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Cells silenced for SDHB expression display characteristic features of the tumor phenotype
Ana M Cervera1, Nadezda Apostolova, Francisco Luna Crespo
1Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
Abstract:
Recently, enzymes of the tricarboxylic acid (TCA) cycle have emerged as novel tumor suppressors. In particular, mutations in the nuclear-encoded subunits of succinate dehydrogenase (SDHB, SDHC, and SDHD) cause paragangliomas and pheochromocytomas. Although the mechanism(s) by which disruption of mitochondrial metabolism leads to neoplasia is largely unknown, increasing evidence points to an activation of pseudohypoxia. In this study, we have shown that silencing of SDHB using DNA-based small interfering RNA resulted in major impairments in cellular proliferation, respiration, and a corresponding shift to glycolysis. The levels of reactive oxygen species, however, were unchanged. As expected, hypoxia-inducible factor-1 alpha (HIF-1 alpha) and HIF-2alpha were up-regulated in chronically silenced cells, suggesting that a pseudohypoxic state was attained. In addition, the c-Jun amino-terminal kinase and p38 kinase stress signaling proteins were hyperphosphorylated in SDHB-silenced cells. Microarray analysis showed that >400 genes were influenced (6-fold or more up-regulation or down-regulation) by silencing of SDHB, confirming the importance of the TCA cycle in cellular metabolism. Examples of dysregulated genes included those involved in proliferation, adhesion, and the hypoxia pathway. Of interest, SDHB-silenced cells had a greater capacity to adhere to extracellular matrix components, including fibronectin and laminin, than control cells, thus suggesting a possible mechanism of tumor initiation. Although transient silencing of the HIF-1 alpha transcription factor in SDHB-silenced cells had little effect on the expression of a subset of up-regulated genes, it partially reversed the adhesion phenotype to fibronectin, pointing to a potentially important role for HIF-1 in this process.
Insights
Succinate dehydrogenase (SDHB) silencing impairs cell growth and respiration, inducing a pseudohypoxic state. This metabolic shift enhances cell adhesion, suggesting a role in tumor initiation.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Enzymes in the tricarboxylic acid (TCA) cycle are emerging as tumor suppressors.
- Mutations in succinate dehydrogenase (SDHB, SDHC, SDHD) subunits are linked to paragangliomas and pheochromocytomas.
- Disruption of mitochondrial metabolism may lead to neoplasia via pseudohypoxia activation.
Purpose of the Study:
- To investigate the effects of SDHB silencing on cellular metabolism and tumor-associated phenotypes.
- To explore the role of pseudohypoxia and associated signaling pathways in SDHB-deficient cells.
Main Methods:
- DNA-based small interfering RNA (siRNA) was used to silence SDHB expression.
- Cellular proliferation, respiration, glycolysis, reactive oxygen species (ROS), and kinase phosphorylation were assessed.
- Microarray analysis was performed to identify gene expression changes.
- Hypoxia-inducible factor-1 alpha (HIF-1 alpha) silencing was used to evaluate its role in observed phenotypes.
Main Results:
- SDHB silencing significantly impaired cellular proliferation and respiration, shifting metabolism towards glycolysis.
- A pseudohypoxic state was confirmed by up-regulation of HIF-1 alpha and HIF-2alpha.
- Stress signaling proteins (c-Jun N-terminal kinase, p38 kinase) were hyperphosphorylated.
- Microarray analysis revealed dysregulation of >400 genes involved in proliferation, adhesion, and hypoxia pathways.
- SDHB-silenced cells exhibited increased adhesion to fibronectin and laminin.
- HIF-1 alpha partial silencing reversed the fibronectin adhesion phenotype.
Conclusions:
- SDHB deficiency disrupts cellular metabolism, leading to impaired proliferation and respiration.
- Pseudohypoxia and stress kinase activation are consequences of SDHB loss.
- Altered gene expression and enhanced cell adhesion in SDHB-silenced cells suggest a mechanism for tumor initiation.
- HIF-1 alpha plays a role in the adhesion phenotype associated with SDHB deficiency.
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