Cells Silenced forSDHBExpression Display Characteristic Features of the Tumor Phenotype

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, hypoxiainducible factor-1A (HIF-1A) and HIF-2A 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-1A transcription factor in SDHB-silenced cells had little effect on the expression of a subset of upregulated genes, it partially reversed the adhesion phenotype to fibronectin, pointing to a potentially important role for HIF-1 in this process. [Cancer Res 2008;68(11):4058-67]

Mitochondrial respiratory chain complexes: apoptosis sensors mutated in cancer?

“…Both reagents target the SDHA succinate-binding site and equally inhibit the SDH and SQR activities (Brusque et al, 2002;Lemarie et al, 2011). When the tumour-suppressor gene function of complex II subunits was discovered for SDHD, SDHC and SDHB (Baysal et al, 2000;Niemann and Muller, 2000;Astuti et al, 2001), two mechanisms were evoked to explain the oncogenesis process associated with complex II deficiency: (1) the establishment of a pseudo-hypoxic state leading to HIF1a stabilization in tumours through succinate inhibition of the HIF1a prolyl hydroxylase (PHD), which favours the glycolytic pathway and promotes tumour formation (GimenezRoqueplo et al, 2001;Pollard et al, 2005Pollard et al, , 2006Selak et al, 2005;Lehtonen et al, 2007;Cervera et al, 2008) and (2) the long-term generation of sublethal superoxides at the complex II level contributing to either genomic instability and subsequent tumoral development or HIF1a stabilization (Senoo-Matsuda et al, 2001;Ishii et al, 2005;Slane et al, 2006;Guzy et al, 2008). Various key publications subsequently brought to light the possible role of complex II as a proapoptotic sensor, through the production of deleterious high levels of ROS, which could provide a third explanation as to why complex II subunits are disabled in tumourigenesis.…”

“…Moreover, in PGL/PH tumours, which exhibit high levels of HIF1a, this transcription factor was shown to exert a negative regulatory loop on complex II through the downregulation of SDHB expression. This then amplified, in a feed-back loop, the effect leading to further pseudo-hypoxia and HIF1a stabilization (Dahia et al, 2005;Cervera et al, 2008). Moreover, hypoxia was shown to further inhibit complex II activity and generate mitochondrial ROS (Paddenberg et al, 2003;Lluis et al, 2007).…”

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

“…Immunocytochemistry was performed using a standard protocol [23]. Cells were incubated with a 1:500 dilution of a-actinin antibody (Sigma-Aldrich) for 2 hours at room temperature (RT), washed again with Tris buffered saline (TBS) and incubated with a 1:100 dilution of fluorescein isothiocyanate-conjugated secondary antibody (Dako Chemicals, Hamburg, Germany, http:// www.dako.com).…”

Succinate dehydrogenase subunit B inhibits the AMPK-HIF-1¿ pathway in human ovarian cancer in vitro

“…Hypoxia is a characteristic of many malignancies arising from various sites [45] and HIF-1α is a hypoxia responsive factor [16]. Research reported that HIF-1α was upregulated in chronically SDHB-silenced Hep3B cells [24], HIF-1α was overexpressed in SDH-deficient leiomyomas and renal cell cancer (HLRCC) [15]. While there was also report that increased HIF-1α was not associated with loss of SDHB expression on a series of familial and sporadic tumours [11].…”

“…The target sequences were as follows: siSucA 5'-GAT TAA GAA TGA AGT TGA CTC-3' [24], siSucC 5'-GCT CAG AGC TGA ACA TAA TT-3' [24]. As a control for silencing, we constructed a negative control (NC) siRNA (5'-UUC UCC GAA CGU GUC ACG UTT-3') that did not affect SDHB expression.…”