Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP₃/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.     

Role of regulatory miRNAs of the PI3K/AKT/mTOR signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the common malignant human tumors with high morbidity worldwide. Aberrant activation of the oncogenic phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling is related to clinicopathological features of HCC. Emerging data revealed that microRNAs (miRNAs) have prominent implications for regulating cellular proliferation, differentiation, apoptosis, and metabolism through targeting the PI3K/AKT/mTOR signaling axis. The recognition of the crucial role of miRNAs in hepatocarcinogenesis represents a promising area to identify novel anticancer therapeutics for HCC. The present study summarizes the major findings about the regulatory role of miRNAs in the PI3K/AKT/mTOR pathway in the pathogenesis of HCC.     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

Effect of orthodontic force on the expression of PI3K,Akt, and P70S6 K in the human periodontal ligament during orthodontic loading

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinases involved in the regulation of cell growth, proliferation, and differentiation through the PI3K/Akt/mTOR/P70S6 K signalling pathway. P70S6 K as a downstream molecule of mTOR is activated by phosphorylation and subsequently promotes the synthesis of ribosomal and translational proteins. In this study, we investigated the role of PI3K, Akt, and P70S6 K in human periodontal tissue remodelling during orthodontic loading. The prepared tissue specimens taken from 4 extracted premolars were processed for immunolabelling. The changes in the expression of PI3K, Akt, and P70S6 K in the periodontal tissues were detected by real‐time quantitative‐polymerase chain reaction and Western blot analysis. The results from real‐time quantitative‐polymerase chain reaction and Western blot both showed that the expression of PI3K, Akt, and P70S6 K in the experimental group began to increase at 3 days and increased significantly at 10 days, then decreased approaching the control group level at 28 days. Our findings showed that the expression of PI3K, Akt, and P70S6 K in human periodontal ligament demonstrated a variability during the orthodontic loading, which suggested that the PI3K/Akt/mTOR/P70S6 K signal pathway was involved in orthodontic tooth movement and played a role in the process of periodontium remodelling.     

mTOR is the rapamycin-sensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k)

Mechanical stretch induces phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k) through a rapamycin-sensitive (RS) pathway that involves a unique PI3K-independent mechanism. Rapamycin is considered to be a highly specific inhibitor of the protein kinase mTOR; however, mTOR is also considered to be a PI3K-dependent signaling molecule. Thus, questions remain as to whether mTOR is the RS element that confers mechanically-induced signaling to p70(S6k)(389). In this study, rapamycin-resistant mutants of mTOR along with mechanical stretch were used to address this question. The results indicate that mTOR is the RS element and reveal that mTOR signaling can be activated through a PI3K-independent mechanism.     

The mechanisms of EGFR in the regulation of axon regeneration

To understand the relationship between epidermal growth factor receptor (EGFR) and axon regeneration and the mechanisms of how EGFR regulates the neuronal intrinsic regenerative ability, we evaluated the levels of mRNA and protein of EGFR、total mammalian target of rapamycin (mTOR), p‐mTOR^Ser2448, total Akt and p‐Akt^Ser473 in rats of different developmental stage by using Western blot and real‐time polymerase chain reaction analysis. Axon protein tau and neuron proteins β‐tubulin/neurofilament (NF) were assessed to evaluate the extent of the axon regeneration in cultured neuron cells. Expressions of EGFR、total mTOR, p‐mTOR^Ser2448, total Akt and p‐Akt^Ser473 in cultured neuron cells were also detected using Western blot analysis. Our results showed that the expressions of EGFR and mTOR dropped off with the ageing of the rats, and Ser473 phosphorylation of Akt and Ser2448 phosphorylation of mTOR were highly expressed in foetal and newborn rats but decreased obviously in adult rats. tau, β‐tubulin and NF were upregulated when EGFR was overexpressed and down‐regulated after EGFR was blocked. The phosphorylation of mTOR and Akt was apparently elevated when EGFR was overexpressed and decreased when EGFR was blocked, which suggested that EGFR has the potential to regulate the neuronal intrinsic regeneration and mTOR and PI3K/Akt pathway activation may have an important role in it. Copyright © 2013 John Wiley & Sons, Ltd.     

PI3K-PKB-mTOR hyperactivation in relation to nasopharyngeal carcinoma progression and prognosis

Nasopharyngeal carcinoma (NPC) has a unique and complex etiology, which is not completely understood. The aim of this study is to investigate the expression patterns of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and mammalian target of rapamycin (mTOR) proteins in patients with NPC and their relationship with NPC progression and prognosis. Between January 2008 and March 2010, PI3K, PKB, and mTOR protein expressions were detected using immunohistochemistry among 119 patients with NPC and 30 healthy people. A 5-year follow-up was conducted for all patients. Correlations of PI3K, PKB, and mTOR proteins with the clinicopathological features and prognosis of NPC were evaluated using Spearman's rank correlation coefficient and Kaplan-Meier curve. Cox's regression analysis was performed to analyze the risk factors for the prognosis of NPC. First, PI3K, PKB, and mTOR were highly expressed in patients with NPC. The expressions of PI3K, PKB, and mTOR proteins were associated with T stage, N stage, clinical stage, relapse, and distant metastasis. Meanwhile, PI3K is positively correlated with PKB and PKB is positively correlated with mTOR in NPC. Higher PI3K, PKB, and mTOR protein expressions were related to a shorter survival time and a lower survival rate in NPC. Cox regression analysis revealed that age, T stage, N stage, PI3K, PKB, and mTOR were independent risk factors for NPC patient survival. Altogether, our data suggest that overexpression of PI3K, PKB, and mTOR proteins is an important indicator of poor survival in NPC. In addition, inhibition of PI3K-PKB-mTOR signaling may also contribute to the development of new therapeutic strategies for NPC.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.     

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr³⁰⁸), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser⁴¹⁷), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser²⁵⁶). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.     

Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.     

The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Feedback loops blockade potentiates apoptosis induction and antitumor activity of a novel AKT inhibitor DC120 in human liver cancer

The serine/threonine kinase AKT is generally accepted as a promising anticancer therapeutic target. However, the relief of feedback inhibition and enhancement of other survival pathways often attenuate the anticancer effects of AKT inhibitors. These compensatory mechanisms are very complicated and remain poorly understood. In the present study, we found a novel 2-pyrimidyl-5-amidothiazole compound, DC120, as an ATP competitive AKT kinase inhibitor that suppressed proliferation and induced apoptosis in liver cancer cells both in vitro and in vivo. DC120 blocked the phosphorylation of downstream molecules in the AKT signal pathway in dose- and time-dependent manners both in vitro and in vivo. However, unexpectedly, DC120 activated mammalian target of rapamycin complex 1 (mTORC1) pathway that was suggested by increased phosphorylation of 70KD ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The activated mTORC1 signal was because of increase of intracellular Ca²⁺ via Ca²⁺/calmodulin (CaM)/ signaling to human vacuolar protein sorting 34 (hVps34) upon AKT inhibition. Meanwhile, DC120 attenuated the inhibitory effect of AKT on CRAF by decreasing phosphorylation of CRAF at Ser259 and thus activated the mitogen-activated protein kinase (MAPK) pathway. The activation of the mTORC1 and MAPK pathways by DC120 was not mutually dependent, and the combination of DC120 with mTORC1 inhibitor and/or MEK inhibitor induced significant apoptosis and growth inhibition both in vitro and in vivo. Taken together, the combination of AKT, mTORC1 and/or MEK inhibitors would be a promising therapeutic strategy for liver cancer treatment.     

Role and therapeutic potential of PI3K-mTOR signaling in de novo resistance to BRAF inhibition

BRAF inhibition is highly active in BRAF-mutant melanoma, but the degree and duration of responses is quite variable. Improved understanding of the mechanisms of de novo resistance may lead to rational therapeutic strategies with improved efficacy. Proteomic analysis of BRAF-mutant, PTEN-wild-type human melanoma cell lines treated with PLX4720 demonstrated that sensitive and de novo resistant lines exhibit similar RAS-RAF-MEK-ERK pathway inhibition, but the resistant cells exhibited durable activation of S6 and P70S6K. Treatment with the mTOR inhibitor rapamycin blocked activation of P70S6K and S6, but it also increased activation of AKT and failed to induce cell death. Combined treatment with rapamycin and PX-866, a PI3K inhibitor, blocked the activation of S6 and AKT and resulted in marked cell death when combined with PLX4720. The results support the rationale for combined targeting of BRAF and the PI3K-AKT pathways and illustrate how target selection will be critical to such strategies.     

A Small Molecule Inhibits Akt through Direct Binding to Akt and Preventing Akt Membrane Translocation

The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)³⁰⁸ and Ser(P)⁴⁷³) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt.     

Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2-ones as potent and selective mTOR kinase inhibitors

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).