Oncogene (2013) 32, 352 --362 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration R Palorini1, D De Rasmo2,3, M Gaviraghi1, L Sala Danna1, A Signorile2, C Cirulli1, F Chiaradonna1, L Alberghina1 and S Papa2,3 The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs. Oncogene (2013) 32, 352--362; doi:10.1038/onc.2012.50; published online 12 March 2012 Keywords: cancer cell metabolism; oncogenic K-ras; PKA; mitochondrial morphology; mitochondrial activity; glucose deprivation INTRODUCTION a depression of genes encoding for components of the cyclic Tumors are generally characterized by a high rate of anaerobic adenosine monophosphate/cAMP-dependent protein kinase 10,26 --31 and aerobic glycolysis and a reduced rate of respiration.1--6 This (cAMP/PKA) signaling pathway has also been reported. switch of energy metabolism is known as the ‘Warburg effect’ and The cAMP/PKA pathway is known to regulate cellular energy it has been reported to be linked to mitochondrial dysfunctions metabolism, exerting a stimulatory effect on glucose transport and 32 --34 that lead cells to depend on glycolysis to generate adenosine utilization, glycolysis, glycogen breakdown and glucose 35 triphosphate (ATP).7--9 The genetic and phenotypic features oxidation. The cAMP/PKA pathway has recently been found to 36 --39 40,41 involved in this general derangement of cellular energy metabo- regulate also mitochondrial respiration, dynamics and 42,43 lism in tumors are not yet fully understood.3,5 However, there are apoptosis. Proteins belonging to the cAMP/PKA pathway are 44 several evidences that alterations in signaling pathways, which associated with various cellular compartments and structures 45 regulate glucose uptake and utilization and mitochondrial including the outer membrane and the inner compartment of 46 --49 functions, may contribute to the Warburg effect.10 --13 In addition, mitochondria. The cytosolic pool of cAMP, produced by the cancer cells mitochondria have been reported to be associated plasma membrane adenylyl cyclase, enhances the functional with mitochondrial DNA mutations,14,15 altered expression capacity of complex I (NADH-ubiquinone oxidoreductase) of the of mitochondrial enzymes,16 reduced levels and activity of respiratory chain and reduces reactive oxygen species (ROS) 37,50 mitochondrial OXPHOS complexes17 --19 and deregulation of production through activation of PKA. mitochondrial fusion and fission.20 In recent years, an in vitro model of cellular transformation In various human tumors (up to 35%), the oncogenic ras gene has been developed. It is represented by two NIH3T3 derived has been found mutated21,22 and these mutations have a critical stable cell lines, NIH3T3 cells expressing an oncogenic K-ras role in the onset of different malignant phenotypes. The ras genes (transformed cell line) and NIH-ras cells expressing a GEF-DN, a encode for guanidine nucleotide binding proteins that mediate protein able to downregulate ras activation and to phenotipically 13,51 cellular signal transduction pathways controlling cell growth and revert transformed cells (reverted cell line). K-ras cell line has differentiation.21,22 Although active form of the ras family been extensively characterized and found to have several members, such as K-ras, have been related to enhancement of metabolic alterations.13,52,53 It has been shown that the enhanced intracellular level of cyclic adenosine monophosphate (cAMP), proliferation potential of K-ras-transformed cells requires high through activation of the plasma membrane adenylyl cyclase,23 --25 initial glucose and glutamine concentrations in the medium. 1Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy; 2Department of Medical Biochemistry, Biology and Physics (DIBIFIM), University of Bari, Bari, Italy and 3Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle Ricerche, Bari, Italy. Correspondence: Dr F Chiaradonna or Professor L Alberghina, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan (MI), Italy. E-mails: [email protected] or [email protected] Received 30 July 2011; revised 27 December 2011; accepted 19 January 2012; published online 12 March 2012 PKA regulation of K-ras cancer cell mitochondria R Palorini et al 353 The selective growth advantage of transformed cells is lost in normal cells that was significantly smaller at 1 mM glucose. Thus, sub-optimal glucose or glutamine growth conditions. Glucose and these data show that in transformed cells the mitochondrial glutamine-dependent growth correlates with an altered metabolic oxidative phosphorylation contributes only marginally to cellular pattern namely increased glucose utilization and lactate produc- ATP production, which is essentially contributed by glycolysis.13 tion, increased utilization of glutamine through the tricarboxylic acid cycle, increased expression of glycolytic genes, depressed cAMP/PKA pathway alterations in transformed fibroblasts expression of mitochondrial genes, altered mitochondrial mor- phology and reduced ability of ATP production.13,52,54 Trans- Direct determination of basal endogenous levels of cAMP along a formed cells produce large amount of ROS associated with time course of 72 h in cells grown at 25 and 1 mM glucose decreased activity of mitochondrial complex I55 and increased cell (Figures 2a and b) showed a time-dependent increase in cAMP death.13 Remarkably, several metabolic phenotypes induced by levels in both cell lines. In transformed cells, at both 48 and 72 h, oncogenic K-ras expression are almost completely reverted by the cAMP levels were higher than in normal cells (Figures 2a and b). GEF-DN expression52 --54 suggesting a main role of oncogenic ras The addition of FSK (an activator of the adenylyl cyclases) signaling in the induction and maintenance of metabolic increased, as expected, the level of cAMP, but such an increase alterations. was much larger in transformed cells as compared with normal This study was aimed at examining alterations of the cAMP/PKA ones (Figures 2a and b). pathway in K-ras-transformed murine NIH3T3 fibroblasts and Kinetic analysis of the PKA activity in cell extracts showed in human breast cancer MDA-MB-231 cells and their impact on cell transformed cells a constitutive (time 0 of the growth curve) growth and energy metabolism. depressed activity of the enzyme as compared with normal cells, The results show that in both cell lines there is an alteration of which slightly increased in 48 h growth and was greatly enhanced the cAMP/PKA system, characterized by enhanced excitability of the by FSK addition to the cultivation medium (Figure 2d). In normal plasma membrane adenylyl cyclase but a reduced PKA activity. This cells, the PKA activity decreased significantly during cell growth alteration of the cAMP/PKA pathway in oncogenic K-ras cells was and was further depressed by FSK (Figure 2c). Direct immuno- associated with a shift of mitochondrial dynamics to a more chemical analysis revealed an increase in the cellular content of fragmented structure, increased apoptosis under glucose depriva- the catalytic subunit of PKA in 48 h of growth of normal and tion, deficiency of complex I activity and ATP production and transformed cells, which in both type of cells was prevented by increase in intracellular ROS levels. Forskolin (FSK), a known FSK addition (Figures 2e and f). These findings indicate that the activator of adenylyl cyclase, leading to an increase in the cytosolic changes in the functional capacity of PKA, rather than being due level of cAMP and hence to PKA activation, induced