PI3K/mTORC1 activation in hamartoma syndromes: Therapeutic prospects

Dysregulated activity of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) is characteristic feature of hamartoma syndromes. Hamartoma syndromes, dominantly inherited cancer predisposition disorders, affect multiple organs and are manifested by benign tumors consisting of various cell types native to the tissues in which they arise. In the past few years, three inherited hamartoma syndromes, Cowden syndrome (CS), tuberous sclerosis complex (TSC) syndrome, and Peutz-Jeghens syndrome (PJS), have all been linked to a common biochemical pathway: the hyperactivation of PI3K/mTORC1 intracellular signaling. Three tumor suppressors, PTEN (phosphatases and tensin homolog), tuberous sclerosis complex TSC1/TSC2, and LKB1, are negative regulators of PI3K/mTORC1 signaling; disease-related inactivation of these tumor suppressors results in the development of PTEN-associated hamartoma syndromes, TSC, and PJS, respectively. The goal of this review is to provide a roadmap for navigating the inherently complex regulation of PI3K/mTORC1 signaling while highlighting the progress that has been made in elucidating the cellular and molecular mechanisms of hamartoma syndromes and identificating potential therapeutic targets for their treatment. Importantly, because the PI3K/mTORC1 pathway is activated in the majority of common human cancers, the identification of novel molecular target(s) for the treatment of hamartoma syndromes may have a broader translational potential, and is critically important not only for therapeutic intervention in hamartoma disorders, but also for the treatment of cancers.     

ERK Inhibition Rescues Defects in Fate Specification of Nf1-Deficient Neural Progenitors and Brain Abnormalities

Germline mutations in the RAS/ERK signaling pathway underlie several related developmental disorders collectively termed neuro-cardio-facial-cutaneous (NCFC) syndromes. NCFC patients manifest varying degrees of cognitive impairment, but the developmental basis of their brain abnormalities remains largely unknown. Neurofibromatosis type 1 (NF1), an NCFC syndrome, is caused by loss-of-function heterozygous mutations in the NF1 gene, which encodes neurofibromin, a RAS GTPase-activating protein. Here, we show that biallelic Nf1 inactivation promotes Erk-dependent, ectopic Olig2 expression specifically in transit-amplifying progenitors, leading to increased gliogenesis at the expense of neurogenesis in neonatal and adult subventricular zone (SVZ). Nf1-deficient brains exhibit enlarged corpus callosum, a structural defect linked to severe learning deficits in NF1 patients. Strikingly, these NF1-associated developmental defects are rescued by transient treatment with an MEK/ERK inhibitor during neonatal stages. This study reveals a critical role for Nf1 in maintaining postnatal SVZ-derived neurogenesis and identifies a potential therapeutic window for treating NF1-associated brain abnormalities.     

The inhibition of glycogen synthase kinase 3beta by a metabotropic glutamate receptor 5 mediated pathway confers neuroprotection to Abeta peptides

Activation of glycogen synthase kinase 3beta (Gsk3beta) has been shown to be a key component in signaling pathways that underlie neurodegeneration and neurodegenerative disease. Conversely, inactivation of Gsk3beta by phosphoinositide 3-kinase (PI3K)/Akt is an important neuroprotective mechanism. Previous studies have shown that agonist activation of group I metabotropic glutamate receptors (mGluRs) can increase neuronal survival and prevent apoptosis. However, little is known about the signaling pathways that couple mGluR5 to neuroprotection. In this report, we investigated whether activation of the PI3K/Akt/Gsk3beta pathway, which has been shown to have an important neuroprotective mechanism, is required for mGluR5 activation mediated neuroprotection against beta-amyloid. We found that brief incubations of mouse hippocampal slices with (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in increased phosphorylation of Akt and Gsk3beta. The PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increased phosphorylation of Akt and Gsk3beta. Similar results were observed in rat primary hippocampal cultures. Finally, we found that the PI3K inhibitor LY294002 can block (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) mediated neuroprotection against beta-amyloid. Thus, these findings suggest that mGluR5 can modulate the PI3K/Akt/Gsk3beta pathway in the hippocampus, and that modulation of this signaling pathway can reverse beta-amyloid-induced neuronal toxicity.     

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.     

Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.     

Neuro-kardio-fazio-kutane Syndrome

Recently, neuro-cardio-facio-cutaneous (NCFC) syndromes were defined as a group of hereditary diseases with phenotypical overlap based on a similar pathogenetic mechanism. These syndromes are associated with increased signal transduction down the RAS-MAPK (RAS: rat sarcoma, MAPK: mitogen-activated protein kinase) pathway. Noonan, LEOPARD, cardio-facio-cutaneous, and Costello syndrome as well as neurofibromatosis type 1 and Legius syndrome belong to the NCFC diseases. Interdisciplinary health management is essential to determine and establish adequate medical diagnostics and treatment and to provide psychosocial support for affected patients and their families.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.     

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.     

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. Here, we reveal a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicated that ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies showed that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo.     

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.     

Leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) is a modulator of FGFR1

Fibroblast growth factor receptors (FGFRs) play critical roles in craniofacial and skeletal development via multiple signaling pathways including MAPK, PI3K/AKT, and PLC-γ. FGFR-mediated signaling is modulated by several regulators. Proteins with leucine-rich repeat (LRR) and/or immunoglobulin (IG) superfamily domains have been suggested to interact with FGFRs. In addition, fibronectin leucine-rich repeat transmembrane protein 3 (FLRT3) has been shown to modulate the FGFR-mediated signaling via the fibronectin type III (FNIII) domain. Therefore proteins with LRR, IG, and FNIII are candidate regulators of the FGFRs. Here we identify leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) as a regulator of the FGFRs.     

PI3K: a potential therapeutic target for cancer

Phosphatidylinositol 3-kinase (PI3K), one member of lipid kinase family, has been demonstrated to play a key role in regulating cell proliferation, adhesion, survival, and motility. Recent studies indicate that PI3K related signaling pathway is one of the most commonly activated pathways in human cancers. Accordingly, pharmacological inhibition of key nodes in this signaling cascade has been a focus in developmental therapeutics. To date, Inhibitors targeting PI3K or nodes in this pathway, AKT and mTOR, are best studied and have reached clinical trials. In this review, we will focus on recent progress on understanding of PI3Ks signaling pathway and the development of PI3K inhibitors.     

Interleukin-6 up-regulates the expression of interleukin-15 is associated with MAPKs and PI3-K signaling pathways in the human keratinocyte cell line,HaCaT

Interleukin (IL)-15 is an important inflammatory cytokine and plays a key role in autoimmune disease. At present, IL-15 gene expression and regulation related to many innate immunity trigger signals have been clarified in some specific cell types, but the relationship of IL-6 and IL-15 in the human keratinocyte cell line (HaCaT) is unknown. In this study, we investigated the effect of IL-6 on the expression of IL-15 and selected signaling pathways in HaCaT cells. Results demonstrated that IL-6 up-regulated the expression of IL-15 both at the mRNA and protein levels. Meanwhile, IL-6 was able to activate MAPKs-ERK1/2 and PI3K-AKT signaling pathways. Furthermore, the high expression of IL-15 induced by IL-6 was down-regulated while MAPKs-ERK1/2 and PI3K-AKT signaling pathways were, respectively, blocked by PD98059 and LY294002. These findings indicate that the expression of IL-15 up-regulated by IL-6 is associated with MAPKs-ERK1/2 and PI3K-AKT signaling pathways in HaCaT cells.     

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.     

Flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for Alzheimer disease

There is increasing evidence that the consumption of flavonoid-rich foods can beneficially influence normal cognitive function. In addition, a growing number of flavonoids have been shown to inhibit the development of Alzheimer disease (AD)-like pathology and to reverse deficits in cognition in rodent models, suggestive of potential therapeutic utility in dementia. The actions of flavonoid-rich foods (e.g., green tea, blueberry, and cocoa) seem to be mediated by the direct interactions of absorbed flavonoids and their metabolites with a number of cellular and molecular targets. For example, their specific interactions within the ERK and PI3-kinase/Akt signaling pathways, at the level of receptors or kinases, have been shown to increase the expression of neuroprotective and neuromodulatory proteins and increase the number of, and strength of, connections between neurons. Concurrently, their effects on the vascular system may also lead to enhancements in cognitive performance through increased brain blood flow and an ability to initiate neurogenesis in the hippocampus. Additional mechanisms have been suggested for the ability of flavonoids to delay the initiation of and/or slow the progression of AD-like pathology and related neurodegenerative disorders, including a potential to inhibit neuronal apoptosis triggered by neurotoxic species (e.g., oxidative stress and neuroinflammation) or disrupt amyloid β aggregation and effects on amyloid precursor protein processing through the inhibition of β-secretase (BACE-1) and/or activation of α-secretase (ADAM10). Together, these processes act to maintain the number and quality of synaptic connections in key brain regions and thus flavonoids have the potential to prevent the progression of neurodegenerative pathologies and to promote cognitive performance.     

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.     

Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors

Neuronal precursor cells in the developing cerebellum require activity of the sonic hedgehog (Shh) and phosphoinositide-3-kinase (PI3K) pathways for growth and survival. Synergy between the Shh and PI3K signaling pathways are implicated in the cerebellar tumor medulloblastoma. Here, we describe a mechanism through which these disparate signaling pathways cooperate to promote proliferation of cerebellar granule neuron precursors. Shh signaling drives expression of mRNA encoding the Nmyc1 oncoprotein (previously N-myc), which is essential for expansion of cerebellar granule neuron precursors. The PI3K pathway stabilizes Nmyc1 protein via inhibition of GSK3-dependent Nmyc1 phosphorylation and degradation. The effects of PI3K activity on Nmyc1 stabilization are mimicked by insulin-like growth factor, a PI3K agonist with roles in central nervous system precursor growth and tumorigenesis. These findings indicate that Shh and PI3K signaling pathways converge on N-Myc to regulate neuronal precursor cell cycle progression. Furthermore, they provide a rationale for therapeutic targeting of PI3K signaling in medulloblastoma.