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Cited by 232Adrenal and Paraganglionic TumorsCancer, Hypoxia, and MetabolismEar and Head Tumors

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The Warburg Effect Is Genetically Determined in Inherited Pheochromocytomas

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Favier, Judith, Brière, Jean-Jacques, Burnichon, Nelly, Rivière, Julie, Vescovo, Laure, Benit, Paule, Giscos-Douriez, Isabelle, De Reyniès, Aurélien, Bertherat, Jérôme, Badoual, Cécile, Tissier, Frédérique, Amar, Laurence, Libé, Rosella, Plouin, Pierre-François, Jeunemaitre, Xavier, Rustin, Pierre, Gimenez-Roqueplo, Anne-Paule·2009·PLoS ONE
DOI 10.1371/journal.pone.0007094PMC PMC2738974
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Abstract

The Warburg effect describes how cancer cells down-regulate their aerobic respiration and preferentially use glycolysis to generate energy. To evaluate the link between hypoxia and Warburg effect, we studied mitochondrial electron transport, angiogenesis and glycolysis in pheochromocytomas induced by germ-line mutations in VHL, RET, NF1 and SDH genes. SDH and VHL gene mutations have been shown to lead to the activation of hypoxic response, even in normoxic conditions, a process now referred to as pseudohypoxia. We observed a decrease in electron transport protein expression and activity, associated with increased angiogenesis in SDH- and VHL-related, pseudohypoxic tumors, while stimulation of glycolysis was solely observed in VHL tumors. Moreover, microarray analyses revealed that expression of genes involved in these metabolic pathways is an efficient tool for classification of pheochromocytomas in accordance with the predisposition gene mutated. Our data suggest an unexpected association between pseudohypoxia and loss of p53, which leads to a distinct Warburg effect in VHL-related pheochromocytomas.

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Figures

Figure 1 Pseudohypoxia in SDH and VHL-related PH/PGL. (A) HIF-1α and HIF-2α immunohistochemistry were performed to evaluate activation of the hypoxic pathway in all samples. Histogreen was used as a chromogen for detection (blue labeling). Calibration bar: 100 µm. Microarray evaluation of HIF-1α (B) and HIF-2α (C) expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM. ***p<0.001.
Figure 1 Pseudohypoxia in SDH and VHL-related PH/PGL. (A) HIF-1α and HIF-2α immunohistochemistry were performed to evaluate activation of the hypoxic pathway in all samples. Histogreen was used as a chromogen for detection (blue labeling). Calibration bar: 100 µm. Microarray evaluation of HIF-1α (B) and HIF-2α (C) expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM. ***p<0.001.CC BY · PubMed Central
Figure 2 Angiogenesis in SDH and VHL-related PH/PGL. (A) CD34 immunohistochemistry was performed to evaluate angiogenesis in all samples. Diaminobenzidin was used as a chromogen for detection (brown labeling). Calibration bar: 200 µm. (B) Quantification of vascular density showing an increased number of blood vessels in SDH, and VHL tissues. Data are means±SEM. *p<0.05, **p<0.01, ***p<0.001. (C) Correlation between vascular density and SCCR enzymatic values for individual patients.
Figure 2 Angiogenesis in SDH and VHL-related PH/PGL. (A) CD34 immunohistochemistry was performed to evaluate angiogenesis in all samples. Diaminobenzidin was used as a chromogen for detection (brown labeling). Calibration bar: 200 µm. (B) Quantification of vascular density showing an increased number of blood vessels in SDH, and VHL tissues. Data are means±SEM. *p<0.05, **p<0.01, ***p<0.001. (C) Correlation between vascular density and SCCR enzymatic values for individual patients.CC BY · PubMed Central
Figure 3 Decreased oxidative phosphorylation in SDH and VHL-related PH/PGL. (A) The abundance of proteins of mitochondrial complexes I (20 kDa subunit), II (SDHB and SDHA), III (Core 2) and IV (Cox II) is lower in PH/PGL from SDH and most VHL than from RET and tumor tissues. (B) SDHB immunohistochemistry performed on the adrenal adjacent to a RET-related PH and in RET, VHL and SDHD-mutated PH reveals a strong labeling in the adrenal compared to tumor cells (asterisks). In VHL PH/PGL tumor, expression of SDHB was reduced when compared to RET-related PH while it is absent in SDHD-related tumor. Note that vascular immunostaining was present in all samples (arrows). Calibration bar: 50 µm. (C) Individual values of SCCR activity reveal that low complex II+III enzymatic activity is associated with low protein abundance. (D–F) Mean values for mitochondrial complexes II+III, III and IV reveal a generalized decrease in respiration in SDH and VHL PH/PGL. Data are means±SEM. **p<0.01, ***p<0.001.
Figure 3 Decreased oxidative phosphorylation in SDH and VHL-related PH/PGL. (A) The abundance of proteins of mitochondrial complexes I (20 kDa subunit), II (SDHB and SDHA), III (Core 2) and IV (Cox II) is lower in PH/PGL from SDH and most VHL than from RET and tumor tissues. (B) SDHB immunohistochemistry performed on the adrenal adjacent to a RET-related PH and in RET, VHL and SDHD-mutated PH reveals a strong labeling in the adrenal compared to tumor cells (asterisks). In VHL PH/PGL tumor, expression of SDHB was reduced when compared to RET-related PH while it is absent in SDHD-related tumor. Note that vascular immunostaining was present in all samples (arrows). Calibration bar: 50 µm. (C) Individual values of SCCR activity reveal that low complex II+III enzymatic activity is associated with low protein abundance. (D–F) Mean values for mitochondrial complexes II+III, III and IV reveal a generalized decrease in respiration in SDH and VHL PH/PGL. Data are means±SEM. **p<0.01, ***p<0.001.CC BY · PubMed Central
Figure 4 Increased glycolysis in VHL-related PH/PGL. (A) Hexokinase-II protein is detected in all VHL PH/PGL but is hardly detectable in RET, NF1 and SDH samples. (B) Lactate dehydrogenase is increased in tumors harboring a VHL mutation as compared to SDH, RET and NF1 tumors.
Figure 4 Increased glycolysis in VHL-related PH/PGL. (A) Hexokinase-II protein is detected in all VHL PH/PGL but is hardly detectable in RET, NF1 and SDH samples. (B) Lactate dehydrogenase is increased in tumors harboring a VHL mutation as compared to SDH, RET and NF1 tumors.CC BY · PubMed Central
Figure 5 Microarray analysis of oxidative phosphorylation in hereditary PH/PGL. (A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 200 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The higher bipartition allows to distinguish VHL (white) and SDH (grey) patients from RET and NF1 (black) patients. (B) Principal component analysis of the 68 samples according to the expression of 200 genes. Three groups are focused on, corresponding to the SDH (red), VHL (green) and RET/NF1 (blue) patients. PC1: principal component; PC2: principal component 2; PC3: principal component 3. (C) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.
Figure 5 Microarray analysis of oxidative phosphorylation in hereditary PH/PGL. (A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 200 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The higher bipartition allows to distinguish VHL (white) and SDH (grey) patients from RET and NF1 (black) patients. (B) Principal component analysis of the 68 samples according to the expression of 200 genes. Three groups are focused on, corresponding to the SDH (red), VHL (green) and RET/NF1 (blue) patients. PC1: principal component; PC2: principal component 2; PC3: principal component 3. (C) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.CC BY · PubMed Central
Figure 6 Microarray analysis of glycolysis in hereditary PH/PGL. (A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 38 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The different mutations are localized in three distinct clusters: SDH (grey), VHL (white) and RET/NF1 (black). (B) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.
Figure 6 Microarray analysis of glycolysis in hereditary PH/PGL. (A) Unsupervised hierarchical clustering analysis of the 68 samples according to the expression of 38 genes. Expression profiles are shown as a heat map indicating high (red) and low (blue) expression according to a log2-transformed scale. The different mutations are localized in three distinct clusters: SDH (grey), VHL (white) and RET/NF1 (black). (B) Mean values for genes expression between SDH, VHL, RET and NF1 tumors. Data are means±SEM, represented as relative to NF1 expression values. **p<0.01, ***p<0.001.CC BY · PubMed Central
Figure 7 Representative expression of p53 and TIGAR in PH/PGL. Immunohistochemistry revealed a mild to strong nuclear p53 labeling, and the cystoplasmic expression of TIGAR in the majority of RET, NF1 and SDH tumors. Such labeling was only detected in 2 out of 9 VHL tumor tissues (data not shown). In most VHL PH/PGL p53 protein and its target TIGAR were hardly detectable. Calibration bar: 50 µm.
Figure 7 Representative expression of p53 and TIGAR in PH/PGL. Immunohistochemistry revealed a mild to strong nuclear p53 labeling, and the cystoplasmic expression of TIGAR in the majority of RET, NF1 and SDH tumors. Such labeling was only detected in 2 out of 9 VHL tumor tissues (data not shown). In most VHL PH/PGL p53 protein and its target TIGAR were hardly detectable. Calibration bar: 50 µm.CC BY · PubMed Central

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Authors
Favier, J., Brière, J.-J., Burnichon, N., Rivière, J., Vescovo, L., Benit, P., Giscos-Douriez, I., De Reyniès, A., Bertherat, J., Badoual, C., Tissier, F., Amar, L., Libé, R., Plouin, P.-F., Jeunemaitre, X., Rustin, P., & Gimenez-Roqueplo, A.-P.
Affiliations
  • Inserm (FR)
  • La Ligue Contre le Cancer (FR)
  • Assistance Publique – Hôpitaux de Paris (FR)
  • Hôpital Cochin (FR)
Corresponding author
Favier, Judith
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2009-09-18
Volume
4
Issue
9
Pages
e7094
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0007094
PMC
PMC2738974
OpenAlex
W2063687158
Cited by
232
References
42
Citations by year
2026: 7 · 2025: 3 · 2024: 4 · 2023: 4 · 2022: 10 · 2021: 7 · 2020: 12 · 2019: 13 · 2018: 14 · 2017: 11 · 2016: 15 · 2015: 28 · 2014: 27 · 2013: 16 · 2012: 27
FWCI
20.62
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Association pour la Recherche sur le Cancer
  • Agence Nationale de la Recherche
  • GIS-Institut des Maladies Rares
Topics
  • Health Sciences › Medicine › Surgery › Adrenal and Paraganglionic Tumors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Health Sciences › Medicine › Oncology › Ear and Head Tumors
Keywords
Warburg effect, pheochromocytoma, pseudohypoxia, VHL gene, SDH genes, RET proto-oncogene, NF1 gene, mitochondrial electron transport chain, angiogenesis, glycolysis, p53 deficiency, cancer metabolism, germline mutations, gene expression profiling
MeSH terms
Adolescent, Adult, Aged, Hypoxia, Child, Electron Transport, Female, Glycolysis, Humans, Male, Middle Aged, Mitochondria, Neovascularization, Pathologic, Oxidative Phosphorylation, Pheochromocytoma, Genes, p53, Germ-Line Mutation, Oligonucleotide Array Sequence Analysis

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