Mitochondrial respiration malfunction and increased glycolysis are frequently observed in cancer cells. This metabolic alteration, known as the Warburg effect, is caused by complex biochemical and molecular mechanisms. mtDNA mutations and tissue hypoxia represent genetic and environmental factors contributing to the Warburg effect. Cells deficient in respiration because of mtDNA alterations or hypoxic conditions are forced to produce ATP through glycolysis, which is much less efficient than oxidative phosphorylation. Nevertheless, cancer cells manage to overcome such an apparent metabolic disadvantage, survive in vivo, and eventually emerge as a malignant cell population resistant to anticancer agents at the late stages of the disease progression. Thus, understanding the mechanisms underlying the increased survival capacity in cancer cells with compromised mitochondrial respiratory function is an important research area.
Our study suggests that mitochondrial respiration deficiency leads to activation of the Akt survival pathway through NADH-mediated inactivation of PTEN. This is a novel mechanism contributing to increased survival and drug resistance in cancer cells with compromised mitochondrial respiration. Several lines of evidences support this conclusion, as follows: (a) Cells that lack mitochondrial respiration because of mtDNA deletion, chemical inhibition of the electron transport chain, or exposure to hypoxia all exhibited significant Akt activation. (b) The cellular NADH/NADPH ratio abnormally increased when mitochondrial respiration was suppressed, and this was associated with a decrease in plasma membrane–associated PTEN. (c) Exogenous NADH led to inactivation of PTEN and activation of Akt in vitro. The inactivation of PTEN seems to be caused by redox modulation because NADH competed with NADPH/Trx to keep PTEN in an oxidized (inactive) state. These findings are consistent with previous studies showing inactivation of PTEN by oxidation using hydrogen peroxide (Lee et al., 2002
; Kwon et al., 2004
). (d) Cells lacking functional PTEN did not respond to respiratory inhibition or hypoxia, and exhibited no further Akt activation, indicating the important role of PTEN in this process.
Under physiological conditions, NADH is generated through glycolysis and the tricarboxylic acid cycle, whereas NADPH is produced mainly via the PPP (shunt). The proportion of glucose directed to each pathway is regulated by the cellular energy metabolic state. Mitochondrial defects render cancer cells dependent on glycolysis for ATP supply, and the NADH generated from the tricarboxylic acid cycle is not used effectively because of the decrease in oxidative phosphorylation. These metabolic alterations lead to an accumulation of NADH. At the same time, NADPH production from the PPP decreases because of increased utilization of glucose for glycolysis. Indeed, we consistently observed that the NADH/NADPH ratio was significantly increased in all eight clones of ρ- cells (Fig. S4). Because NADH competes with NADPH and compromises the ability of NADPH/Trx to keep PTEN in a reduced state, the metabolic changes in cancer cells with mitochondrial defects would lead to inactivation of PTEN and activation of Akt. Interestingly, NAC suppressed Akt activation induced by H2O2, but did not decrease rotenone-induced Akt phosphorylation. The likely explanation is that the redox-sensitive PTEN was inactivated when the ratio of NADH/NADPH was significantly increased. This elevated ratio could not be modulated by NAC when cells were treated with rotenone. In contrast, the antioxidant NAC effectively decreased H2O2 and reduced its direct effect on PTEN.
The PI3K–Akt pathway is critical for cell survival (Cantley, 2002
; Vivanco et al., 2002
). Activation of PI3K results in generation of PIP3
, which leads to activation of phosphoinositide-dependent kinase-1 (PDK-1) and phosphorylation of Akt. In contrast, the lipid phosphatase PTEN removes a phosphate from PIP3
, and thus acts as a negative regulator of Akt. Loss of PTEN leads to Akt activation in cancer cells (Wu et al., 1998
). Thus, it is likely that oxidation of PTEN suppresses its phosphatase activity and subsequently leads to Akt activation. Indeed, PTEN is sensitive to oxidative inactivation by H2
(Connor et al., 2005
). The demonstration that respiration defects lead to activation of the Akt pathway caused by the accumulation of NADH and inactivation of PTEN reveals a novel mechanism by which cancer cells survive under respiration-compromised conditions. illustrates a model of this cell survival mechanism.
Figure 9. Schematic illustration of the mechanisms by which Akt is activated in cancer cells with mitochondrial respiration injury. Genetic factors (mtDNA mutations/deletions) and environmental factors (hypoxic conditions) cause defects in mitochondrial respiration (more ...)
The degree of Akt activation among the ρ- clones appeared somewhat heterogeneous. It is possible that during the process of establishing the ρ- clones, the use of ethidium bromide to deplete mtDNA might also cause nuclear DNA mutations, which might affect PTEN function and/or Akt activation. This could also explain the heterogeneous colony formation efficiencies observed among the ρ- clones. Although this heterogeneity reflects the complexity of the experimental systems, the conclusion that mitochondrial respiration defects lead to NADH-mediated PTEN inactivation and Akt activation remains valid. This argument is supported by the observations that Akt activation was observed in all eight ρ- clones, in cells treated with respiratory chain inhibitor rotenone, and in cells under hypoxia in a PTEN-dependent manner.
Because mitochondrial DNA mutations and hypoxia with subsequently increased glycolysis are prevalent in cancer cells (Polyak et al., 1998
; Wallace, 1999
; Fliss et al., 2000
; Copeland et al., 2002
; Gatenby and Gillies, 2004
), activation of the Akt pathway through NADH-mediated PTEN inactivation is likely an important survival mechanism for cancer cells with such metabolic alterations. Additionally, the ability of Akt to promote glucose uptake may also contribute to cell survival (Rathmell et al., 2003
). Interestingly, a recent study showed that Akt activation stimulates cells to use the glycolytic pathway to generate ATP (Elstrom et al., 2004
). The observations that hypoxia caused Akt activation in both HCT116 and LN-229 cells and that respiratory-deficient cells exhibited certain growth advantage in hypoxia conditions further illustrate the clinical relevance of this mechanism in cancer cell survival and growth in vivo. Furthermore, if Akt activation is an important mechanism contributing to decreased drug sensitivity associated with the Warburg effect, it is possible to overcome such drug resistance by inhibition of Akt activation. In fact, our data suggest that this is possible. Further investigation is warranted to evaluate the clinical implications of this therapeutic strategy.