title: Proline-hydroxylated hypoxia-inducible factor 1α (HIF-1α) upregulation in human tumours.
abstract: The stabilisation of HIF-α is central to the transcriptional response of animals to hypoxia, regulating the expression of hundreds of genes including those involved in angiogenesis, metabolism and metastasis. HIF-α is degraded under normoxic conditions by proline hydroxylation, which allows for recognition and ubiquitination by the von-Hippel-Lindau (VHL) E3 ligase complex. The aim of our study was to investigate the posttranslational modification of HIF-1α in tumours, to assess whether there are additional mechanisms besides reduced hydroxylation leading to stability. To this end we optimised antibodies against the proline-hydroxylated forms of HIF-1α for use in formalin fixed paraffin embedded (FFPE) immunohistochemistry to assess effects in tumour cells in vivo. We found that HIF-1α proline-hydroxylated at both VHL binding sites (Pro402 and Pro564), was present in hypoxic regions of a wide range of tumours, tumour xenografts and in moderately hypoxic cells in vitro. Staining for hydroxylated HIF-1α can identify a subset of breast cancer patients with poorer prognosis and may be a better marker than total HIF-1α levels. The expression of unhydroxylated HIF-1α positively correlates with VHL in breast cancer suggesting that VHL may be rate-limiting for HIF degradation. Our conclusions are that the degradation of proline-hydroxylated HIF-1α may be rate-limited in tumours and therefore provides new insights into mechanisms of HIF upregulation. Persistence of proline-hydroxylated HIF-1α in perinecrotic areas suggests there is adequate oxygen to support prolyl hydroxylase domain (PHD) activity and proline-hydroxylated HIF-1α may be the predominant form associated with the poorer prognosis that higher levels of HIF-1α confer.
authors.affiliation: Tumour Pathology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Cameron E
authors.initials: CE
authors.lastname: Snell
authors.affiliation: Tumour Pathology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Turley
authors.affiliation: Molecular Oncology Laboratories, Department of Medical Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Alan
authors.initials: A
authors.lastname: McIntyre
authors.affiliation: Haemato-Oncology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Demin
authors.initials: D
authors.lastname: Li
authors.affiliation: Haemato-Oncology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Massimo
authors.initials: M
authors.lastname: Masiero
authors.affiliation: Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.affiliation: Tumour Pathology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Gatter
authors.affiliation: Molecular Oncology Laboratories, Department of Medical Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
authors.affiliation: Tumour Pathology Group, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pezzella
mesh_terms: D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D053208:Kaplan-Meier Estimate; D061986:MCF-7 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011379:Prognosis; D011392:Proline / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011392:Proline; D011393:Procollagen-Proline Dioxygenase