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Mitogen-activated kinase activating death domain containing protein (MADD) is abundantly expressed in cancer cells and necessary for maintaining cancer cell survival. However, this survival function of MADD is dependent upon its phosphorylation by protein kinase B (Akt). The tumour suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. The downstream targets of PTEN in triggering apoptosis have not yet been completely identified. Here, we report that MADD can act as a pro-apoptotic factor to initiate TRAIL-induced apoptosis when its phosphorylation is attenuated by PTEN. Our data show that tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) induced a reduction in MADD phosphorylation with a concomitant up-regulation of PTEN. Knock down of PTEN using a specific siRNA prevented TRAIL-induced reduction in pMADD levels. Surprisingly, Akt non-phosphorylated MADD translocated from the plasma membrane to cytoplasm where it bound to 14-3-3 and displaced 14-3-3 associated Bax, which translocated to mitochondria resulting in cytochrome c release. Taken together, our data reveal that PTEN can convey the death signal by preventing MADD phosphorylation by Akt., (© 2013 Wiley Periodicals, Inc.)

Tumor cells accumulate high level of reactive oxygen species (ROS) because they are metabolically more active than normal cells. Elevated ROS levels increase tumorigenecity but also render cancer cells more vulnerable to oxidative stress than normal cells. The oncogenic transcription factor Forkhead Box M1 (FOXM1), which is overexpressed in a wide range of human cancers, was reported to protect cancer cells from the adverse effects of oxidative stress by up regulating the expression of scavenger enzymes. We therefore hypothesized that the combination of FOXM1 ablation and ROS inducers could selectively eradicate cancer cells. We show that RNA interference-mediated knockdown of FOXM1 further elevates intracellular ROS levels and increases sensitivity of cancer cells to ROS-mediated cell death after treatment with ROS inducers. We also demonstrate that the combination of ROS inducers with FOXM1/proteasome inhibitors induces robust apoptosis in different human cancer cells. In addition, we show evidence that FOXM1/proteasome inhibitor bortezomib in combination with the ROS inducer β-phenylethyl isothiocyanate efficiently inhibits the growth of breast tumor xenografts in nude mice. We conclude that the combination of ROS inducers and FOXM1 inhibitors could be used as a therapeutic strategy to selectively eliminate cancer cells., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1alpha (HIF1alpha) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1alpha abundance in these cells is attributed to both an increased level and the sustained translation of HIF1alpha mRNA. Sustained glycogen synthase kinase 3beta inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1alpha abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2(-/)(-) cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2(-/)(-) cells to serum deprivation-induced apoptosis.

Akt deficiency causes resistance to replicative senescence, to oxidative stress- and oncogenic Ras-induced premature senescence, and to reactive oxygen species (ROS)-mediated apoptosis. Akt activation induces premature senescence and sensitizes cells to ROS-mediated apoptosis by increasing intracellular ROS through increased oxygen consumption and by inhibiting the expression of ROS scavengers downstream of FoxO, particularly sestrin 3. This uncovers an Achilles' heel of Akt, since in contrast to its ability to inhibit apoptosis induced by multiple apoptotic stimuli, Akt could not inhibit ROS-mediated apoptosis. Furthermore, treatment with rapamycin that led to further Akt activation and resistance to etoposide hypersensitized cancer cells to ROS-mediated apoptosis. Given that rapamycin alone is mainly cytostatic, this constitutes a strategy for cancer therapy that selectively eradicates cancer cells via Akt activation.

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of leukemic cells in vitro and in vivo, but the mechanisms that mediate such effects are not well understood. We provide evidence that the Akt kinase is phosphorylated/activated during treatment of leukemia cells with As(2)O(3), to regulate downstream engagement of mammalian target of rapamycin (mTOR) and its effectors. Using cells with targeted disruption of both the Akt1 and Akt2 genes, we found that induction of arsenic trioxide-dependent apoptosis is strongly enhanced in the absence of these kinases, suggesting that Akt1/Akt2 are activated in a negative feedback regulatory manner, to control generation of As(2)O(3) responses. Consistent with this, As(2)O(3)-dependent pro-apoptotic effects are enhanced in double knock-out cells for both isoforms of the p70 S6 kinase (S6k1/S6k2), a downstream effector of Akt and mTOR. On the other hand, As(2)O(3)-dependent induction of apoptosis is diminished in cells with targeted disruption of TSC2, a negative upstream effector of mTOR. In studies using primary hematopoietic progenitors from patients with acute myeloid leukemia, we found that pharmacological inhibition of mTOR enhances the suppressive effects of arsenic trioxide on leukemic progenitor colony formation. Moreover, short interfering RNA-mediated inhibition of expression of the negative downstream effector, translational repressor 4E-BP1, partially reverses the effects of As(2)O(3). Altogether, these data provide evidence for a key regulatory role of the Akt/mTOR pathway in the generation of the effects of As(2)O(3), and suggest that targeting this signaling cascade may provide a novel therapeutic approach to enhance the anti-leukemic properties of As(2)O(3).

Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin alpha6beta1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type-specific manner and that integrin alpha6beta1-HSPGs can function as receptors to induce p53-dependent apoptosis.

There is accumulating evidence that cell survival and energy metabolism are inexorably linked. As a major mediator of both the metabolic and anti-apoptotic effects of growth factors, the serine/threonine kinase Akt (also known as protein kinase B or PKB) is particularly well-suited to coordinate the regulation of these interrelated processes. Recent demonstrations that growth factors and Akt require glucose (Glc) to prevent apoptosis and promote cell survival are compatible with this contention, as is a positive correlation between Akt-regulated mitochondrial hexokinase (mtHK) association and apoptotic resistance. From a phylogenetic perspective, the ability of Akt to regulate cellular energy metabolism apparently preceded the capacity to control cell survival, suggesting an evolutionary basis for the Glc dependent anti-apoptotic effects of Akt. We speculate that, somewhere in the course of evolution, the metabolic regulatory function of Akt evolved into an adaptive sensing system involving mtHK that ensures mitochondrial homeostasis, thereby coupling metabolism to cell survival. We also propose that this "guardian" function of mtHK may be specifically exploited for therapeutic purposes.

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.

The serine/threonine kinase Akt/protein kinase B inhibits apoptosis induced by a variety of stimuli, including overexpression or activation of proapoptotic Bcl-2 family members. The precise mechanisms by which Akt prevents apoptosis are not completely understood, but Akt may function to maintain mitochondrial integrity, thereby preventing cytochrome c release following an apoptotic insult. This effect may be mediated, in part, via promotion of physical and functional interactions between mitochondria and hexokinases. Here we show that growth factor deprivation induced proteolytic cleavage of the proapoptotic Bcl-2 family member BID to yield its active truncated form, tBID. Activated Akt inhibited mitochondrial cytochrome c release and apoptosis following BID cleavage. Akt also antagonized tBID-mediated BAX activation and mitochondrial BAK oligomerization, two downstream events thought to be critical for tBID-induced apoptosis. Glucose deprivation, which impaired the ability of Akt to maintain mitochondrion-hexokinase association, prevented Akt from inhibiting BID-mediated apoptosis. Interestingly, tBID independently elicited dissociation of hexokinases from mitochondria, an effect that was antagonized by activated Akt. Ectopic expression of the amino-terminal half of hexokinase II, which is catalytically active and contains the mitochondrion-binding domain, consistently antagonized tBID-induced apoptosis. These results suggest that Akt inhibits BID-mediated apoptosis downstream of BID cleavage via promotion of mitochondrial hexokinase association and antagonism of tBID-mediated BAX and BAK activation at the mitochondria.

In B lymphocytes, induction of apoptosis or programmed cell death (PCD) by Fas (CD95/APO-1) is suppressed by the triggering of CD40. This suppression controls various aspects of the humoral immune response, including antibody affinity maturation. The opposing effects of these receptors are also crucial to B-cell homeostasis, autoimmune disease, and cancer. Cytoprotection by CD40 involves activation of protective genes mediated by NF-kappa B transcription factors; however, its basis remains poorly understood. Here, we report that, in B cells, Gadd45 beta is induced by CD40 through a mechanism that requires NF-kappa B and that this induction suppresses Fas-mediated killing. Importantly, up-regulation of Gadd45 beta by CD40 precedes Fas-induced caspase activation, as well as up-regulation of other NF-kappa B-controlled inhibitors of apoptosis such as Bcl-xL and c-FLIPL. In the presence of Gadd45 beta, the Fas-induced apoptotic cascade is halted at mitochondria. However, in contrast to Bcl-xL, Gadd45 beta is unable to hamper the "intrinsic" pathway for apoptosis and in fact appears to block Fas cytotoxicity herein by suppressing a mitochondria-targeting mechanism activated by this receptor. These findings identify Gadd45 beta as a critical mediator of the prosurvival response to CD40 stimulation and provide important new insights into the apoptotic mechanism that is triggered by Fas in B cells.

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-X(L) prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X(L) to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F(1)F(0)-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-X(L), cells devoid of mitochondrial DNA (rho degrees cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax(-/-) bak(-/-) mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature ofNAC, data interpretationmight not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis. [ABSTRACT FROM AUTHOR]

Akt kinases control essential cellular functions, including proliferation, apoptosis, metabolism and transcription, and have been proposed as promising targets for treatment of angiogenesis-dependent pathologies, such as cancer and ischemic injury. But their precise roles in neovascularization remain elusive. Here we show that Akt1 is the predominant isoform in vascular cells and describe the unexpected consequences of Akt1 knockout on vascular integrity and pathological angiogenesis. Angiogenic responses in three distinct in vivo models were enhanced in Akt1−/− mice; these enhanced responses were associated with impairment of blood vessel maturation and increased vascular permeability. Although impaired vascular maturation in Akt1−/− mice may be attributed to reduced activation of endothelial nitric oxide synthase (eNOS), the major phenotypic changes in vascular permeability and angiogenesis were linked to reduced expression of two endogenous vascular regulators, thrombospondins 1 (TSP-1) and 2 (TSP-2). Re-expression of TSP-1 and TSP-2 in mice transplanted with wild-type bone marrow corrected the angiogenic abnormalities in Akt1−/− mice. These findings establish a crucial role of an Akt-thrombospondin axis in angiogenesis. [ABSTRACT FROM AUTHOR]

Akt is perhaps the most frequently activated oncoprotein human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found Chk1 mutants in the Akt phosphorylation sites behave like wild-type Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity insufficient by itself to exert an effect on Chk1 activity. Here report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage. [ABSTRACT FROM AUTHOR]

Summary: Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1. [Copyright &y& Elsevier]