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Open Access Research article

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas

Monika Golinska1, Helen Troy29, Yuen-Li Chung102, Paul M McSheehy112, Manuel Mayr123, Xiaoke Yin123, Lucy Ly3, Kaye J Williams4, Rachel E Airley5, Adrian L Harris6, John Latigo7, Meg Perumal7, Eric O Aboagye7, David Perrett8, Marion Stubbs12* and John R Griffiths12*

Author Affiliations

1 CR UK Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK

2 CR UK Biomedical Magnetic Resonance Research Group, Division of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, UK

3 Cardiac and Vascular Sciences, St. George's, University of London, London SW17 0RE, UK

4 School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Manchester, M13 9PL, UK

5 Division of Pharmacy and Pharmaceutical Sciences, University of Huddersfield, Huddersfield, HD1 3DH,UK

6 Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK

7 Comprehensive Cancer Imaging Centre at Imperial College, Faculty of Medicine, Hammersmith Hospital Campus, London W12 0NN, UK

8 Queen Mary University of London, Barts & The London School of Medicine and Dentistry, William Harvey Research Institute, London EC1M 6BQ,UK

9 Abbott Ireland Diagnostics Division, Pregnancy & Fertility Team, Lisnamuck, Longford, Ireland

10 Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and Royal Marsden Hospital, Sutton, Surrey SM2 5PT, UK

11 Novartis Institutes for Biomedical Research, Oncology Research, Building WKL-125.2.05, CH-4002, Basel, Switzerland

12 Cardiovascular Division, The James Black Centre, King's College, London SE5 9NU, UK

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BMC Cancer 2011, 11:198  doi:10.1186/1471-2407-11-198

Published: 25 May 2011

Abstract

Background

HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1β-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target.

Methods

Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity.

Results

HIF-1β deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1β deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours.

Conclusions

Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Keywords:
HIF-1β; deficiency; Hepa-1 tumours; glycolytic enzymes; glucose uptake; PFK activation; AMP/ATP ratio