David R. Plas

Akt stimulates aerobic glycolysis in cancer cells.

Cancer cells frequently display high rates of aerobic glycolysis in comparison to their nontransformed counterparts, although the molecular basis of this phenomenon remains poorly understood. Constitutive activity of the serine/threonine kinase Akt is a common perturbation observed in malignant cells. Surprisingly, although Akt activity is sufficient to promote leukemogenesis in nontransformed hematopoietic precursors and maintenance of Akt activity was required for rapid disease progression, the expression of activated Akt did not increase the proliferation of the premalignant or malignant cells in culture. However, Akt stimulated glucose consumption in transformed cells without affecting the rate of oxidative phosphorylation. High rates of aerobic glycolysis were also identified in human glioblastoma cells possessing but not those lacking constitutive Akt activity. Akt-expressing cells were more susceptible than control cells to death after glucose withdrawal. These data suggest that activation of the Akt oncogene is sufficient to stimulate the switch to aerobic glycolysis characteristic of cancer cells and that Akt activity renders cancer cells dependent on aerobic glycolysis for continued growth and survival.

Akt-Directed Glucose Metabolism Can Prevent Bax Conformation Change and Promote Growth Factor-Independent Survival

ABSTRACT The serine/threonine kinase Akt is a component of many receptor signal transduction pathways and can prevent cell death following growth factor withdrawal. Here, we show that Akt inhibition of cell death is not dependent on new protein translation. Instead, Akt inhibition of cell death requires glucose hydrolysis through glycolysis. Akt was found to regulate multiple steps in glycolysis via posttranscriptional mechanisms that included localization of the glucose transporter, Glut1, to the cell surface and maintenance of hexokinase function in the absence of extrinsic factors. To test the role of glucose uptake and phosphorylation in growth factor-independent survival, cells were transfected with Glut1 and hexokinase 1 (Glut1/HK1) cells. Glut1/HK1 cells accumulated Glut1 on the cell surface and had high glucose uptake capacity similar to that of cells with constitutively active Akt (mAkt). Unlike mAkt-expressing cells, however, they did not consume more glucose, did not maintain prolonged phosphofructokinase-1 protein levels and activity, and did not maintain pentose phosphate shuttle activity in the absence of growth factor. Nevertheless, expression of Glut1 and HK1 promoted increased cytosolic NADH and NADPH levels relative to those of the control cells upon growth factor withdrawal, prevented activation of Bax, and promoted growth factor-independent survival. These data indicate that Bax conformation is sensitive to glucose metabolism and that maintaining glucose uptake and phosphorylation can promote cell survival in the absence of growth factor. Furthermore, Akt required glucose and the ability to perform glycolysis to prevent Bax activation. The prevention of Bax activation by posttranscriptional regulation of glucose metabolism may, therefore, be a required aspect of the ability of Akt to maintain long-term cell survival in the absence of growth factors.

Growth Factors Can Influence Cell Growth and Survival through Effects on Glucose Metabolism

ABSTRACT Cells from multicellular organisms are dependent upon exogenous signals for survival, growth, and proliferation. The relationship among these three processes was examined using an interleukin-3 (IL-3)-dependent cell line. No fixed dose of IL-3 determined the threshold below which cells underwent apoptosis. Instead, increasing growth factor concentrations resulted in progressive shortening of the G1 phase of the cell cycle and more rapid proliferative expansion. Increased growth factor concentrations also resulted in proportional increases in glycolytic rates. Paradoxically, cells growing in high concentrations of growth factor had an increased susceptibility to cell death upon growth factor withdrawal. This susceptibility correlated with the magnitude of the change in the glycolytic rate following growth factor withdrawal. To investigate whether changes in the availability of glycolytic products influence mitochondrion-initiated apoptosis, we artificially limited glycolysis by manipulating the glucose levels in the medium. Like growth factor withdrawal, glucose limitation resulted in Bax translocation, a decrease in mitochondrial membrane potential, and cytochromec redistribution to the cytosol. In contrast, increasing cell autonomous glucose uptake by overexpression of Glut1 significantly delayed apoptosis following growth factor withdrawal. These data suggest that a primary function of growth factors is to regulate glucose uptake and metabolism and thus maintain mitochondrial homeostasis and enable anabolic pathways required for cell growth. Consistent with this hypothesis, expression of the three genes involved in glucose uptake and glycolytic commitment, those for Glut1, hexokinase 2, and phosphofructokinase 1, was found to rapidly decline to nearly undetectable levels following growth factor withdrawal.

Akt-dependent transformation: there is more to growth than just surviving

Activation of the Akt/PKB protein kinase family triggers increases in cell size, metabolism and survival. Akt coordinately regulates these fundamental cellular processes through phosphorylation-dependent inactivation of tumor suppressors and activation of trophic signaling. Akt signaling stimulates transport and metabolism of both glucose and amino acids, which in turn support mTOR-dependent increases in protein translation. In addition, Akt activation directs cells to undertake a metabolic conversion from oxidative phosphorylation to aerobic glycolysis. Although this conversion promotes cell growth, it also renders cell survival dependent on a continuous supply of extracellular nutrients, which themselves are required regulatory elements in Akt signal transduction.

Akt Activation Promotes Degradation of Tuberin and FOXO3a via the Proteasome

Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Here we report that Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggered proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilized the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggered the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes.

Direct Regulation of ZAP-70 by SHP-1 in T Cell Antigen Receptor Signaling

The threshold at which antigen triggers lymphocyte activation is set by the enzymes that regulate tyrosine phosphorylation. Upon T cell activation, the protein tyrosine phosphatase SHP-1 was found to bind to the protein tyrosine kinase ZAP-70. This interaction resulted in an increase in SHP-1 phosphatase activity and a decrease in ZAP-70 kinase activity. Expression of a dominant negative mutant of SHP-1 in T cells increased the sensitivity of the antigen receptor. Thus, SHP-1 functions as a negative regulator of the T cell antigen receptor and in setting the threshold of activation.

Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology

A comparison of Akt- and Bcl-xL-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic geneBcl-x L . Although both genes prevent cell death, Akt-protected cells can be distinguished fromBcl-x L -protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-xL-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-xL suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-xL maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells.

Cell metabolism in the regulation of programmed cell death

Increased cellular metabolism and resistance to apoptosis are two hallmarks of cell transformation. Recent progress in the understanding of the role of mitochondria in controlling apoptosis has brought attention to the links between elements of the apoptotic machinery and cellular metabolism. Here, we review the coordinated effects of growth factor withdrawal on bioenergetics and programmed cell death, and discuss the metabolic consequences of genes that prevent apoptosis, including the BCL2 family of genes and AKT.

Phosphatidylinositol 3-Kinase/Akt Signaling Is Neither Required for Hypoxic Stabilization of HIF-1α nor Sufficient for HIF-1-dependent Target Gene Transcription

The serine/threonine kinase Akt/PKB and the oxygen-responsive transcription factor HIF-1 share the ability to induce such processes as angiogenesis, glucose uptake, and glycolysis. Akt activity and HIF-1 are both essential for development and implicated in tumor growth. Upon activation by products of phosphatidylinositol 3-kinase (PI3K), Akt phosphorylates downstream targets that stimulate growth and inhibit apoptosis. Previous reports suggest that Akt may achieve its effects on angiogenesis and glucose metabolism by stimulating HIF-1 activity. We report here that, whereas serum stimulation can induce a slight accumulation of HIF-1α protein in a PI3K/Akt pathway-dependent fashion, hypoxia induces much higher levels of HIF-1α protein and HIF-1 DNA binding activity independently of PI3K and mTOR activity. In addition, we find the effects of constitutively active Akt on HIF-1 activity are cell-type specific. High levels of Akt signaling can modestly increase HIF-1α protein, but this increase does not affect HIF-1 target gene expression. Therefore, the PI3K/Akt pathway is not necessary for hypoxic induction of HIF-1 subunits or activity, and constitutively active Akt is not itself sufficient to induce HIF-1 activity.

Homeostatic control of lymphocyte survival: potential origins and implications.

Lymphocytes depend on extracellular ligands to maintain their viability. Structurally diverse lymphocyte receptors transmit survival signals through separate signal transduction cascades, which all share the ability to sustain viability by maintaining the sequestration of apoptogenic factors within mitochondria. Receptors can induce cellular survival either by promoting the expression and/or function of anti-apoptotic Bcl-2 family proteins or by activating the phosphatidylinositol-3 kinase–Akt pathway. Either of these events represses the function of the pro-apoptotic proteins Bax and Bak, which are required for mitochondrial release of cytochrome c. As we discuss here, the apparently redundant functions of Bax and Bak may have evolved to prevent lymphocyte mitochondria from adapting to loss of receptor–mediated signal transduction and thus keep lymphocytes from accumulating in a cell-autonomous manner.