185P - Activation of AKT by hypoxia predicts poor outcome in malignant pleural mesothelioma (MPM)

Date 30 September 2012
Event ESMO Congress 2012
Session Poster presentation II
Topics Mesothelioma
Translational Research
Presenter Kathy Gately
Authors K.A. Gately1, D. Stewart2, S. Heavey3, A. Davies4, K.J. O'Byrne5
  • 1Clinical Medicine, St James's Hospital, 8 - Dublin/IE
  • 2Thoracic Oncology, Glenfield Hospital, Leicester/UK
  • 3Trinity College Dublin/St. James Hospital, 8 - Dublin/IE
  • 4Clinical Medicine, Trinity College Dublin/St. James Hospital, 8 - Dublin/IE
  • 5Clinical Medicine, Trinity College Dublin/St. James's Hospital, Dublin/IE

Abstract

Introduction

The Phosphatidylinositol-3 kinase (PI3K)/Akt (PKB) pathway is activated in a wide range of tumour types, and plays a central role in cell survival. Recent evidence indicates that hypoxia induces upregulation of Akt and activation of the Akt/PKB pathway. Once activated, Akt phosphorylates a variety of downstream substrates, including FOXO3a, which decreases transcription of the pro-apoptotic factor Bim, facilitating cell survival. We have shown that nuclear phospho-Akt (pAkt) expression is associated with more advanced disease or poor prognosis in Non-Small Cell Lung Cancer and MPM. Carbonic Anhydrase (CA)-IX, a tumour-specific member of the carbonic anhydrase family, is a surrogate marker of hypoxia overexpressed in solid tumours. This study examines the expression of CA-IX and phosphorylated Akt (pAkt) in tumour samples from patients with MPM, correlating expression with established prognostic factors. The role of pAkt in the survival of MPM cell lines exposed to both normoxia and hypoxia was also examined with both PI3K and PI3k-mTOR inhibitors.

Methods

Tumour sections were stained using pAkt and CA-IX specific antibodies. The effect of hypoxia on pAkt, Akt and Bim expression in 4 MPM cell lines in the presence or absence of LY294002 (phosphatidylinositol-3-kinase inhibitor) was examined by Western blot. The percentage of apoptotic cells was also quantified in these cells using FACs and High-Content Analysis (HCA). Changes in subcellular localisation of pAkt and FOXO3a were quantified using HCA. The cells were also transfected with siRNAs to PDK1or DNA-PKcs (two kinases known to catalyse the phosphorylation of Akt) and the effect on pAkt expression was determined by Western blot.

Results

A positive association between CA-IX and pAkt staining implies intra-tumoural hypoxia may stimulate Akt phosphorylation. Multivariate analysis showed increased expression of nuclear pAkt was associated with poor survival. Hypoxia induced the activation of Akt in MPM cell lines resulting in changes in the subcellular localisation of pAkt and FOXO3a. A greater increase in the level of apoptosis was seen in cells treated with PI3K inhibitors under hypoxia.

Conclusion

pAkt plays an anti-apoptotic role in MPM and represents a potential therapeutic target particularly in hypoxic tumours.

Disclosure

All authors have declared no conflicts of interest.