Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.

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.

The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53

Activation of the oncogenic kinase Akt stimulates glucose uptake and metabolism in cancer cells and renders these cells susceptible to death in response to glucose withdrawal. Here we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reverses the sensitivity of Akt-expressing glioblastoma cells to glucose deprivation. AICAR's protection depends on the activation of AMPK, as expression of a dominant-negative form of AMPK abolished this effect. AMPK is a cellular energy sensor whose activation can both block anabolic pathways such as protein synthesis and activate catabolic reactions such as fatty acid oxidation to maintain cellular bioenergetics. While rapamycin treatment mimicked the effect of AICAR on inhibiting markers of cap-dependent translation, it failed to protect Akt-expressing cells from death upon glucose withdrawal. Compared to control cells, Akt-expressing cells were impaired in the ability to induce fatty acid oxidation in response to glucose deprivation unless stimulated with AICAR. Stimulation of fatty acid oxidation was sufficient to maintain cell survival as activation of fatty acid oxidation with bezafibrate also protected Akt-expressing cells from glucose withdrawal-induced death. Conversely, treatment with a CPT-1 inhibitor to block fatty acid import into mitochondria prevented AICAR from stimulating fatty acid oxidation and promoting cell survival in the absence of glucose. Finally, cell survival did not require reversal of Akt's effects on either protein translation or lipid synthesis as the addition of the cell penetrant oxidizable substrate methyl-pyruvate was sufficient to maintain survival of Akt-expressing cells deprived of glucose. Together, these data suggest that activation of Akt blocks the ability of cancer cells to metabolize nonglycolytic bioenergetic substrates, leading to glucose addiction.

p53-dependent apoptosis is a major determinant of its tumor suppressor activity and can be triggered by hypoxia. No p53 target is known to be induced by p53 or to mediate p53-dependent apoptosis during hypoxia. We report that p53 can directly upregulate expression of Bnip3L, a cell death inducer. During hypoxia, Bnip3L is highly induced in wild-type p53-expressing cells, in part due to increased recruitment of p53 and CBP to Bnip3L. Apoptosis is reduced in hypoxia-exposed cells with functional p53 following Bnip3L knockdown. In vivo, Bnip3L knockdown promotes tumorigenicity of wild-type versus mutant p53-expressing tumors. Thus, Bnip3L, capable of attenuating tumorigenicity, mediates p53-dependent apoptosis under hypoxia, which provides a novel understanding of p53 in tumor suppression.

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.

Soluble mitogens and adhesion-dependent organization of the actin cytoskeleton are required for cells to enter S phase in fibroblasts. The induction of cyclin A is also required for S-phase entry, and we now report that distinct effects of mitogens and the actin cytoskeleton on the phosphorylation of CREB and pocket proteins regulate the extent and timing of cyclin A promoter activity, respectively. First, we show that CREB phosphorylation and binding to the cyclic AMP response element (CRE) determines the extent, but not the timing, of cyclin A promoter activity. Second, we show that pocket protein inactivation regulates the timing, but not the extent, of cyclin A promoter activity. CREB phosphorylation and CRE occupancy are regulated by soluble mitogens alone, while the phosphorylation of pocket proteins requires both mitogens and the organized actin cytoskeleton. Mechanistically, cytoskeletal integrity controls pocket protein phosphorylation by allowing for sustained ERK signaling and, thereby, the expression of cyclin D1. Our results lead to a model of cyclin A gene regulation in which mitogens play a permissive role by stimulating early G 1 -phase phosphorylation of CREB and a distinct regulatory role by cooperating with the organized actin cytoskeleton to regulate the duration of ERK signaling, the expression of cyclin D1, and the timing of pocket protein phosphorylation.The proliferation of most cell types requires exposure to mitogenic growth factors, adhesion to the extracellular matrix and organization of the actin cytoskeleton. Growth factors, cell adhesion, and the organized actin cytoskeleton are all specifically required for cell cycle progression through the G 1 phase (2). Organization of the actin cytoskeleton is a consequence of integrin-mediated adhesion, but cell adhesion and the organized cytoskeleton have distinguishable effects on G 1 -phase progression (14,17). While many studies have focused on differences in signaling and cell cycle progression in adherent and nonadherent cells, the cell cycle consequence of disrupting the actin cytoskeleton in adherent cells has received much less attention.Cell cycle progression is mediated by the cyclin-dependent kinases (cdk's) with the formation of active cyclin D-cdk4 (or -cdk6) and cyclin E-cdk2 complexes controlling progression through G 1 phase (35). In fibroblasts, the activation of cyclin D1-cdk4 or -cdk6 is typically limited by the mid-G 1 -phase expression of cyclin D1, while the activation of cyclin E-cdk2 is controlled by the mid-to-late G 1 -phase downregulation of p21 family cdk inhibitors (typically p21 cip1 and p27 kip1 ). The G 1 -phase cyclin-cdk's are required for phosphorylation of the retinoblastoma protein (pRb) and p107 (referred to as pocket proteins); this phosphorylation results in the disruption of E2F-pocket protein complexes and the induction of pRb-regulated genes such as cyclin A (15, 40). We and others have shown that the expression of cyclin D1 and the downregulation of p21 cip1 and p27 kip1 are jointly dependent upon ...

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3–23.3 (n=1), 9q33.1–34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31–36.33 (n=6), 17q21.2–q21.31 (n=5) and 17q25.1–25.3 (n=5) and deletions affecting 18p11.31–11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a ‘HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal.