High Akt Activity Research Articles

Cutting Edge: PHLPP Regulates the Development, Function, and Molecular Signaling Pathways of Regulatory T Cells

Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.

Progesterone Receptor-B Induction of BIRC3 Protects Endometrial Cancer Cells from AP1-59-Mediated Apoptosis

Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.

Level of G protein–Coupled Receptor Kinase-2 Determines Myocardial Ischemia/Reperfusion Injury via Pro- and Anti-Apoptotic Mechanisms

Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. We demonstrate, using cardiac-specific GRK2 and βARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via βARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of β(2)ARs abolished βARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

NSAID Sulindac and Its Analog Bind RXRα and Inhibit RXRα-Dependent AKT Signaling

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.

Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.

BackgroundA significant proportion of breast cancer patients face failure of endocrine therapy due to the acquisition of endocrine resistance. We have explored mechanisms involved in such disease progression by using a mouse breast cancer model that is induced by medroxyprogesterone acetate (MPA). These tumors transit through different stages of hormone sensitivity. However, when cells from tumor variants were seeded on plastic, all were stimulated by progestins and inhibited by antiprogestins such as RU486. Furthermore, cells from a RU486-resistant tumor variant recovered antiprogestin sensitivity.HypothesisA three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression.MethodWe compared the behavior of tumors growing in vivo and cancer cells ex vivo (in 3D Matrigel). In this system, we evaluated the effects of kinase inhibitors and hormone antagonists on tumor growth.Principal FindingsLY294002, a PI3K/AKT pathway inhibitor, decreased both tumor growth in vivo and cell survival in Matrigel in MPA-independent tumors with higher AKT activity. Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity. Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant. Finally, while Matrigel reproduced differential responsiveness of MPA-dependent and -independent breast cancer cells, it was not sufficient to preserve antiprogestin resistance of RU486-resistant tumors.ConclusionWe demonstrated that the PI3K/AKT pathway is relevant for MPA-independent tumor growth. Three-dimensional cultures were useful to test the effects of kinase inhibitors on breast cancer growth and highlight the need for in vivo models to validate experimental tools used for selective therapeutic targeting.

FoxOs Inhibit mTORC1 and Activate Akt by Inducing the Expression of Sestrin3 and Rictor

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here, we unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.

Differential regulation of Akt phosphorylation in endometriosis

Protein kinase B (PKB/Akt), a serine/threonine kinase, regulates the function of many cellular proteins involved in apoptosis and proliferation. It was postulated that there is a higher Akt activity in endometriosis compared with normal endometrium, and that oestrogen may be one of the factors responsible for the high Akt activation in endometriotic cells. Phospho-Akt (pAkt) concentrations in normal, eutopic and ectopic endometrial tissues were compared by immunohistochemistry, and a higher pAkt immunoreactivity was revealed in eutopic and ectopic endometrium compared with normal endometrium, in vivo. Higher Akt phosphorylation in stromal cells from eutopic endometrium was observed, when compared with normal, in vitro (P < 0.05). Akt phosphorylation was rapidly (2-10 min) stimulated when endometrial stromal cells from normal and endometriosis patients were treated with 17 beta-oestradiol. In endometrial stromal cells from the endometriosis group, ICI 182,780 (ICI, a specific oestrogen receptor antagonist) failed to antagonize the effect of oestradiol when combined with oestradiol, and revealed a stimulatory effect on Akt phosphorylation when given alone (P < 0.05). In conclusion, since Akt affects cell survival, it is suggested that increased Akt phosphorylation may be related to the altered apoptosis/proliferation harmony in endometriosis, and therefore Akt may play a critical role in the pathogenesis of endometriosis.

Probing Akt-Inhibitor Interaction by Chemical Cross-Linking and Mass Spectrometry

The serine/threonine kinase Akt is a critical enzyme that regulates cell survival. As high Akt activity has been shown to contribute to the pathogenesis of various human malignancies, inhibition of Akt activation is a promising therapeutic strategy for cancers. We have previously demonstrated that changes in Akt interdomain arrangements from a closed to open conformation occur upon Akt-membrane interaction, which in turn allows Akt phosphorylation/activation. In the present study, we demonstrate a novel strategy to discern mechanisms for Akt inhibition based on Akt conformational changes using chemical cross-linking and (18)O labeling mass spectrometry. By quantitative comparison of two interdomain cross-linked peptides, which represent the proximity of the domains involved, we found that the binding of Akt to an inhibitor (PI analog) caused the open interdomain conformation where the PH and regulatory domains moved away from the kinase domain, even before interacting with membranes, subsequently preventing translocation of Akt to the plasma membrane. In contrast, the interdomain conformation remained unchanged after incubating with another type of inhibitor (peptide TCL1). Subsequent interaction with unilamellar vesicles suggested that TCL1 impaired particularly the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. This novel approach based on the conformation-based molecular interaction mechanism should be potentially useful for drug discovery efforts for specific Akt inhibitors or anti-tumor agents.

Abstract 1416: IGF-1 Gene Transfer Promotes Connexin-43 Expression and Enhances Integration of Bone Marrow Stem Cells in the Infarcted Heart

For functional benefits, bone marrow stem cells should engraft and develop functional syncitium with the host myocytes in the infarcted heart. We report that IGF-1 overexpression significantly enhances connexin-43 (Cx-43) in mesenchymal stem cells (MSCs) which promotes their survival and functional integration with host myocardium post-engraftment. IGF-1 gene transduced MSCs ( IGF-1 MSCs) expressed higher IGF-1 ( p &lt; 0.001 ) as compared with GFP gene transduced cells ( GFP MSCs). After 8h anoxia, MSCs, H2C9 and endothelial cells treated with IGF-1 MSCs conditioned medium ( IGF-1 CM) showed higher survival ( p &lt; 0.001 vs GFP MSCs conditioned medium ( GFP CM) supported by higher Akt activation which was abolished by wortmannin. Immunodetection showed Cx-43 plaques in the peripheral areas besides punctate distribution in the cytoplasm around the nucleus in IGF-1 MSCs and IGF-1 CM treated MSCs whereas GFP MSCs and GFP CM treated MSCs showed Cx-43 expression in regions of intimate cell to cell contact only. IGF-1 overexpression or IGF-1 CM treatment of MSCs also induced higher level Akt and p27 activation, and Cx-43 expression as compared with GFP MSCs ( p = 0.02 ) which was abolished by wortmannin. For in vivo studies, 70 μ l DMEM without cells (group-1) or containing 1x10 6 GFP MSC (group-2) or IGF-1 MSC (group-3) were injected intramyocardially in female rat model of coronary artery ligation (n=16/group). The animals were harvested on 7 days and 6 weeks after cell engraftment. FISH and sry -gene analysis on 7 day showed extensive survival of the male donor cells in the heart. Real-time PCR and Western blot on the heart tissue showed higher level of Cx-43 and activation of p27 in group-3 ( p &lt; 0.05 vs group-2). The number of TUNEL + cells was lower in group-3 ( p = 0.01 vs group-2). Immunostaining for α -sarcomeric actin and Cx-43 showed extensive myogenic differentiation of IGF-1 MSCs and gap junction formation between adjacent neofibers. Indices of heart function including ejection fraction (63.05±1.37%) and fractional shortening (28.54±0.92%) were improved in group-3 ( p &lt; 0.001 vs group-2 ) . IGF-1 enhances Cx-43 expression in MSCs and promotes their engraftment and functional integration in the heart and improves heart function.

The Neurogenesis-Controlling Factor, Pax6, Inhibits Proliferation and Promotes Maturation in Murine Astrocytes

Astrocytes serve various important functions in the CNS, but the molecular mechanisms of their generation and maturation are still enigmatic. Here, we show that Pax6, a key transcription factor that controls neurogenesis, also regulates proliferation, differentiation, and migration of astrocytes in the CNS. We first reveal that Pax6 is expressed in astrocytes during development as well as postnatally in the wild-type mouse. Astrocytes derived from Pax6 homozygous mutants (Sey/Sey) mice exhibited aberrant proliferation together with immature differentiation, both in vivo and in vitro, with higher migration potential in scratch-wound assays in vitro. Furthermore, a larger population of Sey/Sey astrocytes expresses neural stem cell markers such as nestin, Sox2, and prominin-1. These phenotypes of Pax6-deficient astrocytes putatively occur via higher Akt activity. Thus, the breakdown of Pax6 function induces the retention of neural stem-like characteristics and inhibits astrocyte maturation.

Direct comparison of endothelial cell and smooth muscle cell response to supercooling and rewarming

Cryoplasty combines mechanical dilatation with the delivery of hypothermia to atherosclerotic plaques. The response of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to supercooling and subsequent rewarming is still not clear. This study investigated the differential effects of vascular cell survival and proliferation in an in vitro model simulating cryoplasty. Bovine aortic ECs and SMCs were cultured separately with medium supplemented with 10% fetal bovine serum. The samples were supercooled to -10 degrees C for 0, 60, or 120 seconds on a cooling stage and then rewarmed in an incubator at 37 degrees C for 0, 6, 12, or 24 hours. Terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) and 5'-bromo-2'-deoxyuridine incorporation were used to measure the degree of apoptosis and proliferation respectively. Activation of protein kinase B (AKT), P70 S6 kinase, and P44/42 mitogen-activated protein kinase (MAPK) were assessed by Western blot and quantified using densitometry. Results are given as mean +/- standard error of mean and analyzed by analysis of variance. SMC and EC apoptosis were significantly increased with increasing supercooling and rewarming time, with a higher rate in SMCs. SMC apoptosis was maximal at 60 seconds cooling, followed by 24 hours rewarming (17.05% +/- 0.44%), whereas maximal EC apoptosis was after 120 seconds cooling, followed by 24 hours rewarming (4.21% +/- 0.22%, P < .05). Higher AKT activation was observed in ECs, with a maximum obtained of 3.34-fold at 120 seconds cooling with 24 hours rewarming (P < .05); only modest activation was found in SMCs. ECs had a decreased proliferation with cooling and rewarming time, and although SMCs maintained their low proliferative rate, ECs still had a higher overall proliferation rate that was statistically significant at 60 and 120 seconds cooling without rewarming compared with noncooling and nonrewarming (P < .05). Both p70S6 kinase and p44/42 MAPK activities decreased in SMCs, with significant drop at 60 seconds cooling, followed by 12 hours rewarming (P < .05). However, ECs showed a significant rise of P70 S6 kinase activity at 60 seconds cooling with 12 hours rewarming by 1.62-fold and P44/42 MAPK at 120 seconds cooling with 24 hours rewarming by 1.74-fold (P < .05). The higher apoptosis and lower proliferation of SMCs compared with ECs demonstrate the different effects of supercooling and rewarming on different vascular cell types. This information may be important in helping to understand the mechanism by which cryoplasty of atherosclerotic lesions may result in less restenosis.

RAD001 Inhibits Human Ovarian Cancer Cell Proliferation, Enhances Cisplatin-Induced Apoptosis, and Prolongs Survival in an Ovarian Cancer Model

mTOR (mammalian target of rapamycin) plays a central role in regulating cell growth and cell cycle progression and is regarded as a promising therapeutic target. We examined whether mTOR inhibition by RAD001 (everolimus) is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with cisplatin. Using four human ovarian cancer cell lines, we determined the effect of RAD001 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Western blot, and apoptosis assays. We evaluated the association between phospho-AKT/mTOR activity and RAD001 sensitivity. We also determined the effect of RAD001 on tumor growth and malignancy using mice inoculated with human ovarian cancer cells. RAD001 markedly inhibited cell proliferation of human ovarian carcinoma cells with high AKT activity (OVCAR10 and SKOV-3), but the effect was minimal in cells with low AKT activity (OVCAR4 and OVCAR5). Sensitivity to RAD001 was independent of p53 expression. RAD001 inhibited the phosphorylation of downstream 4E-BP1 and p70S6 kinase and attenuated the expression of Myc. RAD001 also attenuated the expression of HIF-1 alpha and vascular endothelial growth factor, important factors in angiogenesis and tumor invasiveness. RAD001 enhanced cisplatin-induced apoptosis in cells with high AKT/mTOR activity, with minimal effect in cells with low AKT-mTOR activity. Mouse xenografts of SKOV-3 cells revealed that RAD001 inhibits tumor growth, angiogenesis, and i.p. dissemination and ascites production and prolongs survival. Moreover, treatment with RAD001 significantly enhanced the therapeutic efficacy of cisplatin in vivo. These results indicate that RAD001 could have therapeutic efficacy in human ovarian cancers with hyperactivated AKT/mTOR signaling.

BRAF mutation predicts MEK-dependence in non-small cell lung cancer (NSCLC)

10523 Background: Constitutive ERK signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS and upstream receptors. Missense BRAF kinase domain mutations are frequently observed in melanoma, colon and thyroid cancers and less frequently in lung and other cancer types. The vast majority (&gt;90%) involve a glutamic acid for valine substitution at codon 600 (V600E), which results in elevated BRAF kinase activity. BRAF kinase domain mutations with intermediate and impaired kinase activity have also been identified, most frequently in NSCLC. We have previously reported that tumors with V600E BRAF mutation are selectively sensitive to MEK inhibition. Methods: Using the potent and selective MEK1/2 inhibitor PD0325901 (Pfizer), we examined a panel of NSCLC cell lines with mutant EGFR, KRAS, and/or low, intermediate and high-activity BRAF kinase domain mutations, with regard to MEK dependence. In all but one case, EGFR, KRAS and BRAF mutations were mutually exclusive. Results: Consistent with our prior results, NSCLC cells with V600E BRAF mutation were exquisitely sensitive to MEK inhibition (PD0325901 IC50 of 2nM). The proliferation of cells with non-V600E mutations, including those with high (G469A), intermediate (L597V) and impaired (G466V) kinase activities, was also MEK dependent with IC50's ranging between 2.7 and 80 nM. Inhibition of MEK in these cells resulted in downregulation of cyclin D1 and G1 growth arrest, with variable induction of apoptosis. Despite high basal ERK activity, NSCLC tumor cells with EGFR mutation were uniformly resistant to MEK inhibition (at doses of up to 500nM), despite effective inhibition of ERK phosphorylation. Tumor cells with RAS mutation had a more variable response, with some cell lines demonstrating sensitivity, while others were completely resistant. There was no correlation between basal ERK activity and sensitivity to MEK inhibition. A strong inverse correlation between AKT phosphorylation/ activity and PD0325901 sensitivity was observed. Conclusions: These results suggest that MEK inhibition may be useful therapeutically in tumors with V600E and non-V600E BRAF kinase domain mutations. The results also suggest that inhibition of both MEK and AKT signaling may be required in NSCLC tumors with high basal AKT activity. No significant financial relationships to disclose.

RETRACTED ARTICLE: Knockdown of astrocyte-elevated gene-1 inhibits prostate cancer progression through upregulation of FOXO3a activity

Astrocyte-elevated gene-1 (AEG-1) has been reported to be upregulated in several malignancies and play a critical role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase/AKT signaling pathway. However, the role of AEG-1 in prostate cancer (PC) has never been reported. We now show that AEG-1 is overexpressed in clinical PC tissue samples and cultured PC cells compared to benign prostatic hyperplasia tissue samples and normal prostate epithelial cells. Interestingly, AEG-1 knockdown induced cell apoptosis through upregulation of forkhead box (FOXO) 3a activity. This alteration of FOXO3a activity was dependent on reduction of AKT activity in LNCaP and PC-3 cells with high constitutive AKT activity, but not in DU145 cells with low constitutive AKT activity, although AEG-1 knockdown had no impact on phosphatase and tensin homolog expression in these cells. AEG-1 knockdown also attenuated the constitutive activity of the nuclear factor kappaB (NF-kappaB) and the activator protein 1 (AP-1) with a corresponding depletion in the expression of NF-kappaB and AP-1-regulated genes (interleukin (IL)-6, IL-8 and matrix metalloproteinase-9) and significantly decreased cell invasion properties of PC-3 and DU145 cells. Overall, our findings suggest that aberrant AEG-1 expression plays a dominant role as a positive auto-feedback activator of AKT and as a suppressor of FOXO3a in PC cells. AEG-1 may therefore represent a novel genetic biomarker to serve as an attractive molecular target for new anticancer agents to prevent PC cell progression and metastasis.

Signaling induced by hop/STI-1 depends on endocytosis

The co-chaperone hop/STI-1 is a ligand of the cell surface prion protein (PrP C), and their interaction leads to signaling and biological effects. Among these, hop/STI-1 induces proliferation of A172 glioblastoma cells, dependent on both PrP C and activation of the Erk pathway. We tested whether clathrin-mediated endocytosis affects signaling induced by hop/STI-1. Both hyperosmolarity induced by sucrose and monodansyl-cadaverine blocked Erk activity induced by hop/STI-1, without affecting the high basal Akt activity typical of A172. The endocytosis inhibitors also affected the sub-cellular distribution of phosphorylated Erk, consistent with blockade of the latter’s activity. The data indicate that signaling induced by hop/STI-1 depends on endocytosis. These findings are consistent with a role of sub-cellular trafficking in signal transduction following engagement by PrP C by ligands such as hop/STI-1, and may help help unravel both the functions of the prion protein, as well as possible loss-of-function components of prion diseases.

Akt-Induced Tamoxifen Resistance is Associated with Altered FKHR Regulation

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses, is associated with a poor pathophenotype in breast carcinoma, and is involved in hormone and chemotherapy resistance, including resistance to the antiestrogen, tamoxifen. Akt promotes cell survival by phosphorylating and inactivating proapoptotic proteins and increasing the transcription of survival genes. To explore the role that specific components of the Akt kinase pathway play in the cellular response to tamoxifen, we transfected MCF-7 cells with an expression plasmid for a constitutively active Akt. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Initial analysis of the molecular responses in the Akt/MCF-7 xenografts to tamoxifen suggests that high Akt activity alters apoptotic responses to tamoxifen. Control MCF-7 xenografts demonstrated activation of the proapoptotic forkhead (FKHR) transcription factor in response to tamoxifen treatment, while the xenografts expressing the constitutively active Akt transgene demonstrated no alterations in FKHR expression. In addition, TUNEL analysis demonstrated higher levels of apoptosis in the control xenografts in response to tamoxifen treatment compared to the Akt xenografts. Inhibition of Akt activity in vitro restored apoptotic responses to tamoxifen in the Akt/MCF-7 cells to those observed in the control cells. These data suggest that alteration of survival responses is an important mechanism by which Akt confers resistance to tamoxifen.

Peptide Aptamers with Binding Specificity for the Intracellular Domain of the ErbB2 Receptor Interfere with AKT Signaling and Sensitize Breast Cancer Cells to Taxol

The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol.

Prognostic significance of clinical factors and Akt activation in patients with bronchioloalveolar carcinoma

Lung cancer is the leading cause of cancer related mortality in the world. Bronchioloalveolar carcinoma (BAC) is a subset of NSCLC that has recently gained attention because of distinct biological and clinical features, increased incidence, and enhanced responsiveness to new therapies such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). However, prognostic features for BAC have not been well defined. Because activation of Akt is highly prevalent and a poor prognostic factor for other types of NSCLC, we assessed the prognostic significance of clinical features and Akt activation in patients with BAC. Forty-six cases of BAC in Iceland were classified according to WHO 1999 criteria. Akt activation was assessed using two phospho-specific antibodies against Akt (S473 and T308) in immunohistochemical (IHC) analysis. Associations between ordered Akt levels and other dichotomous parameters were evaluated using an exact Cochran-Armitage test for trend. Survival was analyzed by the Kaplan-Meier method and log-rank test, with hazard ratios (HR) determined by Cox proportional hazard models. The Cox model was also used to assess the joint effect of multiple factors on survival when they are considered simultaneously. Age and histology (mucinous versus non-mucinous) were not associated with survival. Activation of Akt was highly prevalent in BAC, with only 2 out of 46 patients exhibiting negative staining with either antibody. Moderate to high Akt activation was observed in 63% of cases and was associated with non-mucinous histology. Akt activation was not associated with differences in survival or smoking status. In contrast, Cox model analysis revealed that male gender (HR 2.24, 95% CI, 1.07-4.71, p=0.032), advanced stage (III or IV) (HR 2.17, 95% CI, 1.004-4.71, p=0.049) and smoking status (HR 6.89, 95% CI, 1.49-31.88, p=0.013) were associated with a worse prognosis. Male gender, advanced stage, and especially smoking status (but not Akt activation) are potentially important prognostic features for BAC. These features should be considered in the design and interpretation of clinical trials that enroll BAC patients.

P85-PI3K and PTEN Regulator

The liver is a key regulator of fuel utilization, and insulin acts to inhibit hepatic gluconeogenesis and activate lipogenesis, thereby preventing excessive glucose release during the fed state. Phosphatidylinositol 3-kinase (PI3K) is a key mediator of insulin signaling. PI3K is a dimer of a catalytic subunit (p110) and a regulatory subunit (p85). Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), the product of PI3K, is metabolized by the lipid phosphatase PTEN. Taniguchi et al . created a liver-specific knockout of the p85α subunit in mice (L-Pik3r1KO) and found that, contrary to expectations, the knockout mice showed increased liver responsiveness to insulin and had lower circulating glucose, free fatty acids, and triglyceride concentrations than wild-type littermates. Peripheral tissue (muscle and adipose) glucose utilization was also increased in the knockout mice. Although the knockout mice showed decreased total hepatic PI3K activity and decreased abundance of the p110 subunit (p85 stabilizes p110), insulin stimulated a prolonged elevation in hepatic PIP 3 abundance and higher activation of Akt, a kinase activated by PIP 3 , compared with wild-type mice. The increase in PIP 3 abundance appeared to be due to decreased PTEN activity in the livers of the knockout mice, suggesting that the p85 subunit regulates not only the production of PIP 3 but also its metabolism. C. M. Taniguchi, T. T. Tran, T. Kondo, J. Luo, K. Ueki, L. C. Cantley, C. R. Kahn, Phosphoinositide 3-kinase regulatory subunit p85α suppresses insulin action via positive regulation of PTEN. Proc. Natl. Acad. Sci. U.S.A. 103 , 12093-12097 (2006). [Abstract] [Full Text]

Akt regulates nuclear/cytoplasmic localization of tuberin

The autosomal dominantly inherited disease tuberous sclerosis (TSC) affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. TSC1, encoding hamartin, and TSC2, encoding tuberin are tumor suppressor genes responsible for TSC. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR)/p70S6K cascade. Phosphorylation of S939 and T1462 by Akt downregulates tuberin's potential to inhibit mTOR/p70S6K. Here, we show that this tuberin phosphorylation by Akt does not affect tuberin-mediated control of p27 protein amounts. This demonstrates that regulating p27 protein amounts and mTOR/p70S6K are separable functions of tuberin. Furthermore, we found that phosphorylation by Akt triggers upregulation of cytoplasmic and downregulation of nuclear tuberin. In cycling cells with high Akt activity, tuberin is predominantly localized to the cytoplasm. In arrested G0 cells with downregulated Akt activity, a significant proportion of tuberin is localized to the nucleus. Upon re-entry into the normal ongoing cell cycle, nuclear localization of tuberin is downregulated parallel to the activation of Akt. Recently, the mTOR/p70S6K cascade has been demonstrated to exist in both the cytoplasm and nucleus. We here also found that tuberin harbors the potential to regulate p70S6K activity in both the cytoplasm and nucleus. This description of functional tuberin in the cytoplasm and the nucleus together with our observation of Akt-controlled and cell cycle-regulated tuberin localization are of particular interest for a further understanding of tuberin's function as a gate keeper of the G0 cell status as well as of Akt's activity to control cell proliferation.