If tumours are to survive and grow, cancer cells need to adapt to environments high in metabolic stress and to avoid programmed cell death (apoptosis).
The molecular mechanisms through which cell death and survival are regulated under these conditions are not yet well understood.
However, a signalling pathway involving two kinases – the liver kinase B1 (LKB1) and the AMP-activated protein kinase (AMPK) is known to be involved in the adaptation of cells to energy stress.
The loss of function of either of these proteins makes cells more sensitive to apoptosis in low glucose environments, and cells deficient in either AMPK or LKB1 have been shown to be resistant to oncogenic transformation.
Nissam Hay from the University of Illinois College of Medicine, Chicago, Illinois, USA and his co-workers Sang-Min Jeon and Navdeep Chandel have now investigated the mechanism through which these proteins regulate the balance between cancer cell growth and apoptosis under energy stress.
The researchers incubated LKB1-deficient cancer cells in the presence of either glucose or one of the non-metabolizable glucose analogues 2-deoxyglucose (2DG) and 5-thioglucose (5TG). They found that 2DG, but not 5TG, induced the activation of AMPK and protected the cells from apoptosis, even in cells that were deficient in LKB1, and showed this to occur through a hexokinase-dependent mechanism.
The researchers speculated that oxidative stress might be increased in glucose-deficient environments through decreasing the production of NADPH, which is required for the elimination of hydrogen peroxide (H2O2), and that AMPK acts in this pathway to decrease this oxidative stress.
They tested this hypothesis by, firstly, showing that glucose deprivation depleted NADPH levels, increased H2O2 levels and increased cell death, and that this was accelerated in cells deficient in the enzyme glucose-6-phosphate dehydrogenase. Anti-oxidants were also found to inhibit cell death in cells deficient in either AMPK or LKB1.
After establishing that AMPK protects cells from apoptosis through redox regulation, Hay and his co-workers set out to establish its mechanism of action. They found that knockdown or knockout of either LKB1 or AMPK in cancer cells significantly increased levels of H2O2 but not of peroxide (O2-) during glucose depletion. The glucose analogue 2DG was able to activate AMPK and maintain high levels of NADPH and low levels of H2O2 in these cells.
The nucleotide coenzyme NADPH is generated in the pentose phosphate pathway and mitochondrial metabolism, and consumed in H2O2 elimination and fatty acid synthesis. If glucose is limited mitochondrial metabolism becomes the major source of NADPH, supported by fatty acid oxidation. AMPK is known to be a regulator of fatty acid metabolism through inhibition of two acetyl-CoA carboxylases, ACC1 and ACC2.
Inhibition of these enzymes inhibits fatty acid synthesis and activates fatty acid oxidation respectively, thus maintaining or increasing NADPH levels. Hay and co-workers used short interfering RNAs (siRNAs) to knock down levels of both ACC1 and ACC2 in A549 cancer cells and found that only ACC2 knockdown significantly increased peroxide accumulation and apoptosis.
However, knockdown of either carboxylase was found to have the same effect in some other cancer cell lines, Over-expression of mutant ACC1 and ACC2 in cell lines proficient in LKB1 was found to increase H2O2 and apoptosis, implying that the inhibition of these carboxylases by AMPK is necessary for redox regulation and the promotion of tumour growth in glucose-deficient conditions.
Cancer cells are often detached from their matrix and starved of glucose during the early stages of solid tumour development. Hay and co-workers tested the effect of AMPK and ACC on tumour formation and found that AMPK activation promoted tumour growth while ACC activation restricted it.
Taken together, these results suggest that AMPK acts to promote early tumour growth and prevent apoptosis in conditions of energy stress through inhibiting acetyl-CoA carboxylase activity, thus maintaining NADPH levels and preventing the build-up of peroxide.
Jeon, S-M., Chandel, N.S. and Hay, N. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature, published online ahead of print 10 May 2012. doi:10.1038/nature11066
(13 May 2013)
(13 May 2013)
(13 May 2013)