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

<div><p>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.</p> </div

Tumor tissue levels of oxidative phosphorylation complex activity and oxidative stress.

<p><b>A</b> Oxidative phosphorylation complex complex activity in SDHB- (n = 4) and VHL-derived (n = 4) PHEO/PGL tissue. * indicates p<0.05. <b>B</b> Malondialdehyde level in SDHB (n = 5) and VHL (n = 5) tissue as a measure of lipid oxidation. <b>C</b> Integrated density of DHE fluorescence in VHL (n = 5), SDHB (n = 6).</p

Oxidative phosphorylation complex activity, reactive oxygen species production, and expression of selected oxidative phosphorylation complex subunits in MPC and MTT. A

<p>Ratio of ADP-stimulated (S3) and baseline oxygen consumption (S4) in MPC and MTT. <b>B</b> Baseline hydrogen peroxide levels (left) and those observed after addition of substrate (glutamate/malate, complex I (center); succinate, complex II (right). ** indicates p<0.01. <b>C</b> protein expression of selected subunits of OXPHOS complexes (complex I: NADH dehydrogenase 1 beta subcomplex subunit 8 (NDUFB8); complex II: succinate dehydrogenase B (SDHB); complex III: ubiquinol-cytochrome-c reductase complex core protein 2 (Uqcrc2); complex V: ATP synthase α (ATPsynα)).</p

Patient Information.

<p>ID (identifier): B followed by a number indicates SDHB, V followed by a number indicates VHL cases. Genetic Background: SDHB^p: SDHB polymorphism, VHL^c: VHL-Chuvash. Gender: F: female, M: male. Type: bp: bilateral primary, mm: metastatic metastases, mltp: multiple primary, pm: primary metastatic, sp: solitary primary. Location: R: right, L: left. Biochem. (biochemical phenotype): A: adrenergic, DA: dopaminergic, NA: noradrenergic, nk: not known. The 4 right columns indicate which samples have been used in the experiments specified by the column headings. Subscript letters are used if more than one tumor from the same patient was available, to indicate which sample has been used (R: right, L: left, A and B: as specified under the heading “location”. OXP (oxidative phosphorylation complex activity), DHE (dihydroethidium fluorescence), MDA (malondialdehyde), WB (western blot). In the western blot column, samples used for LDHA and B and LDHB blots are indicated by . Samples used for all other proteins are indicated by *.</p

Expression of selected glycolysis, oxidative phosphorylation, and oxidative stress related genes. A

<p>mRNA expression of glucose transporter 1 (GLUT1), hexokinase-1 (HK1), hexokinase-2 (HK2), pyruvate kinase isozyme M2 (PKM2), NADH-ubiquinone oxidoreductase chain 1 (ND1), ubiquinol-cytochrome-c reductase complex core protein (UQCRC1), cytochrome c oxidase subunit 1 (CO1) in SDHB- and VHL-derived PHEOs/PGLs relative to normal adrenal medulla. Significant differences are indicated where appropriate by * for p<0.05, ** for p<0.01, and *** for p<0.001. <b>B</b> Western blot of selected proteins in SDHB- and VHL-derived PHEOs/PGLs (complex I: NADH dehydrogenase 1 beta subcomplex subunit 8 (NDUFB8); complex III: ubiquinol-cytochrome-c reductase complex core protein 2 (Uqcrc2); complex IV: CO1; complex V: ATP synthase (ATPsyn) α and β, cytochrome C (CytC), superoxide dismutase (SOD) 1, and 2).</p

Protein expression in MPC compared to MTT.

<p><b>A</b> Representative silver staining of 2D-gel. Spots of interest are circled in red. Magnification of spots containing ATP synthase subunits are shown below for all four MPC and MTT gels. <b>B</b> Western blot for validation of differential expression of selected proteins of interest.</p