Akt3-Mediated Resistance to Apoptosis in B-RAF–Targeted Melanoma Cells
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Melanoma cells are highly resistant to anoikis, a form of apoptosis induced in nonadherent/inappropriate adhesion conditions. Depleting B-RAF or the prosurvival Bcl-2 family protein Mcl-1 renders mutant B-RAF melanoma cells susceptible to anoikis. In this study, we examined the effect of targeting B-RAF on the survival of primary stage melanoma cells cultured in three-dimensional type I collagen gels, which partially mimics the dermal microenvironment. Depletion/inhibition of B-RAF with small interfering RNA or the mutant B-RAF inhibitor, PLX4720, induced apoptosis of mutant B-RAF melanoma cells in three-dimensional collagen. Apoptosis was dependent on two upregulated BH3-only proteins, Bim-EL and Bmf, and was inhibited by ectopic Mcl-1 expression. Akt3 activation has been associated with the survival of melanoma cells. Mutant B-RAF melanoma cells ectopically expressing a constitutively activated form of Akt3 or endogenously expressing mutant Akt3 were protected from apoptosis induced by B-RAF knockdown or PLX4720 treatment. Furthermore, intrinsically resistant metastatic melanoma cells displayed elevated Akt phosphorylation in three-dimensional collagen and were rendered susceptible to PLX4720 by Akt3 knockdown. Importantly, myristylated Akt3 prevented B-RAF targeting-induced upregulation of Bim-EL and Bmf in three-dimensional collagen and partially protected Mcl-1-depleted cells from apoptosis. These findings delineate how mutant B-RAF protects melanoma cells from apoptosis and provide insight into possible resistance mechanisms to B-RAF inhibitors.Keywords:
Anoikis
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Ectopic expression
Background The Akt/PKB family of serine/threonine kinases plays a key role in neuronal cell function. Mutations in human AKT genes are found in human schizophrenic subjects, and other neurodevelopmental and psychiatric disorders. Genes encoding the three Akt isoforms ( Akt1, Akt2, Akt3 ) are expressed in the mouse brain suggesting that Akt ‐mutant mice are suitable models to understand the consequences of AKT gene dysfunction in the human brain. Methods To model the impact of Akt genotypes on fear memory processing and depression‐like behaviors, we used knockout strains for mouse Akt1 , Akt2 and Akt3 to examine behavioral and biochemical phenotypes. Results Mice deficient for Akt genes had specific behavioral phenotypes during the acquisition and processing of fear memories. To complement our results with a pharmacological approach, we also inhibited Akt with an allosteric inhibitor. Baseline anxiety levels in naive Akt ‐mutant mice or wild‐type littermates with inhibited Akt signaling were not altered. Acute stress exposure of Akt ‐mutant or inhibitor‐treated mice resulted in depression‐like behavioral responsiveness. Akt knockout mice exhibited an increased susceptibility to chronic stress and decreased responsiveness to antidepressant treatment. Discussion Our data in Akt ‐mutant mice converge with genetic findings in human subjects to support a critical role for Akt signaling in cognition and mood regulation. Our findings define the physiological contribution of selected Akt kinase isoforms to fear memory processing and the resilience to chronic stress. Our results support the study of Akt isoforms as diagnostic markers and therapeutic targets for treatment of neurodevelopmental and psychiatric disorders. This work is supported in part by NIH grants DA032280 and OD018339, and AFSP.
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Deregulated Akt activity leading to apoptosis inhibition, enhanced proliferation and drug resistance has been shown to be responsible for 35-70% of advanced metastatic melanomas. Of the three isoforms, the majority of melanomas have elevated Akt3 expression and activity. Hence, potent inhibitors targeting Akt are urgently required, which is possible only if (a) the factors responsible for the failure of Akt inhibitors in clinical trials is known; and (b) the information pertaining to synergistically acting targeted therapeutics is available. Areas covered: This review provides a brief introduction of the PI3K-Akt signaling pathway and its role in melanoma development. In addition, the functional role of key Akt pathway members such as PRAS40, GSK3 kinases, WEE1 kinase in melanoma development are discussed together with strategies to modulate these targets. Efficacy and safety of Akt inhibitors is also discussed. Finally, the mechanism(s) through which Akt leads to drug resistance is discussed in this expert opinion review. Expert opinion: Even though Akt play key roles in melanoma tumor progression, cell survival and drug resistance, many gaps still exist that require further understanding of Akt functions, especially in the (a) metastatic spread; (b) circulating melanoma cells survival; and
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The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.
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Akt (PKB) is a serine/threonine protein kinase that plays an important role in the transduction of signals affecting apoptosis, cell proliferation and survival. The Akt gene is frequently hyperactivated in tumors and has been shown to be amplified in a number of types of human cancers. Furthermore, Akt activity is elevated in cell lines with the mutated PTEN tumor suppressor gene. These studies establish Akt as an attractive target for cancer therapy. To determine the roles of Akt1, Akt2 and Akt3 in signal transduction, constitutively active Akt1, Akt2 and Akt3 was ectopically overexpressed in human pancreatic MiaPaCa-2 cells. The three Akt stable clones were characterized to determine their effects on transformation and proliferation. Compared to a vector control, the three Akt clones were able to drive cellular proliferation even in reduced serum conditions. Furthermore, in soft-agar assays, the Akt clones showed an 25-38% increase in colony formation in 2% serum. Our results indicate that all three forms of Akt may have protective effects within the cell depending on the type of apoptotic stimuli. Using 2D-PAGE comparisons between parental and Akt overexpressing cells, we attempted to determine novel targets of Akt phosphorylation. In this study, we identified prohibitin as a substrate for Akt both in vitro and in vivo. These studies suggest that Akt may regulate the cellular function of prohibitin via its phosphorylation.
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This article describes recent advances in the development and biological evaluation of allosteric and ATP-competitive small molecule inhibitors for the serine/threonine kinase Akt (protein kinase B, PKB). Unregulated activation of the PI3K/Akt/PTEN pathway is a prominent feature of many human cancers and Akt is over-expressed or activated in all major cancers making Akt an exciting new target for cancer therapy. The development of Akt inhibitors has been complicated and hampered by the presence of three Akt isozymes, (Akt1, Akt2 and Akt3) which differ in function and tissue distribution, as well as a lack of Akt specific inhibitors. In the past 18 months, a large number of reports have appeared describing the discovery and development of allosteric Akt kinase inhibitors and classical ATP-competitive Akt kinase inhibitors. This review will discuss the PI3K/Akt/PTEN pathway, allosteric and ATP-competitive Akt kinase inhibitors, their biological evaluation and progress towards target validation.
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Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBα/Akt1, PKBβ/Akt2 and PKBγ/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.
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Dyregulation of serine/threonine protein kinase Akt/PKB activity is a prominent feature of many human cancers.A large number of studies have demonstrated that Akt plays a key role in mediating cell growth and proliferation,promoting cell migration and invasion,stimulating neoangiogenesis,and protecting from pro-apoptotic stimuli.In view of the highly pleiotropic biologic effects of Akt signaling in multiple aspects of tumor pathophysiology,development of clinically applicable drugs specifically targeting Akt has been the subject of intense research.Up to now,a number of Akt specific inhibitors such as API2,Akt-I-1,Akt-I-2 and DCIEL have been developed and investigated in pre-clinical studies.These compounds have been shown to potently inhibit growth of tumors with aberrantly expressed or activated Akt,therefore,are likely to be developed as promising targeted anticancer drugs.
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AbstractAKT is a key serine/threonine kinase in the PTEN/PI3K/AKT pathway1 and activation of AKT is often observed in human cancers. To explore the role of AKT in cell survival in different tumor cells, we tested 20 human tumor cell lines for response to knockdown of AKT by small interference RNA (siRNA) and/or a kinase-dead mutant AKT. siRNA-mediated knockdown of all three AKT isoforms in tumor cell lines led to a reduction of phosphorylation of AKT substrates. Knockdown of AKT resulted in apoptosis in 6 out of 11 tumor cells with activated AKT. In contrast, knockdown of AKT induced apoptosis in 3 out of 9 cell lines with a low level of active AKT. The responsiveness of the cells to knockdown of AKT was not affected by mutational status of p53 but appeared correlated with overexpression of HER2. To assess the role of individual AKT isoforms, five of the cell lines responsive to knockdown of AKT were further characterized. In ZR-75 cells, AKT1 is the predominant isoform responsible for cell proliferation and survival. Conversely, in IGROV1 cells, AKT2 plays a major role in cell proliferation, but no single isoform is essential for cell survival. Thus, the relative importance of the AKT isoforms is cell linespecific. Our data suggest that inhibiting all three AKT isoforms is necessary to elicit maximal apoptotic response in tumor cells, and the level of activated AKT is a favorable but not always reliable biomarker for pre-selection of responsive tumor cell lines to AKT inhibitors.
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Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent, isozyme-specific activity. In this report, we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds, maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin, a downstream substrate of Akt, is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally, we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells, but not normal cells, to apoptotic stimuli.
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The Akt (PKB) protein kinases are critical regulators of human physiology that control an impressive array of diverse cellular functions, including the modulation of growth, survival, proliferation and metabolism. The Akt kinase family is comprised of three highly homologous isoforms: Akt1 (PKBα), Akt2 (PKBβ) and Akt3 (PKBγ). Phenotypic analyses of Akt isoform knockout mice documented Akt isoform specific functions in the regulation of cellular growth, glucose homeostasis and neuronal development. Those studies establish that the functions of the different Akt kinases are not completely overlapping and that isoform-specific signaling contributes to the diversity of Akt activities. However, despite these important advances, a thorough understanding about the specific roles of Akt family members and the molecular mechanisms that determine Akt isoform functional specificity will be essential to elucidate the complexity of Akt regulated cellular processes and how Akt isoform-specific deregulation might contribute to disease states. Here, we summarize recent advances in understanding the roles of Akt isoforms in the regulation of metabolism and cancer, and possible mechanisms contributing to Akt isoform functional specificity.
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