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PNC1) Regulates Mitochondrial Biogenesis and the Invasive Phenotype of Cancer Cells

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PNC1) Regulates Mitochondrial Biogenesis and the Invasive Phenotype of Cancer Cells Oncogene (2010) 29, 3964–3976 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells C Favre1, A Zhdanov2, M Leahy1, D Papkovsky2 and R O’Connor1 1Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College Cork, Cark, Ireland and 2Laboratory of Biophysics and Bioanalysis, Department of Biochemistry, University College Cork, Cork, Ireland The insulin-like growth factor (IGF-I) signalling path- occur with mitochondrial DNA (mtDNA) mutations way is essential for metabolism, cell growth and survival. (Penta et al., 2001). However, the Warburg hypothesis It induces expression of the mitochondrial pyrimidine has been revisited notably because observed increases in nucleotide carrier 1 (PNC1) in transformed cells, but the glycolysis in cancer cells can occur along with normal consequences of this for cell phenotype are unknown. Here mitochondrial function or respiration. There is now we show that PNC1 is necessary to maintain mitochondrial considerable evidence to indicate that enhanced glyco- function by controlling mitochondrial DNA replication and lysis in cancer cells cooperates with oxidative metabo- the ratio of transcription of mitochondrial genes relative to lism, and that the anabolic functions of mitochondria in nuclear genes. PNC1 suppression causes reduced oxidative glutamine and fatty acid metabolism are essential for phosphorylation and leakage of reactive oxygen species tumour cell proliferation (reviewed by Deberardinis (ROS), which activates the AMPK-PGC1a signalling path- et al., 2008; Jones and Thompson, 2009). way and promotes mitochondrial biogenesis. Overexpression The insulin-like growth factor (IGF-I) signalling of PNC1 suppresses mitochondrial biogenesis. Suppression pathway through PI3-kinase, Akt and mTOR is of PNC1 causes a profound ROS-dependent epithelial– important in promoting cell growth and proliferation. mesenchymal transition (EMT), whereas overexpression This signalling pathway can directly enhance both of PNC1 suppresses both basal EMT and induction of glycolysis and oxidative phosphorylation (OxPhos). EMT by TGF-b. Overall, our findings indicate that PNC1 It controls the expression and trafficking of glucose is essential for mitochondria maintenance and suggest that transporters (Quon et al., 1995; Hajduch et al., 1998; its induction by IGF-I facilitates cell growth whereas Huang et al., 2005), enhances glycolysis (Elstrom et al., protecting cells from an ROS-promoted differentiation 2004; Pankratz et al., 2009) and may also directly programme that arises from mitochondrial dysfunction. enhance OxPhos (Unterluggauer et al., 2008). IGF-I- Oncogene (2010) 29, 3964–3976; doi:10.1038/onc.2010.146; mediated activation of the mTOR pathway is regulated published online 10 May 2010 by AMP-activated kinase (AMPK), a serine or threo- nine kinase that is considered to be a master regulator Keywords: PNC1; IGF-I; mitochondria; ROS; EMT of cellular and systemic metabolism by acting to restore in cancer; metabolism energy balance (Marshall, 2006). However, although AMPK has an important role in mitochondrial bio- genesis, the links between the IGF-I signalling pathway and mitochondrial function and biogenesis are not clear. Introduction The Warburg effect suggests that increased glycolysis in cancer cells harbouring mitochondria with mtDNA The importance of mitochondria in cancer cell proli- mutations confers a growth advantage in hypoxic feration has been studied since Warburg (1956) conditions. However, damaged mitochondria may also proposed that tumour cells are less dependant on directly participate in increasing metastatic potential by mitochondria for ATP production than normal cells. activating a reactive oxygen species (ROS)-mediated Increased glycolysis and rapid production of ATP could retrograde signalling pathway (Ferraro et al., 2006; allow tumour cells to survive in low oxygen conditions Owusu-Ansah et al., 2008). Earlier studies have corre- found inside solid tumours in vivo (Pedersen, 1978; lated elevated levels of ROS in cancer cells with Gatenby and Gillies, 2004). It has long been suggested carcinogenesis (Klaunig et al., 1998; Mori et al., 2004). that this adaptation is due to an energy deficiency Numerous studies have implicated ROS in DNA caused by non-functional mitochondria such as may mutation and apoptosis, low levels of ROS produced in response to growth factor signalling may also Correspondence: Professor R O’Connor, Department of Biochemistry, function as a secondary messenger to activate signalling University College Cork, BioSciences Institute, College Road, pathways (Finkel, 2000). Cork, Ireland. The intense interest in targeting the IGF-I pathway E-mail: [email protected] Received 28 July 2009; revised 23 February 2010; accepted 25 February for cancer therapy (reviewed in Pollak, 2008) highlight 2010; published online 10 May 2010 the need for better insights into the mechanistic links PNC1 regulates mitochondrial function and EMT C Favre et al 3965 between IGF-I or insulin signalling, mitochondrial (which inhibit glycolytic production of ATP resulting in function, ROS signalling and metabolic regulation. reliance on respiration (OxPhos) (Marroquin et al., For these reasons we investigated the role of IGF-I 2007)). No significant difference was observed between signalling in mitochondrial activity in tumour cells by PNC1-deficient cells and control cells cultured in glucose analyzing the function of the mitochondrial pyrimidine (Figure 2a). No difference was also observed in control nucleotide carrier 1 (PNC1), which we previously cells after 2 h culture in galactose. In contrast, PNC1- identified as an IGF-I and insulin-responsive protein deficient clones showed a reduction of 15–20% in in transformed cells (Floyd et al., 2007). Here we show cellular ATP level in the presence of galactose. This that PNC1 is essential for maintenance of mtDNA and reduction in mitochondrial ATP production suggests respiration, and that suppression of PNC1 leads to that cells with PNC1 suppressed are deficient in OxPhos activation of an ROS-mediated signalling pathway that and cannot compensate for inhibition of glycolysis by controls nuclear gene expression. The findings show galactose as efficiently as controls. that IGF-I signalling is integrated with mitochondrial To further assess the effect of PNC1 suppression on function to determine the phenotype of cancer cells. respiration, we investigated the effect of PNC1 suppres- sion on oxygen consumption using the phosphorescent probe MitoXpress (Luxcel Biosciences, Cork, Ireland), whose fluorescence emission is quenched by oxygen. Results When cell cultures are isolated from air, the cells consume dissolved oxygen and the fluorescence emission PNC1 suppression is associated with increased of the probe increases (Will et al., 2006). As can be seen mitochondrial ROS production in Figure 2b, HeLa cells with suppressed PNC1 grown We have previously shown that PNC1 expression levels in glucose show decreased oxygen consumption com- are inversely correlated with cellular ROS levels (Floyd pared with controls. This effect on oxygen consumption et al. , 2007). To investigate the mechanism of this, we is consistent with the decrease in mitochondrial ATP generated HeLa cell lines in which PNC1 expression was production in PNC1-deficient cells grown in galactose stably suppressed using shRNA. Clones showing an (Figure 2a). approximately 40% (ShRNA PNC1 clone 1) and 60% Cells with suppressed PNC1 could compensate for the (ShRNA PNC1 clone 2) reduction, respectively in PNC1 loss of mitochondrial ATP by increasing activation of transcription were isolated (Figure 1a). Increased basal the glycolytic pathway for ATP production. We there- expression of cellular ROS was observed in these clones fore investigated glycolytic activity in PNC1-deficient compared with controls (Figure 1b). The subcellular cells by measuring the rate of extracellular acidification, origin of the observed ROS was investigated using which is linked to lactic acid production during immunofluorescence with the ROS-specific H2DCF-DA glycolysis. We found that cells with suppressed PNC1 dye. As shown in Figure 1c, when compared with have increased extracellular acidification (increased controls, cells with suppressed PNC1 expression show emission of the pH-sensitive probe). This indicates that increased accumulation of the H2DCF-DA dye in glycolysis is increased in these cells compared with regions in which it is colocalized with the mitochondrial controls (Figure 2c). marker MitoTracker. We then used inhibitors that Altogether our study data indicate that in conditions target different complexes of the mitochondrial respira- of normal oxygen supply suppression of PNC1 causes a tory chain to determine which complex may contribute defect in the respiratory chain and increases the rate of to this ROS production. Rotenone (blocks transfer of glycolysis to compensate for overall cellular ATP electron from complex I to complex II) induced ROS production. production in both control and shRNA clones whereas antimycin A (complex III inhibitor) and CCCP (Elec- tron Transport Chain, uncoupler; unpublished data) did PNC1 expression regulates mitochondrial mass not increase ROS production in cells with suppressed and mtDNA replication PNC1 (Figure 1d). The observation that antimycin A We next turned our
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