Hypoxia Genomics

Deciphering gene expression regulation by hypoxia

Hypoxia results in the changes in expression of many genes. The protein products of these genes have a wide variety of critical roles in important processes such as energy metabolism, angiogenesis, growth and apoptosis. The majority of changes in gene expression under hypoxia are mediated via the transcriptional activity of the hypoxia inducible factor (HIF) complex. In the presence of oxygen, HIF-a undergo prolyl hydroxylation which is catalysed by three homologous 2-oxoglutarate dependent dioxygenases, PHD1, PHD2 and PHD3. This is the first step in the process leading to the proteasomal degradation process of HIF-a molecules. We have performed comprehensive gene array studies that have emphasised the dominant role of the HIF transcriptional system and the HIF peptidyl hydroxylases in the regulation of gene expression by hypoxia (Elvidge et al., 2006). This work involved the expression profiling of cell lines grown under conditions of normoxia (21% Oxygen), hypoxia (1% Oxygen), drug induced hypoxia response and RNAi to block the expression of key genes in the response to hypoxia.

Recently we and others showed that hypoxia also regulates microRNA (miRNA) expression. MicroRNAs are a class of short (25bp) single strand non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Using microarrays, we have detected the induction of a small set of miRNAs in the breast cancer cell line MCF7 under hypoxic conditions (1% oxygen for 16 hours). Furthermore, we have characterized the hypoxic induction of one of these miRNAs (hsa-miR-210). We have shown that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1a and VHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor (Camps et al., 2008).

Identifying target genes for these miRNAs is therefore important in understanding the cellular pathways involved in tumour growth so that in the future there may be the possibility of more targeted drugs which inhibit specific miRNAs.

We used gene expression profiling results from breast cancer samples and several miRNA target prediction programs to generate a list of potential targets for miR-210 and several miRNAs differentially expressed in hypoxia. We are currently using luciferase reporter constructs with the 3'-UTRs of the potential mRNA targets co-transfected with either miRNAs mimic or anti-miRNAs to demonstrate that specific miRNAs can directly repress translation process.

Indeed, miRNAs have been implicated in the regulation of a variety of cellular processes like cell cycle, cell death, and cell metabolism that are keys in determining the adaptation to the hypoxic microenvironment. We are also studying the effect of mimic and anti-miRNAs on cell proliferation, apoptosis and mitochondria activity and assessing target regulations by real-time PCR and western blot analysis.

People

Carme Camps
Christine Blancher
Elena Favaro

Gareth Elvidge, Louisa Glenny (former members of the group and initiators of this research area)

To discuss potential collaborative projects please contact: Jiannis Ragoussis

Collaborators

Pr. Adrian L Harris (Cancer Research UK Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK)
(link http://www.imm.ox.ac.uk/wimm-research/molecular-oncology/adrian-l.-harris).

Dr. Jonathan Gleadle (current address: Renal Unit, Flinders Medical Centre, South Australia 5042, Australia) and Professor Peter Ratcliffe (The Nephrology Oxygen Sensing Group)
(link http:// www.well.ox.ac.uk/renal).

Publications

Gee H. E., Camps C., Buffa F. M., Colella S., Sheldon H., Gleadle J. M., Ragoussis J., and Harris A. L. (2008). MicroRNA-10b and breast cancer metastasis. Nature 455: E8-9;

Camps C., Buffa F. M., Colella S., Moore J., Sotiriou C., Sheldon H., Harris A. L., Gleadle J. M., and Ragoussis J. (2008). hsa-miR-210 Is Induced by Hypoxia and Is an Independent Prognostic Factor in Breast Cancer. Clin Cancer Res 14: 1340-1348.

Eldvidge. G.P., Glenny, L., Appelhoff, R.J., Ratcliffe, P.J., Ragoussis, J., and Gleadle, J.M. (2006) JBC, vol. 281, 22, 15215-15226.