2-Aminothiadiazole inhibitors of AKT1 as potential cancer therapeutics

A series of 2-aminothiadiazole of inhibitors of AKT1 is described. SAR relationships are discussed, along with selectivity for protein kinase A (PKA) and cyclin-dependent kinase 2 (CDK2). Moderate selectivity observed in several compounds for AKT1 versus PKA is rationalized by X-ray crystallographic analysis. Key compounds showed activity in cellular assays measuring phosphorylation of two AKT substrates, PRAS40 and FKHRL1. Compound 30 was advanced to a mouse liver PD assay, where it showed dose-dependent inhibition of AKT activity, as measured by the inhibition of phospho-PRAS40.     

Distinct roles of AKT isoforms in regulating β1-integrin activity, migration, and invasion in prostate cancer

AKT1 and AKT2 kinases have been shown to play opposite roles in breast cancer migration and invasion. In this study, an RNA interference screen for integrin activity inhibitors identified AKT1 as an inhibitor of β1-integrin activity in prostate cancer. Validation experiments investigating all three AKT isoforms demonstrated that, unlike in breast cancer, both AKT1 and AKT2 function as negative regulators of cell migration and invasion in PC3 prostate cancer cells. Down-regulation of AKT1 and AKT2, but not AKT3, induced activation of cell surface β1-integrins and enhanced adhesion, migration, and invasion. Silencing of AKT1 and AKT2 also resulted in increased focal adhesion size. Importantly, the mechanisms involved in integrin activity regulation were distinct for the two AKT isoforms. Silencing of AKT1 relieved feedback suppression of the expression and activity of several receptor tyrosine kinases, including EGFR and MET, with established cross-talk with β1-integrins. Silencing of AKT2, on the other hand, induced up-regulation of the microRNA-200 (miR-200) family, and overexpression of miR-200 was sufficient to induce integrin activity and cell migration in PC3 cells. Taken together, these data define an inhibitory role for both AKT1 and AKT2 in prostate cancer migration and invasion and highlight the cell type-specific actions of AKT kinases in the regulation of cell motility.     

Ca(2+)/calmodulin directly interacts with the pleckstrin homology domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane.     

The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C

The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.     

Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)

CK1delta, a member of the casein kinase 1 family of serine/threonine specific kinases, has been shown to be involved in the regulation of microtubule dynamics. We have now identified a 176 aa fragment of the light chain LC2 of MAP1A (termed LC2-P16) specifically interacting with CK1delta. Two CK1delta interacting domains of LC2 were identified, located between aa 2629 and 2753 close to aa 2683 and between aa 2712 and 2805 of LC2. The two regions necessary for the interaction of LC2 with CK1delta have been mapped between aa 76-103 and aa 351-375 of CK1delta. Furthermore, LC2 has been identified as a new substrate of CK1delta. We therefore propose a model in which CK1delta could modulate microtubule dynamics by changing the phosphorylation status of the light chain LC2 of MAP1A.     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Identification of functional domains in AKT responsible for distinct roles of AKT isoforms in pressure-stimulated cancer cell adhesion

Cell adhesion is a critical step in cancer metastasis, activated by extracellular forces such as pressure and shear. Reducing AKT1, but not AKT2, ablates the increase in cancer cell adhesion associated with 15 mm Hg increased extracellular pressure. To identify the determinants of this AKT isoform specificity, we exchanged the pleckstrin homology (PH) domains and/or hinge regions of AKT1 and AKT2. Wild type isoforms or these chimeras were overexpressed in Caco-2 cells in the absence or presence of isoform-specific siRNA to suppress endogenous AKT1. Pressure-induced AKT translocation and phosphorylation to the membrane were compared, along with the stimulation of cell adhesion by pressure. Pressure stimulated translocation of AKT1, but not AKT2 to the plasma membrane. Among our chimeras, only the chimeric AKT2 (chimera2), in which both the AKT2 PH domain and hinge region had been replaced by those of AKT1, translocated to the membrane in response to pressure. Similarly, only chimera2 rescued the function of AKT1 in mediating pressure-stimulated adhesion after endogenous AKT1 had been reduced. Pressure also promoted phosphorylation of AKT1 but not AKT2, and expression of a nonphosphorylatable double point mutant prevented pressure-stimulated adhesion. Among the chimeras, pressure promoted only chimera2 phosphorylation. These results identify the AKT1 PH domain and hinge region as functional domains which jointly permit AKT1 translocation and phosphorylation in response to extracellular pressure and distinguish determine the specificity of AKT1 in mediating the effects of extracellular pressure on cancer cell adhesion. These may be useful targets for interventions to inhibit metastasis.     

Protein Kinase B (AKT) Mediates Phospholipase D Activation via ERK1/2 and Promotes Respiratory Burst Parameters in Formylpeptide-stimulated Neutrophil-like HL-60 Cells

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K_m 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67^phox and p47^phox, ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47^phox, p67^phox, and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.     

Inward rectification of the AKT2 channel abolished by voltage-dependent phosphorylation

The Arabidopsis K(+) channel AKT2 possesses the remarkable property that its voltage threshold for activation can be either within the physiological range (gating mode 1), or shifted towards considerably more positive voltages (gating mode 2). Gating mode 1 AKT2 channels behave as delayed K(+)-selective inward rectifiers; while gating mode 2 AKT2 channels are K(+)-selective 'open leaks' in the physiological range of membrane potential. In the present study we have investigated modulation of AKT2 current by effectors of phosphatases/kinases in COS cells and Xenopus oocytes. These experiments show that (i) dephosphorylation can result in AKT2 channel silencing; and (ii) phosphorylation by protein kinase A (PKA) favors both recruitment of silenced AKT2 channels and transition from gating mode 1 to gating mode 2. Interestingly, phosphorylation of AKT2 by PKA in COS cells and Xenopus oocytes is favored by hyperpolarization. Two PKA phosphorylation sites (S210 and S329) were pinpointed in the region of the pore inner mouth. The role of these phosphorylation sites in the switch between the two gating modes was assessed by electrophysiological characterization of mutant channels. The molecular aspects of AKT2 regulation by phosphorylation, and the possible physiological meaning of such regulation in the plant context, are discussed.     

AKT2 inhibition of cisplatin-induced JNK/p38 and Bax activation by phosphorylation of ASK1: implication of AKT2 in chemoresistance

Cisplatin and its analogues have been widely used for treatment of human cancer. However, most patients eventually develop resistance to treatment through a mechanism that remains obscure. Previously, we found that AKT2 is frequently overexpressed and/or activated in human ovarian and breast cancers. Here we demonstrate that constitutively active AKT2 renders cisplatin-sensitive A2780S ovarian cancer cells resistant to cisplatin, whereas phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 sensitizes A2780S and cisplatin-resistant A2780CP cells to cisplatin-induced apoptosis through regulation of the ASK1/JNK/p38 pathway. AKT2 interacts with and phosphorylates ASK1 at Ser-83 resulting in inhibition of its kinase activity. Accordingly, activated AKT2 blocked signaling down-stream of ASK1, including activation of JNK and p38 and the conversion of Bax to its active conformation. Expression of nonphosphorylatable ASK1-S83A overrode the AKT2-inhibited JNK/p38 activity and Bax conformational changes, whereas phosphomimic ASK1-S83D inhibited the effects of cisplatin on JNK/p38 and Bax. Cisplatin-induced Bax conformation change was inhibited by inhibitors or dominant negative forms of JNK and p38. In conclusion, our data indicate that AKT2 inhibits cisplatin-induced JNK/p38 and Bax activation through phosphorylation of ASK1 and thus, plays an important role in chemoresistance. Further, regulation of the ASK1/JNK/p38/Bax pathway by AKT2 provides a new mechanism contributing to its antiapoptotic effects.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Phosphomimetic substitution of heterogeneous nuclear ribonucleoprotein A1 at serine 199 abolishes AKT-dependent internal ribosome entry site-transacting factor (ITAF) function via effects on strand annealing and results in mammalian target of rapamycin complex 1 (mTORC1) inhibitor sensitivity

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.     

Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target GSK3β.     

Involvement of protein kinase B/AKT in early development of mouse fertilized eggs

The activation of AKT (also called protein kinase B) is thought to be a critical step in the phosphoinositide 3-kinase pathway that regulates cell growth and differentiation. In this report, we investigated the role of AKT in the regulation of mouse early embryo development. Injection of mRNA coding for a constitutively active myristoylated AKT (myr-Akt1) into one-cell stage fertilized eggs induced cell division more effectively than injection of wild-type AKT (Akt1-WT) mRNA, whereas microinjection of mRNA of kinase-deficient AKT (Akt1-KD) delayed the first mitotic division. Meanwhile, microinjection of different kinds of mRNA of AKT affected the phosphorylation status of CDC2A-Tyr15 and the activation of M-phase promoting factor (MPF). To investigate the intermediate factor between AKT and MPF, we then injected one-cell stage eggs first with Akt1-WT mRNA or myr-Akt1 mRNA and then with mRNA encoding either wild-type CDC25B (Cdc25b-WT) or a AKT-nonphosphorylatable Ser351 to Ala CDC25B mutant (Cdc25b-S351A). Cdc25b-S351A strongly inhibited the effect of AKT. Therefore, AKT causes the activation of MPF and strongly promotes the development of one-cell stage mouse fertilized eggs by inducing AKT-dependent phosphorylation of CDC25B, a member of the CDC25 phosphatase family. Our finding that CDC25B acts as a potential target of AKT provides new insight into the effect of AKT in the regulation of early development of mouse embryos.     

Anti-Diabetic Effects and Molecular Mechanisms of Amide Alkaloids from Piper longum Based on Network Pharmacology Integrated with Cellular Assays

Piper longum is a well-known spice and traditional medicine. It was revealed to possess anti-diabetic activity, but few information about its active component and underlying mechanism could be available. In this study, retrofractamides A ( 1 ) and C ( 2 ) isolated from P. longum showed potent inhibitory activity against PTP1B. Therefore, the potential mechanism was predicted by network pharmacology and molecular docking. PI3K/AKT was obtained as the most remarkable pathway against type 2 diabetes mellitus (T2DM), and AKT1 and GSK3β were yielded as the top two core targets of retrofractamides A ( 1 ) and C ( 2 ). Molecular docking of compounds with AKT1 and GSK3β showed strong binding affinity between them. Additionally, cellular experiments with a L6 cell model was conducted to further verify the above predictions. Results indicated that retrofractamides A ( 1 ) and C ( 2 ) exerted anti-diabetic effect via activating PI3K/AKT pathway, and they promoted glucose consumption, glucose uptake, glycogen synthesis and glycolysis.     

Receptor-specific mechanisms regulate phosphorylation of AKT at Ser473: role of RICTOR in β1 integrin-mediated cell survival

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells.