Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype.

Cellular senescence permanently arrests cell proliferation, often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). Loss of mitochondrial function can drive age-related declines in the function of many post-mitotic tissues, but little is known about how mitochondrial dysfunction affects mitotic tissues. We show here that several manipulations that compromise mitochondrial function in proliferating human cells induce a senescence growth arrest with a modified SASP that lacks the IL-1-dependent inflammatory arm. Cells that underwent mitochondrial dysfunction-associated senescence (MiDAS) had lower NAD+/NADH ratios, which caused both the growth arrest and prevented the IL-1-associated SASP through AMPK-mediated p53 activation. Progeroid mice that rapidly accrue mtDNA mutations accumulated senescent cells with a MiDAS SASP in vivo, which suppressed adipogenesis and stimulated keratinocyte differentiation in cell culture. Our data identify a distinct senescence response and provide a mechanism by which mitochondrial dysfunction can drive aging phenotypes.

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title: Mitochondrial Dysfunction Contributes to Oncogene-Induced Senescence
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title: Feedback between p21 and reactive oxygen production is necessary for cell senescence
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title: Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging
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title: Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice.
abstract: Type 2 diabetes (T2D) has become epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD(+) biosynthesis, and the NAD(+)-dependent protein deacetylase SIRT1. Here, we show that NAMPT-mediated NAD(+) biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD(+) intermediate, ameliorates glucose intolerance by restoring NAD(+) levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD(+) and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a potential nutriceutical intervention against diet- and age-induced T2D.

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abstract: Seven mammalian sirtuins are nicotinamide adenine dinucleotide (NAD)(+)-dependent deacetylases and are important modulators of energy metabolism and stress resistance. Two new studies by Du et al. (2011) and Peng et al. (2011) identify a new enzymatic activity for SIRT5, expanding the cellular repertoire of posttranslational modifications targeted by the sirtuins.

title: Sirt6 regulates TNFα secretion via hydrolysis of long chain fatty acyl lysine
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title: Mammalian sirtuins: biological insights and disease relevance.
abstract: Aging is accompanied by a decline in the healthy function of multiple organ systems, leading to increased incidence and mortality from diseases such as type II diabetes mellitus, neurodegenerative diseases, cancer, and cardiovascular disease. Historically, researchers have focused on investigating individual pathways in isolated organs as a strategy to identify the root cause of a disease, with hopes of designing better drugs. Studies of aging in yeast led to the discovery of a family of conserved enzymes known as the sirtuins, which affect multiple pathways that increase the life span and the overall health of organisms. Since the discovery of the first known mammalian sirtuin, SIRT1, 10 years ago, there have been major advances in our understanding of the enzymology of sirtuins, their regulation, and their ability to broadly improve mammalian physiology and health span. This review summarizes and discusses the advances of the past decade and the challenges that will confront the field in the coming years.

title: p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes
abstract: In addition to allowing immune cells to signal tissue damage, HMBG1 is secreted by senescent cells to initiate inflammatory cytokine secretion.

title: Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice
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abstract: SIRT1 regulates a variety of cellular functions, including cellular stress responses and energy metabolism. SIRT1 activity is negatively regulated by DBC1 (Deleted in Breast Cancer 1) through direct binding. However, how the DBC1-SIRT1 interaction is regulated remains unclear. We found that the DBC1-SIRT1 interaction increases following DNA damage and oxidative stress. The stress-induced DBC1-SIRT1 interaction requires the ATM-dependent phosphorylation of DBC1 at Thr 454, which creates a second binding site for SIRT1. Finally, we showed that the stress-induced DBC1-SIRT1 interaction is important for cell fate determination following genotoxic stress. These results revealed a novel mechanism of SIRT1 regulation during genotoxic stress.

title: A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen
abstract: Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that “senescent” mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-α. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

title: p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype
abstract: Cellular senescence suppresses cancer by forcing potentially oncogenic cells into a permanent cell cycle arrest. Senescent cells also secrete growth factors, proteases, and inflammatory cytokines, termed the senescence‐associated secretory phenotype (SASP). Much is known about pathways that regulate the senescence growth arrest, but far less is known about pathways that regulate the SASP. We previously showed that DNA damage response (DDR) signalling is essential, but not sufficient, for the SASP, which is restrained by p53. Here, we delineate another crucial SASP regulatory pathway and its relationship to the DDR and p53. We show that diverse senescence‐inducing stimuli activate the stress‐inducible kinase p38MAPK in normal human fibroblasts. p38MAPK inhibition markedly reduced the secretion of most SASP factors, constitutive p38MAPK activation was sufficient to induce an SASP, and p53 restrained p38MAPK activation. Further, p38MAPK regulated the SASP independently of the canonical DDR. Mechanistically, p38MAPK induced the SASP largely by increasing NF‐κB transcriptional activity. These findings assign p38MAPK a novel role in SASP regulation—one that is necessary, sufficient, and independent of previously described pathways.

title: Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent
abstract: Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.

title: Cell surface-bound IL-1α is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network
abstract: Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1α is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1α mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1α antibodies, and IL-1α depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1α neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1α depletion reduced the DNA binding activity of NF-κB and C/EBPβ, which stimulate IL-6/IL-8 transcription. IL-1α was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1α blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1α-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1α regulates the biological effects of these cytokines. Thus, cell surface IL-1α is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

title: Muscle or liver-specific Sirt3 deficiency induces hyperacetylation of mitochondrial proteins without affecting global metabolic homeostasis
abstract: Sirt3 is a mitochondrial sirtuin, predominantly expressed in highly metabolic tissues. Germline ablation of Sirt3 has major metabolic consequences, including increased susceptibility to metabolic damage and oxidative stress after high fat feeding. In order to determine the contribution of liver and skeletal muscle to these phenotypes, we generated muscle-specific Sirt3 (Sirt3skm−/−) and liver-specific Sirt3 (Sirt3hep−/−) knock-out mice. Despite a marked global hyperacetylation of mitochondrial proteins, Sirt3skm−/− and Sirt3hep−/− mice did not manifest any overt metabolic phenotype under either chow or high fat diet conditions. Similarly, there was no evidence for increased oxidative stress in muscle or liver when Sirt3 was ablated in a tissue-specific manner. These observations suggest that the mitochondrial hyperacetylation induced by Sirt3-deletion in a tissue specific manner is not necessarily linked to mitochondrial dysfunction and does not recapitulate the metabolic abnormalities observed in the germline Sirt3 knock-out mice.

title: Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells.
abstract: Ethidium bromide (EB), at concentrations of 0.1 to 5 μg/ml, was found to inhibit cell growth and mitochondrial DNA synthesis of cultured L cells (mouse) and BHK cells (hamster), as indicated by cell counts and the incorporation of 3 H-thymidine per μg purified DNA. In contrast, nuclear DNA synthesis of L cells, BHK and polyoma virus transformed BHK cells was actually stimulated to up to 250% of control values by treatment of cells with 0.1 to 2 μg/ml EB for 1 to 2 days. Double-labeling experiments were performed to follow the differential synthesis of mitochondrial and nuclear DNA synthesis and the decay of radioactivity in pre-existing DNA during up to 4 days of treatment with EB. These results and analytical determinations of mitochondrial DNA showed a slightly lower yield of mitochondrial DNA after 4 days of treatment with EB than in controls. The content of protein in mitochondrial fractions was increased to up to 190% of control values. In all three cell lines EB treatment led to a structural alteration of covalently closed mitochondrial DNA, consisting in part of a change to an increased degree of supercoiling and in addition breakage of circular DNA without re-closing. This change involved the preexisting as well as the traces of newly synthesized DNA. These changes were reversible by subsequent growth of cells in EB-free medium.

title: Involvement of Mortalin in Cellular Senescence from the Perspective of its Mitochondrial Import, Chaperone, and Oxidative Stress Management Functions
abstract: Mortalin (mtHSP70/GRP75) is a heat uninducible member of hsp70 family of proteins. Some of the established features of mortalin include its various subcellular sites, multiple binding partners, and differential subcellular distribution in normal and immortal cells. Overexpression of mortalin leads to extended life span in nematode and normal human cells. On the other hand, it serves as a major target for oxidation and was shown to be involved in old age pathologies including Parkinson's and Alzheimer's disease. Since mortalin interacts with many proteins, its modifications in response to stress and damage caused by intracellular oxidation are likely to generate pleiotropic effects. For example, (a) inefficient import of mitochondrial proteins by mortalin–Tim complexes may result into inefficient mitochondrial genesis, energy generation, and functional decline and (b) inefficient chaperoning of proteins can result into a garbage catastrophe.

title: Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA.
abstract: We have examined the contribution of the mitochondrial genome to the tumorigenic phenotype expressed by human cell lines derived from an ovarian and a cervical carcinoma and from an osteogenic sarcoma. All these continuous cell lines are anchorage-independent in soft agar and form tumors in athymic nude mice. Long-term exposure of the cells to ethidium bromide, an intercalating agent which inhibits mitochondrial DNA replication, gave rise to subclones depleted of mitochondrial DNA and RNA molecules and displaying either anchorage independence or dependence. These respiratory-deficient subclones contain disorganized and enlarged mitochondria, are auxotrophic for uridine and pyruvate, and grow in vitro at a rate nearly identical or moderately slower than their respective parent. The tumor-forming ability of both anchorage-independent and -dependent cell lines was tested by s.c. and intramuscular implantation of the cells in nude mice. We found that the tumorigenic capacity was influenced by the route of inoculation. Subcutaneously, mitochondrial DNA-less cell lines are either poorly or nontumorigenic, while all but one cell line form tumors when implanted into the hind leg muscle. The relative in vivo growth rate of the parent and the mitochondrial DNA-less subclones reflects their respective in vitro rate of growth. All intramuscular tumors introduced into culture mimic the molecular and phenotypic traits of the injected cells, with the exception of the anchorage-dependent cell lines which give rise to anchorage-independent tumor cell lines. The present observations indicate that human cells without mitochondrial DNA have the capacity to proliferate and form tumors in vivo.

title: Growth of rho 0 human Namalwa cells lacking oxidative phosphorylation can be sustained by redox compounds potassium ferricyanide or coenzyme Q10 putatively acting through the plasma membrane oxidase.
abstract: Pyruvate is conventionally used as a key growth supplement for mammalian rho 0 cells that lack mitochondrial DNA and are thereby devoid of oxidative phosphorylation. We have tested the proposition that cultured rho 0 human cells can be grown using redox compounds other than pyruvate. The results show that potassium ferricyanide and coenzyme Q10 can each be used to replace pyruvate to support the growth of rho 0 Namalwa cells (a lymphoblastoid cell line). Ferricyanide and coenzyme Q10 have both been reported as substrates for a plasma membrane NADH oxidase system which is capable of re-oxidising cytosolic NADH to NAD+. These compounds are also known to stimulate the activity of this enzyme system. We interpret our data to indicate that redox support for growth of rho 0 human cells can be achieved by external electron acceptors such as ferricyanide (a plasma membrane impermeant compound), or coenzyme Q10 (an integral component of the plasma membrane oxidase), through the enhanced conversion of cytosolic NADH to NAD+. This re-oxidation of NADH enables glycolysis to function efficiently as the sole source of cellular ATP, in the absence of mitochondrial oxidative phosphorylation in rho 0 cells. This has important implications for the development of new strategies for the amelioration of the bioenergy decline that occurs in mitochondrial disease and during the human ageing process.

title: AMPK: a nutrient and energy sensor that maintains energy homeostasis
abstract: AMP-activated protein kinase (AMPK) is a crucial cellular energy sensor. Once activated by falling energy status, it promotes ATP production by increasing the activity or expression of proteins involved in catabolism while conserving ATP by switching off biosynthetic pathways. AMPK also regulates metabolic energy balance at the whole-body level. For example, it mediates the effects of agents acting on the hypothalamus that promote feeding and entrains circadian rhythms of metabolism and feeding behaviour. Finally, recent studies reveal that AMPK conserves ATP levels through the regulation of processes other than metabolism, such as the cell cycle and neuronal membrane excitability.

title: HuR Uses AUF1 as a Cofactor To Promote p16INK4 mRNA Decay
abstract: In this study, we show that HuR destabilizes p16INK4 mRNA. Although the knockdown of HuR or AUF1 increased p16 expression, concomitant AUF1 and HuR knockdown had a much weaker effect. The knockdown of Ago2, a component of the RNA-induced silencing complex (RISC), stabilized p16 mRNA. The knockdown of HuR diminished the association of the p16 3′ untranslated region (3′UTR) with AUF1 and vice versa. While the knockdown of HuR or AUF1 reduced the association of Ago2 with the p16 3′UTR, Ago2 knockdown had no influence on HuR or AUF1 binding to the p16 3′UTR. The use of EGFP-p16 chimeric reporter transcripts revealed that p16 mRNA decay depended on a stem-loop structure present in the p16 3′UTR, as HuR and AUF1 destabilized EGFP-derived chimeric transcripts bearing wild-type sequences but not transcripts with mutations in the stem-loop structure. In senescent and HuR-silenced IDH4 human diploid fibroblasts, the EGFP-p16 3′UTR transcript was more stable. Our results suggest that HuR destabilizes p16 mRNA by recruiting the RISC, an effect that depends on the secondary structure of the p16 3′UTR and requires AUF1 as a cofactor.

title: Nutlin-3 inhibits the NFκB Pathway in a p53 Dependent Manner: Implications in Lung Cancer Therapy
abstract: Nutlins were identified as the first potent and specific small molecule Mdm2 antagonists that inhibit the p53- Mdm2 interaction. We show in this study that Nutlin-3 can downregulate TNFα induced activation of the NF-κB reporter in lung cancer cells. Activation of p53 dependent transcription is not compromised when Nutlin-3 is combined with TNFα. Instead, this combination treatment decreases cell viability in a p53 dependent manner. We show that Nutlin-3 strikingly inhibits the protein expression of NF-κB target genes ICAM-1 and MCP-1 while other targets like Bcl-xL and FLIP are not affected, thereby suggesting that the inhibition is promoter specific. This inhibition of ICAM-1 and MCP-1 by Nutlin-3 is again dependent on the p53 status in cells. Furthermore, we show that Nutlin-3 strongly inhibits protein expression of ICAM-1 and MCP-1 induced by IL1, another NF-κB activating stimuli. Nutlin-3 does not inhibit Akt phosphorylation, IκBα phosphorylation, IκBα degradation, p65 modification or p65 DNA binding in the cell lines tested. This study suggests the potential of Nutlin-3 as a bitargeted anti-cancer drug by simultaneously causing p53 activation and NF-κB suppression. It also suggests that Nutlin-3 could be evaluated for treatment of lung cancer as a single agent or in combination therapy by targeting its effect on ICAM-1 and MCP-1 which are known to be critical for cancer cell invasion, thereby downregulating tumor formation and metastasis. This study also suggests biomarkers of response for evaluation of Nutlin-3 in the clinic.

title: Transcriptional cross talk between NF-kappaB and p53.
abstract: Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-kappaB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-kappaB has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF-kappaB inhibit each other's ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF-kappaB subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF-kappaB will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregulating NF-kappaB-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF-kappaB can contribute to tumorigenesis and disease.

title: Mitochondrial DNA mutations and aging: devils in the details?
abstract: Although several lines of evidence support a role for accumulating somatic mitochondrial DNA (mtDNA) mutations in the etiology of aging, it remains unclear if they are a major cause of age-related deterioration and death. Mouse models that harbor elevated mtDNA mutation frequencies age prematurely; these findings were thought to provide conclusive evidence for a causal role of such mutations in aging. Yet, the presence of several conflicting reports has sparked controversy in the field and this is further aggravated by discrepancies in the estimates of mtDNA mutant fractions, which disagree by orders of magnitude. Here, we briefly review the evidence and some of the unresolved questions surrounding a causative role for accumulating mtDNA mutations in aging.

title: SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress.
abstract: The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

title: Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts
abstract: Most mammalian cells do not divide indefinitely, owing to a process termed replicative senescence. In human cells, replicative senescence is caused by telomere shortening, but murine cells senesce despite having long stable telomeres. Here, we show that the phenotypes of senescent human fibroblasts and mouse embryonic fibroblasts (MEFs) differ under standard culture conditions, which include 20% oxygen. MEFs did not senesce in physiological (3%) oxygen levels, but underwent a spontaneous event that allowed indefinite proliferation in 20% oxygen. The proliferation and cytogenetic profiles of DNA repair-deficient MEFs suggested that DNA damage limits MEF proliferation in 20% oxygen. Indeed, MEFs accumulated more DNA damage in 20% oxygen than 3% oxygen, and more damage than human fibroblasts in 20% oxygen. Our results identify oxygen sensitivity as a critical difference between mouse and human cells, explaining their proliferative differences in culture, and possibly their different rates of cancer and ageing.

title: Extension of Murine Life Span by Overexpression of Catalase Targeted to Mitochondria
abstract: To determine the role of reactive oxygen species in mammalian longevity, we generated transgenic mice that overexpress human catalase localized to the peroxisome, the nucleus, or mitochondria (MCAT). Median and maximum life spans were maximally increased (averages of 5 months and 5.5 months, respectively) in MCAT animals. Cardiac pathology and cataract development were delayed, oxidative damage was reduced, H2O2 production and H2O2-induced aconitase inactivation were attenuated, and the development of mitochondrial deletions was reduced. These results support the free radical theory of aging and reinforce the importance of mitochondria as a source of these radicals.

title: INACTIVATION OF AMPK ALTERS GENE EXPRESSION AND PROMOTES GROWTH OF PROSTATE CANCER CELLS
abstract: AMP-activated protein kinase (AMPK) serves as a fuel-sensing enzyme that is activated by binding of AMP and subsequent phophorylation by upstream kinases such as the tumor suppressor LKB1, when cells sense an increase in the ratio of AMP to ATP. Acute activation of AMPK stimulates fatty acid oxidation to generate more ATP and simultaneously inhibits ATP-consuming processes including fatty acid and protein syntheses, thereby preserving energy for acute cell-surviving program, whereas chronic activation leads to inhibition of cell growth. The goal of the present study is to explore the mechanisms by which AMPK regulates cell growth. Toward this end, we established stable cell lines by introducing a dominant-negative mutant of AMPK α1 subunit or its shRNA into the prostate cancer C4-2 cells and other cells, or wild type LKB1 into the lung adenocarcinoma A549 and breast MB-MDA-231 cancer cells, both of which lack functional LKB1. Our results showed that the inhibition of AMPK accelerated cell proliferation and promoted malignant behavior such as increased cell migration and anchorage-independent growth. This was associated with decreased G1 population, downregulation of p53 and p21, and upregulation of S6K, IGF-1 and IGF1R. Conversely, treatment of the C4-2 cells with 5-aminoimidazole-4-carboxamide 1-D-ribonucleoside (AICAR), a prototypical AMPK activator, caused opposite changes. In addition, our study using microarray and RT–PCR revealed that AMPK regulated gene expression involved in tumor cell growth and survival. Thus, our study provides novel insights into the mechanisms of AMPK action in cancer cells and presents AMPK as an ideal drug target for cancer therapy.