Identification of hypoxia-sensing/signalling pathways that are dysregulated in cancer.

Project Overview

Hypoxia is a physiological stress that leads to widespread changes in cellular gene expression and behaviour in part through the activation of cellular pathways that operate to sense and signal changes in oxygen concentration. In the cancer setting, tumour hypoxia can also lead to the activation of these pathways contributing to tumour heterogeneity.

Hypoxia inducible factor (HIF) has been defined as the key factor mediating transcriptional responses to hypoxia. HIF is a heterodimer and its hypoxia-inducible behaviour is conferred by O2-dependent enzymatic post-translational modifications of the HIF-a subunits by a series of HIF hydroxylase enzymes. The HIF hydroxylases are members of the wider oxygenase superfamily and the inhibition of their activity under hypoxia leads to an upregulation of HIF and downstream transcriptional effects on HIF target genes. In cancer, tumour hypoxia and other mechanisms (including loss of tumour suppressors/activation of oncogenes, increased redox stress, altered metabolism) leads to an abnormal activation of the HIF transcriptional response (Masson and Ratcliffe, 2014).

The laboratory has a long-standing interest in HIF hydroxylase system, and is now focusing on the definition of other ‘oxygen sensing’ pathways that are important in human physiology e.g.  Simpson et al., 2015, or in human pathologies such as cancer and cardiovascular disease.

Following evidence for its involvement in angiogenesis and cardiovascular disease, the laboratory is investigating a novel mechanism of oxygen sensing in the Arg/Cys branch of the proteolytic N-end rule pathway that enables the oxygen-dependent degradation of protein substrates containing an N-terminal Cys residue in both plant and animal kingdoms (Licausi et al. Nature 2011, Hu et al. Nature 2005). In plants oxygen-dependent regulation has been shown to be dependent upon cysteine oxidase activity (Weits et al. Nature Comm 2013).

The studentship will build on our investigation of the Arg/Cys N-end rule pathway in mammalian cell lines to (i) test candidate human enzymes for involvement in the oxygen-dependent regulation. (ii). to identify additional mammalian proteins that may undergo similar oxygen-dependent regulation; and (iii) to identify the physiological relevance of these oxygen-sensing pathway(s). The project provides the opportunity to undertake training in a wide range of physiological and molecular methodologies. In parallel to the pathway dissection, the student will also assess the consequences of pathway dysregulation in both physiology and cancer using both publicly available bioinformatics/proteomics data resources and study in relevant cell lines. We are looking for an exceptional and motivated student with a desire to understand hypoxia physiology/pathology and a willingness to compete at the highest international level. The successful candidate will have a strong track record in a relevant subject and is expected to have good communication skills, attention to detail and an ability to work both independently and as part of a team. Interest and experience in molecular biology (and a basic interest in mass spectrometry) would be considered advantageous.

Training Opportunities

The research project will involve an extensive range of molecular biology techniques together with some basic mass spectrometry, including:

Theme

Physiology, Cellular & Molecular Biology and Cancer Biology

Admissions

Project reference number: 988

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Sir Peter J Ratcliffe FRS Target Discovery Institute Oxford University, NDM Research Building GBR peter.ratcliffe@ndm.ox.ac.uk
Dr Norma Masson Centre for Cellular and Molecular Physiology Oxford University, Henry Wellcome Building for Molecular Physiology GBR norma.masson@well.ox.ac.uk

Masson N, Ratcliffe PJ. 2014. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways. Cancer Metab, 2 (1), pp. 3. Read abstract | Read more

Both tumor hypoxia and dysregulated metabolism are classical features of cancer. Recent analyses have revealed complex interconnections between oncogenic activation, hypoxia signaling systems and metabolic pathways that are dysregulated in cancer. These studies have demonstrated that rather than responding simply to error signals arising from energy depletion or tumor hypoxia, metabolic and hypoxia signaling pathways are also directly connected to oncogenic signaling mechanisms at many points. This review will summarize current understanding of the role of hypoxia inducible factor (HIF) in these networks. It will also discuss the role of these interconnected pathways in generating the cancer phenotype; in particular, the implications of switching massive pathways that are physiologically 'hard-wired' to oncogenic mechanisms driving cancer. Hide abstract

Simpson PD, Eipper BA, Katz MJ, Gandara L, Wappner P, Fischer R, Hodson EJ, Ratcliffe PJ, Masson N. 2015. Striking Oxygen Sensitivity of the Peptidylglycine α-Amidating Monooxygenase (PAM) in Neuroendocrine Cells. J. Biol. Chem., 290 (41), pp. 24891-901. Read abstract | Read more

Interactions between biological pathways and molecular oxygen require robust mechanisms for detecting and responding to changes in cellular oxygen availability, to support oxygen homeostasis. Peptidylglycine α-amidating monooxygenase (PAM) catalyzes a two-step reaction resulting in the C-terminal amidation of peptides, a process important for their stability and biological activity. Here we show that in human, mouse, and insect cells, peptide amidation is exquisitely sensitive to hypoxia. Different amidation events on chromogranin A, and on peptides processed from proopiomelanocortin, manifest similar striking sensitivity to hypoxia in a range of neuroendocrine cells, being progressively inhibited from mild (7% O2) to severe (1% O2) hypoxia. In developing Drosophila melanogaster larvae, FMRF amidation in thoracic ventral (Tv) neurons is strikingly suppressed by hypoxia. Our findings have thus defined a novel monooxygenase-based oxygen sensing mechanism that has the capacity to signal changes in oxygen availability to peptidergic pathways. Hide abstract

Licausi F, Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LA, Perata P, van Dongen JT. 2011. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature, 479 (7373), pp. 419-22. Read abstract | Read more

The majority of eukaryotic organisms rely on molecular oxygen for respiratory energy production. When the supply of oxygen is compromised, a variety of acclimation responses are activated to reduce the detrimental effects of energy depletion. Various oxygen-sensing mechanisms have been described that are thought to trigger these responses, but they each seem to be kingdom specific and no sensing mechanism has been identified in plants until now. Here we show that one branch of the ubiquitin-dependent N-end rule pathway for protein degradation, which is active in both mammals and plants, functions as an oxygen-sensing mechanism in Arabidopsis thaliana. We identified a conserved amino-terminal amino acid sequence of the ethylene response factor (ERF)-transcription factor RAP2.12 to be dedicated to an oxygen-dependent sequence of post-translational modifications, which ultimately lead to degradation of RAP2.12 under aerobic conditions. When the oxygen concentration is low-as during flooding-RAP2.12 is released from the plasma membrane and accumulates in the nucleus to activate gene expression for hypoxia acclimation. Our discovery of an oxygen-sensing mechanism opens up new possibilities for improving flooding tolerance in crops. Hide abstract

Hu RG, Sheng J, Qi X, Xu Z, Takahashi TT, Varshavsky A. 2005. The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators. Nature, 437 (7061), pp. 981-6. Read abstract | Read more

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues--aspartate, glutamate and cysteine--are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well. Hide abstract

Weits DA, Giuntoli B, Kosmacz M, Parlanti S, Hubberten HM, Riegler H, Hoefgen R, Perata P, van Dongen JT, Licausi F. 2014. Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway. Nat Commun, 5 pp. 3425. Read abstract | Read more

In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants. Hide abstract