Proline-Hydroxylated Hypoxia-Inducible Factor 1α (HIF-1α) Upregulation in Human Tumours

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 reco...

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Published in:	PloS one Vol. 9; no. 2; p. e88955
Main Authors:	Snell, Cameron E., Turley, Helen, McIntyre, Alan, Li, Demin, Masiero, Massimo, Schofield, Christopher J., Gatter, Kevin C., Harris, Adrian L., Pezzella, Francesco
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 12.02.2014 Public Library of Science (PLoS)
Subjects:	Angiogenesis Animals Antibodies Binding Sites Biology Brain cancer Breast cancer Breast Neoplasms - pathology Cancer Cell Line, Tumor Degradation Female Gene expression Gene Expression Regulation, Neoplastic Humans Hydroxylation Hypoxia Hypoxia-Inducible Factor 1, alpha Subunit - metabolism Hypoxia-inducible factors Immunoglobulins Immunohistochemistry Kaplan-Meier Estimate Laboratories MCF-7 Cells Medical research Medicine Metabolism Metastases Metastasis Mice Mice, Inbred BALB C Neoplasm Transplantation Neoplasms - metabolism Oncology Oxygen Paraffin Pathology Procollagen-Proline Dioxygenase - metabolism Prognosis Proline Proline - metabolism Prolyl hydroxylase Proteins Stability analysis Transcription Tumors Ubiquitin-protein ligase Ubiquitination Up-Regulation VHL protein Xenografts
ISSN:	1932-6203, 1932-6203
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Conceived and designed the experiments: CES HT AM DL MM CJS KCG ALH FP. Performed the experiments: CES HT AM DL MM. Analyzed the data: CES HT AM DL MM. Contributed reagents/materials/analysis tools: CES HT AM DL MM CJS KCG ALH FP. Wrote the paper: CES HT AM DL MM CJS KCG ALH FP. Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0088955