Mechanism Of Akt Activation Research Articles

Gain of function mutant p53 protein activates AKT through the Rac1 signaling to promote tumorigenesis

ABSTRACT Tumor suppressor p53 is the most frequently mutated gene in human cancer. Mutant p53 (mutp53) not only loses the tumor suppressive activity of wild type p53, but often gains new oncogenic activities to promote tumorigenesis, defined as mutp53 gain of function (GOF). While the concept of mutp53 GOF is well-established, its underlying mechanism is not well-understood. AKT has been suggested to be activated by mutp53 and contribute to mutp53 GOF, but its underlying mechanism is unclear. In this study, we found that the activation of the Rac1 signaling by mutp53 mediates the promoting effect of mutp53 on AKT activation. Blocking Rac1 signaling by RNAi or a Rac1 inhibitor can inhibit AKT activation by mutp53. Importantly, targeting Rac1/AKT can greatly compromise mutp53 GOF in tumorigenesis. Results from this study uncover a new mechanism for AKT activation in tumors, and reveal that activation of AKT by mutp53 via the Rac1 signaling contributes to mutp53 GOF in tumorigenesis. More importantly, this study provides Rac1 and AKT as potential targets for therapy in tumors containing mutp53.

Mutant ASXL1 Promotes Expansion of the Phenotypic Hematopoietic Stem Cell Compartment

Somatic mutations of the ASXL1 gene are recurrently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations causes CH are not understood. Here, using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we investigated the effect of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). To examine the influence of ASXL1-MT on hematopoiesis, we bred the ASXL1-MT-KI mice with Vav-Cre transgenic mice. Young ASXL1-MT-KI mice (6-12 weeks) did not show significant changes in hematological parameters and differentiation status of peripheral blood. We observed the decreased frequency of hematopoietic stem and progenitor cells (HSPCs), including long-term HSCs (LT-HSCs). Competitive transplantation assays showed the reduced repopulation ability in ASXL1-MT-KI HSPCs. Thus, ASXL1-MT decreased the number and impaired the function of HSPCs in young mice. Next, we examined age-related changes in hematopoiesis caused by ASXL1-MT. Aged ASXL1-MT-KI mice displayed a myeloid-biased differentiation and hypocellular bone marrow, indicating the dysfunction of hematopoiesis. Interestingly, ASXL1-MT markedly increased the frequency of phenotypic LT-HSCs (pLT-HSCs) in aged mice (20-24 months). Competitive transplantation assays showed the impaired repopulation potential of pLT-HSCs from aged ASXL1-MT-KI mice. These data demonstrate that the increased pLT-HSCs in aged ASXL1-MT-KI mice are not functional HSCs with long-term repopulation potential. To elucidate how ASXL1-MT drives HSPC dysfunction, we conducted RNA-Seq analysis using HSPCs from young mice. This analysis revealed upregulation of mitochondrial genes in ASXL1-MT-KI HSPCs. In addition, MitoTracker staining, extracellular flux analyses and metabolome analyses demonstrated the enhanced mitochondrial metabolism in ASXL1-MT-KI HSPCs. We also found that the aberrantly elevated mitochondrial activity induced ROS overproduction and increased DNA damage, resulting in HSPC dysfunction. As a mechanism underlying the enhanced mitochondrial activity of ASXK1-MT-KI HSPCs, we revealed that ASXL1-MT activated the Akt/mTOR pathway in HSPCs. Treatment with an Akt inhibitor perifosine or an mTOR inhibitor rapamycin normalized the mitochondrial membrane potential and ROS levels in ASXL1-MT-KI HSPCs. Moreover, rapamycin treatment improved engraftment of ASXL1-MT-KI bone marrow cells after transplantation. These data indicate that the activated Akt/mTOR signaling leads to the enhanced mitochondrial activity, elevated ROS levels, and HSPC dysfunction in ASXL1-MT-KI mice. To assess the impact of the enhanced Akt/mTOR signaling on age-related changes in ASXL1-MT-KI mice, we administered rapamycin to aged ASXL1-MT-KI mice. Intriguingly, rapamycin treatment decreased the frequency of pLT-HSCs, and normalized the bone marrow cellularity in aged ASXL1-MT-KI mice. Cell cycle analysis revealed that pLT-HSCs in G0 phase were decreased in aged ASXL1-MT-KI mice, which was normalized by rapamycin treatment. These data demonstrate that the activated Akt/mTOR pathway provokes the aberrant expansion of pLT-HSCs in aged ASXL1-MT-KI mice. We next attempted to clarify the underlying mechanism of Akt activation in ASXL1-MT-KI mice. Immunoprecipitation experiments revealed that ASXL1-MT/BAP1 complex deubiquitinated AKT in 293T cells. To determine the role of endogenous Bap1 on Akt signaling, we assessed the effect of Bap1 deletion in murine bone marrow cells transformed by combined expression of SETBP1-D868N and ASXL1-MT (cSAM cells). A time course experiments showed Akt phosphorylation induced by IL-3 stimulation was attenuated and shortened in Bap1-depleted cSAM cells. These data suggest that ASXL1-MT/BAP1 complex deubiquitinate and stabilize phosphorylated Akt. In summary, we demonstrated that ASXL1-MT cooperated with BAP1 to promote AKT deubiquitination and activation. The activated Akt/mTOR pathway led to enhanced mitochondrial metabolism, elevated ROS levels and increased DNA damage. These molecular bases underlie the age-associated expansion of the pLT-HSC compartment. Our results underscore the possibility that CH can originate from a pLT-HSC with a limited repopulation potential. A pharmacological inhibition of the Akt/mTOR pathway could be a promising therapeutic intervention to individuals with CH harboring ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Abstract LB-277: A small molecule compound targets mutant Ras driven cancers via changes in mitochondrial morphology and mTOR-dependent execution

Abstract In a recent report, we presented evidence that a small molecule compound selectively induces autophagy-associated execution of mutant RAS driven cancer cells via Akt-dependent mitochondrial ROS production (Iskandar, K. et al. Antiox. Redox Sig., 2016). During our efforts to explore the underlying mechanism of RAS dependent execution, we unraveled a novel mechanism of action involving changes in proteins responsible for maintaining mitochondrial morphology, Drp-1 and Opa-1. Exposure of cells to the small molecule, C1, results in specific phosphorylation of Drp-1 (serine 616) associated with mitochondrial fission as well as cleavage of the fusion associated protein Opa-1. Gene knockdown of KRAS significantly abrogated drug-induced phosphorylation of Drp-1 and rescued mitochondrial morphology. Interestingly, siKRAS also resulted in the complete inhibition of mitophagy induced upon exposure to the small molecule compound; mitophagy in this model is driven by the activation of Drp-1. We also provide evidence that KRAS is recruited to the mitochondria, upstream of Drp-1 activation and mitophagy induction in response to drug treatment. Furthermore, as Akt activation was linked to an increase in ROS production and knockout of Akt1/2 salvaged cells from the death inducing activity of C1, we studied the mechanism of Akt activation in this model. We present evidence that Akt activation might be downstream of Torin-sensitive but not Rapamycin-sensitive mTOR activation. Furthermore, a priori treatment with Torin completely blocked the effect of C1 on tumor colony forming ability. These data provide an interesting insight into the molecular mechanism(s) of a small molecule compound that triggers RAS dependent execution with potential therapeutic implications. Citation Format: Jonathan C. Foo, Kartini B. Iskandar, Shazib Pervaiz. A small molecule compound targets mutant Ras driven cancers via changes in mitochondrial morphology and mTOR-dependent execution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-277.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Substance P Inhibits Hyperosmotic Stress-Induced Apoptosis in Corneal Epithelial Cells through the Mechanism of Akt Activation and Reactive Oxygen Species Scavenging via the Neurokinin-1 Receptor.

Hyperosmolarity has been recognized as an important pathological factor in dry eye leading to ocular discomfort and damage. As one of the major neuropeptides of corneal innervation, substance P (SP) has been shown to possess anti-apoptotic effects in various cells. The aim of this study was to determine the capacity and mechanism of SP against hyperosmotic stress-induced apoptosis in cultured corneal epithelial cells. The cells were exposed to hyperosmotic stress by the addition of high glucose in the presence or absence of SP. The results showed that SP inhibited hyperosmotic stress-induced apoptosis of mouse corneal epithelial cells. Moreover, SP promoted the recovery of phosphorylated Akt level, mitochondrial membrane potential, Ca2+ contents, intracellular reactive oxygen species (ROS) and glutathione levels that impaired by hyperosmotic stress. However, the antiapoptotic capacity of SP was partially suppressed by Akt inhibitor or glutathione depleting agent, while the neurokinin-1 (NK-1) receptor antagonist impaired Akt activation and ROS scavenging that promoted by SP addition. In conclusion, SP protects corneal epithelial cells from hyperosmotic stress-induced apoptosis through the mechanism of Akt activation and ROS scavenging via the NK-1 receptor.

FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.

Abstract LB-35: Site specific activation of AKT protects cells from death induced by glucose deprivation .

Abstract The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKT phosphorylation at Thr308 and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKT Thr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose-deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKT at Thr308 but not Ser473. Our results reveal a novel mechanism of AKT activation under prolonged metabolic stress that protects cells from metabolic stress. The selective activation of AKT phosphorylation at Thr308 that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. Citation Format: Meng Gao, Jiyong Liang, Yiling Lu, Huifang Guo, Peter German, Shanshan Bai, Eric Jonasch, Xingsheng Yang, Gordon B. Mills, Zhiyong Ding. Site specific activation of AKT protects cells from death induced by glucose deprivation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-35. doi:10.1158/1538-7445.AM2013-LB-35

Site-specific activation of AKT protects cells from death induced by glucose deprivation

The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKTThr308 phosphorylation and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose-deprivation-induced AKTThr308 phosphorylation is dependent on PDK1 and PI3K but not EGF receptor or IGF1R. Prolonged glucose deprivation induces the formation of a complex of AKT, PDK1 and the GRP78 chaperone protein, directing phosphorylation of AKTThr308 but not AKTSer473. Our results reveal a novel mechanism of AKT activation under prolonged glucose deprivation that protects cells from metabolic stress. The selective activation of AKTThr308 phosphorylation that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling.

High glucose upregulation of early-onset Parkinson's disease protein DJ-1 integrates the PRAS40/TORC1 axis to mesangial cell hypertrophy

The Akt kinase signaling pathway is frequently deregulated in many human diseases including cancer, autoimmune disease and diabetes. In nephropathy, associated with diabetes, increased Akt signal transduction results in glomerular especially mesangial cell hypertrophy. The mechanism of Akt activation by elevated glucose is poorly understood. The oncogene DJ-1 prevents oxidative damage and apoptosis of dopaminergic neurons in animal models of Parkinson's disease and in culture. We identified DJ-1 to increase in response to high glucose in renal glomerular mesangial cells concomitant with an increase in phosphorylation of Akt in a time-dependent manner. Plasmid-derived overexpression as well as downregulation of DJ-1 by siRNA showed the requirement of this protein in high glucose-stimulated Akt phosphorylation. The tumor suppressor protein PTEN acts as a negative regulator of Akt activation. Interestingly, DJ-1 was associated with PTEN and this interaction was significantly increased in response to high glucose. High glucose-induced increase in DJ-1 promoted phosphorylation of the PRAS40, a negative regulator of TORC1 kinase activity, resulting in activating and inactivating phosphorylation of S6 kinase and 4EBP-1, respectively. Furthermore, DJ-1 increased protein synthesis and hypertrophy of mesangial cells. Our results provide evidence for a unique mechanism whereby DJ-1 induces Akt/PRAS40/TORC1-mediated hypertrophy in response to high glucose.

PKC-β1 Mediates Glucose-Induced Akt Activation and TGF-β1 Upregulation in Mesangial Cells

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. The serine/threonine kinase Akt mediates glucose-induced upregulation of collagen I in mesangial cells through transactivation of the EGF receptor (EGFR). In addition, in renal tubular cells, glucose-induced secretion of TGF-beta requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-beta expression, but the mechanisms of Akt activation and its involvement in TGF-beta regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C beta (PKC-beta)-dependent manner. Glucose led to PKC-beta1 membrane translocation and association with Akt, and PKC-beta1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-beta1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-beta and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-beta1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-beta1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-beta1. In vivo, the PKC-beta inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-beta1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-beta1 transcriptional upregulation requires EGFR/PKC-beta1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation.

Akt signaling: a damaging interaction makes good

Phosphorylation of the protein kinase Akt at Ser473 is a key regulatory event in the induction of cellular survival mechanisms. A new study provides a novel mechanism for Akt activation. In cells and mice exposed to DNA-damaging insults, Ser473 phosphorylation is mediated by the DNA-dependent protein kinase at sites of DNA double-strand breaks. This event triggers the induction of a transcriptional program that promotes survival in response to DNA damage.

Contribution of constitutive activation of Akt to the pathogenesis of mantle cell lymphoma (MCL) independent of PTEN protein expression

8101 Background: To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt in primary MCL cases and cell lines. We also sought to determine whether loss of the phosphatase PTEN is the mechanism of Akt activation in MCL. Methods: We evaluated the protein levels of Akt, phosphorylated Akt and PTEN in 15 primary MCL cases and 2 cell lines by immunoblotting Results: Akt was phosphorylated in 3 of 15 MCL cases and in 2 of 2 mantle cell lymphoma cell lines. PTEN protein was expressed in 15 of 15 primary mantle cell lymphoma cases and in 2 of 2 MCL cell lines. Conclusions: We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL. Loss of PTEN protein expression is not the responsible mechanism for Akt activation. Alternative mechanisms of Akt activation are being evaluated to identify markers predictive for response to PI3K/Akt inhibitors in MCL. No significant financial relationships to disclose.

PYK2 mediates anti-apoptotic AKT signaling in response to benzo[a]pyrene diol epoxide in mammary epithelial cells

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. The carcinogenicity of BaP has been partially attributed to the formation of the BaP diol epoxide (BPDE), which has been shown to stably bind DNA and act as an initiator. BaP is a complete carcinogen, but the mechanisms for tumor promotion are less well characterized. Previous studies have demonstrated that BPDE enhanced anti-apoptotic signaling through Akt; however, mechanisms for Akt activation by BPDE are not well defined. In the current studies, we found that BPDE increased intracellular Ca2+ concentration in the human mammary epithelial cell line MCF-10A. A peak in Ca2+ concentration at 20 min was followed by increased phosphorylation of Pyk2 at Tyr881 and increased total tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Consistent with activation of the EGFR, Akt and ERK1/2 phosphorylation was detected in MCF-10A cells treated with BPDE. Pharmacological methods to prevent Ca2+ elevation and EGFR activity, and small-interfering RNA against Pyk2, prevented Akt phosphorylation by BPDE, which suggested that Ca2+, Pyk2 and EGFR activation lay upstream of Akt. In addition, we found that BPDE increased p53 activity and apoptosis in MCF-10A; however, transient transfection of constitutively active Akt attenuated both BPDE-dependent apoptosis and p53 activity. In contrast, apoptosis was enhanced by inhibitors of phosphatidyl inositol 3-kinase (PI3-K). This work demonstrates a novel mechanism for Akt activation by BPDE that occurs through increased Ca2+ concentration, and implicates Ca2+, Pyk2, EGFR and Akt as a potential pathway by which BPDE can inhibit apoptosis and act as a promoter of carcinogenesis.

Constitutive Activation of AKT Contributes to the Pathogenesis and Survival of Blastoid Variants of Mantle Cell Lymphoma.

The serine/threonine protein kinase AKT [protein kinase B (PKB)] plays a pivotal role in the tumorigenesis of many human malignancies, including hematopoietic neoplasms. AKT mediates cancer cell survival and cell growth through the phosphorylation of key regulatory proteins such as FRKHL-1, MDM2 and p27. We wished to determine whether the AKT signaling pathway might be involved in the pathogenesis of mantle cell lymphoma (MCL).The activated form of AKT (pAKT) and phosphorylation of several AKT targets, including FRKHL-1, MDM2 and p27 as well as the upstream modulators PTEN and TCL-1 were analyzed by immunoblotting in 31 MCL cases (16 classic MCL and 15 MCL of the blastoid variant) and in four MCL cell lines (Granta 519, NCEB, REC-1 and Z138). PI3KCA mutation analysis at two hotspot regions (Exons 9 and 20) were completed for all cases and cell lines. Subsequently, inhibition of the PIK3/AKT-pathway with LY294002, Wortmannin and AKT-inhibitor was performed in the four cell lines. pAKT was expressed in all 15 blastoid variants of MCL and in the 4 cell lines, with high expression in 13/15 cases and low expression in 2/15 cases. In contrast, only 2/16 typical MCL expressed pAKT, at low levels. Furthermore, pAKT expression in the blastoid variants and cell lines was accompanied by the phosphorylation of multiple downstream targets of activated AKT, including the cell cycle regulatory proteins p27, FRKHL-1, MDM-2 and the apoptosis associated protein bad. 42% of the blastoid MCL cases showed loss of PTEN, while only 2 (12%) of the typical cases showed diminished PTEN expression.TCL-1 expression levels were not correlated with AKT activation and no mutations of PI3KCA were detected. Functional studies of all four MCL lines demonstrated that the phosphorylation of the downstream effectors were regulated by activated AKT:Inhibition of the PI3K/AKT pathway by both 20 μM LY294002 and 8 μM Wortmannin or 40 μM AKT-inhibitor abrogated the phosphorylation of AKT, p27, FRKHL-1, bad and MDM2 after 24 to 48 hours of treatment, and resulted in G1 arrest after 24 hours of treatment and measurable apoptosis by 48 hours. We conclude that constitutive activation of AKT contributes to the pathogenesis of the blastoid variants of MCL. One possible mechanism of AKT activation is the loss of PTEN expression. The inhibition of the PIK3/AKT pathway in MCL cell lines leads to induction of cell cycle arrest and apoptosis, raising the possibility that PI3K/AKT inhibitors may be effective in the treatment of aggressive (blastoid) variants of MCLs.

Akt activation in renal cell carcinoma: contribution of a decreased PTEN expression and the induction of apoptosis by an Akt inhibitor

Background:: Akt has been implicated in the oncogenesis of human malignant tumors, because Akt regulates many key effector molecules involved in cell survival. PTEN (phosphatase and tensin homolog deleted on chromosome 10) negatively regulates Akt activation.Materials and methods:: The expression of phosphorylated Akt (p-Akt), total Akt and PTEN was analyzed by Western blotting in 45 renal cell carcinoma (RCC) patients. The Bad and phosphorylated Bad (p-Bad) statuses were analyzed in 20 RCC patients. A phosphatidylinositol ether analog was used as an Akt inhibitor to treat four RCC cell lines, namely Caki-1, KU19-20, SW839 and Caki-2.Results:: The PTEN expression in RCC was observed to decrease and p-Akt expression to increase significantly in comparison with that in the corresponding normal kidney tissue. The PTEN expression inversely correlated with the p-Akt expression. These alterations were specific for clear cell type RCC, but not for papillary or chromophobe type RCC. Alterations in Bad phosphorylation were also specifically observed in clear cell type. The Akt inhibitor induced apoptosis in KU19-20 and Caki-2 cells with a high Akt activity.Conclusions:: A decreased expression of PTEN may be an underlying mechanism for Akt activation. An Akt inhibitor may be a therapeutic option for a subset of RCC with an elevated Akt activity.

Akt signaling in thyroid neoplasia

Purpose of review The purpose of this paper is to review the recent data defining the mechanisms of Akt activation, and the role of Akt activity in thyroid oncogene signaling and thyroid cancer development and progression. Recent findings During the past decade, the genetic causes of approximately 50% of thyroid cancers have been defined. These abnormalities all result in constitutive activation of receptor tyrosine kinase-mediated signaling pathways, including the Akt and MAP kinase pathways. Cell signaling in thyroid cancers correlate with the presence of specific oncogenes. Secondary changes, in particular, further enhancement of Akt activation and nuclear expression of Akt, correlate with thyroid cancer invasion in vitro and in vivo. These data, therefore, define the Ras/MAP kinase and the PI3 kinase/Akt pathways as potential therapeutic targets for thyroid cancer therapy. Summary The pathogenetic mechanisms for thyroid tumorigenesis are being defined, as are the critical pathways responsible for aggressive cancer behavior. The role of the Akt kinase pathway in thyroid oncogene signaling, and the potential unique role for this pathway in thyroid cancer progression define it as a potential molecular target for new therapies.

Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A.

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.

Activation of Akt/Protein Kinase B in Epithelial Cells by theSalmonella typhimurium Effector SigD

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308). We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.