Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

Stephanie Fliedner ,Alfred L Yergey,Robert Wesley,Andrea Limpuangthip,Hendrik Lehnert,Nina Kaludercic,Peter S Backlund,Forbes D Porter,Nazareno Paolocci,Arthur S Tischler,Lucia Martiniova,Nikoletta Lendvai ,Jiřı́ Zeman ,Xiao Jiang ,Jana Sládková ,Hana Hansı́ková ,J Breza ,Zuzana Hájková ,Ivana Jochmanová ,Karel Pacák

doi:10.1371/journal.pone.0040949

Abstract

A glycolytic profile unifies a group of pheochromocytomas and paragangliomas (PHEOs/PGLs) with distinct underlying gene defects, including von Hippel-Lindau (VHL) and succinate dehydrogenase B (SDHB) mutations. Nevertheless, their tumor aggressiveness is distinct: PHEOs/PGLs metastasize rarely in VHL-, but frequently in SDHB-patients. To date, the molecular mechanisms causing the more aggressive phenotype in SDHB-PHEOs/PGLs remain largely unknown. Recently, however, an excellent model to study aggressive PHEOs (mouse tumor tissue (MTT) cells) has been developed from mouse PHEO cells (MPC). We employed this model for a proteomics based approach to identify changes characteristic for tumor aggressiveness, which we then explored in a homogeneous set of human SDHB- and VHL-PHEOs/PGLs. The increase of glucose transporter 1 in VHL, and of hexokinase 2 in VHL and SDHB, confirmed their glycolytic profile. In agreement with the cell model and in support of decoupling of glycolysis, the Krebs cycle and oxidative phosphorylation (OXPHOS), SDHB tumors showed increased lactate dehydrogenase levels. In SDHB-PGLs OXPHOS complex activity was increased at complex III and, as expected, decreased at complex II. Moreover, protein and mRNA expression of all tested OXPHOS-related genes were higher in SDHB- than in VHL-derived tumors. Although there was no direct evidence for increased reactive oxygen species production, elevated superoxide dismutase 2 expression may reflect elevated oxidative stress in SDHB-derived PHEOs/PGLs. For the first time, we show that despite dysfunction in complex II and evidence for a glycolytic phenotype, the Warburg effect does not seem to fully apply to SDHB-PHEOs/PGLs with respect to decreased OXPHOS. In addition, we present evidence for increased LDHA and SOD2 expression in SDHB-PHEOs/PGLs, proteins that have been proposed as promising therapeutic targets in other cancers. This study provides new insight into pathogenic mechanisms in aggressive human PHEOs/PGLs, which may lead to identifying new diagnostic and prognostic markers in the near future.

Highlights

After going unnoticed for decades, Warburg’s hypothesis of a glycolytic phenotype in tumors is increasingly recognized
Differential Expression in Human PHEOs/PGLs In the more aggressive mouse tumor tissue (MTT) cells, we found an increase in overall LDH due to an increased expression of LDHA, while LDHB expression was decreased compared to mouse PHEO cells (MPC)
It is becoming clear that aggressive tumors acquire dependence on glycolysis, often related to pseudo-hypoxia and/or oxidative phosphorylation (OXPHOS) dysfunction [35]

Summary

Introduction

After going unnoticed for decades, Warburg’s hypothesis of a glycolytic phenotype in tumors is increasingly recognized. In highly aggressive tumors a shift from efficient ATP synthesis via oxidative phosphorylation (OXPHOS) to increased glycolysis has been observed [1,2,3]. Increased glycolysis has been shown to result from suppression of OXPHOS due to a hypoxic state in tumor areas with poor blood and oxygen supply, from mutation of key regulatory genes, or from increased reactive oxygen species (ROS) levels. The latter two lead to a pseudo-hypoxic state under normoxic conditions [5,6,7,8]. Hypoxia has been recognized as a predictive marker for metastatic disease, therapy resistance, and poor outcome in several types of cancer [9,10]

Methods

Results

Conclusion