF-1a (clone 54, Transduction Laboratories, Oxford, UK), HIF-2a (NB-100 132, Novus Biologicals, Littleton, CO, USA), CA9 (M75) (Pastorekova et al, 1992), PHD3 (188e) (Appelhoff et al, 2004) and b-tubulin (clone 2-28-33, Sigma, Gillingham, UK).…”

“…They have a highly conserved domain architecture, including sites of prolyl and asparaginyl hydroxylation, and strongly promote transcription from similar hypoxia response elements (HREs). However, there is increasing evidence for the functional non-equivalence of HIF-1a and HIF-2a, and in cancer, several recent studies have indicated that, at least in certain settings, they have contrasting effects on tumour growth (Maranchie et al, 2002;Kondo et al, 2002Kondo et al, , 2003Zimmer et al, 2004;Covello et al, 2005;Raval et al, 2005). In VHL-defective renal carcinoma cells (RCC), inactivation of HIF-a proteolysis upregulates both HIF-1a and HIF-2a with global induction of HIF target gene expression (Maxwell et al, 1999), stimulating investigations of the role of the HIF pathway in tumour development (Maranchie et al, 2002;Kondo et al, 2002Kondo et al, , 2003Zimmer et al, 2004;Raval et al, 2005).…”