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Educational Session: Hypoxia: Impact on Cancer Biology

Regulation of HIF-1; enzymatic hydroxylation, the role of VHL and activation in renal cell carcinoma.

Imperial College of Science, Technology and Medicine, London, United Kingdom

Abstract

A fundamental challenge for complex multicellular organisms is to co-ordinate oxygen supply and demand. HIF-1 is centrally involved in meeting this challenge, altering the transcription of a broad array of genes when oxygen tension is reduced. Regulation of HIF is via the alpha subunit, of which two oxygen responsive isoforms have been extensively characterised — HIF-1 and HIF-2. These are regulated very similarly, but there is increasing evidence for differential effector roles in the overall response to hypoxia.

Control of HIF alpha subunits by oxygen is mainly post-transcriptional. This involves tight regulation of stability, with HIF alpha protein being essentially undetectable in most cells under standard conditions but accumulating rapidly under low oxygen. In addition, transactivation is prevented by oxygen. These involve enzymatic hydroxylation of specific prolyl and asparaginyl residues. The enzymes are recently identified oxygenases, which have a ferrous ion at the active site and use 2-oxoglutarate as co-substrate. The reactions require oxygen to proceed, and the reaction rate depends on the concentration of oxygen, thus providing a molecular "oxygen-sensor". To date, three HIF prolyl hydroxylases have been identified termed PHD1-3 (also referred to as HIF-PH1-3 or EGLN1-3), and one asparaginyl hydroxylase (FIH-1).

Hydroxylation of Asn803 prevents HIF-1 from interacting with P300, and thereby blocks transactivation. In contrast, the consequence of hydroxylating Pro402 or Pro564 is to enable an interaction - with the VHL tumor suppressor protein. This acts as the recognition component of a ubiquitin E3 ligase complex, and capture results in ubiquitylation and destruction of the HIF alpha subunit. The HIF-PHD-VHL system is conserved from C. elegans to man, and appears to operate in all mammalian cell types. With evolution, considerable scope for refining the basic oxygen response system appears to have developed; C. elegans has a single HIF alpha subunit with a single prolyl hydroxylation site and a single identified HIF prolyl hydroxylase, whereas mammals have at least two alpha subunits, each with two prolyl hydroxylation sites, and three prolyl hydroxylases. The PHD genes show marked variation in expression between tissues, and in response to a variety of stimuli including hypoxia. This is likely to provide a potent means of adjusting the HIF response to different circumstances, and provides an element of feedback inhibition. As well as the concentration of hydroxylase enzymes other parameters, such as the availability of ferrous iron in the cell, can influence the rate of the reaction. This is likely to be important in cancer and in some cancer cell lines HIF prolyl hydroxylase activity is limiting under normal culture conditions, resulting in partial activation of HIF-1, which can be corrected by ascorbate supplementation. Germline mutations in VHL cause a hereditary cancer syndrome, whose main manifestations are a high risk of clear cell renal cell carcinoma, haemangioblostomas of the eye and central nervous system, and phaeochromocytoma. In accordance with Knudson's two hit hypothesis, biallelic inactivation of VHL occurs in the great majority of clear cell renal carcinomas (CCRC) and many sporadic haemangioblastomas. Most clear cell tumours and CCRC cell lines show constitutive HIF activation, and in the latter case this can be corrected by re-expressing VHL which also suppresses growth as xenografts. HIF activation accounts for many phenotypic aspects of CCRC, but it remains possible that deregulation of another target of VHL is pivotal in initiating tumor development. On the other hand, it is clear that HIF-2 alpha inactivation is necessary for VHL to suppress CCRC tumour growth, and antagonising HIF activation seems a particularly attractive strategy in these tumours.