Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.     

mTOR inhibitor for the treatment of hepatocellular carcinoma

Mammalian target of rapamycin (mTOR) plays a central role in the regulation of cellular growth, proliferation, and survival via a cytoplasmic serine/threonine kinase. mTOR also works as a nutrition sensor to monitor cellular metabolism. mTOR is located downstream in the PI3K/Akt pathway, in which Akt and the tuberous sclerosis complex (TSC) 1/2 are involved, to form a signal transduction pathway. New anticancer agents that target mTOR in the PI3K/Akt pathway of the signal transduction pathways involved in cell proliferation control have recently been developed and are already commercially available. A phase III clinical trial of mTOR inhibitor for hepatocellular carcinoma (HCC) is now ongoing worldwide to expand indications. RAD001 is a signal-transduction inhibitor (STI) that targets mTOR (more specifically, mTORC1). mTORC1 signaling is intricately regulated by mitogens, growth factors, energy, and nutrients. mTORC1 is a regulator essential for general protein synthesis, located downstream of the PI3K/AKT/mTOR pathway, which is dysregulated in most human cancers. Inhibiting mTOR with molecules, such as RAD001, generates additive effects that accompany upstream and downstream target inhibition; alternatively, upstream receptor inhibition is compensated for by inhibiting the downstream pathway, even if some resistance develops against receptor inhibition regardless of initial or acquired resistance. In conclusion, RAD001 is a potential targeted agent for HCC and therefore final results of a phase III study are awaited.     

The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish

Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.     

Target of rapamycin-mediated amino acid signaling in mosquito anautogeny

Mosquitoes generate an enormous burden on human health worldwide. Disease-transmitting species use a reproductive strategy, termed anautogeny, that requires a blood meal to initiate egg maturation. Whereas this strategy is important for driving disease transmission, the molecular mechanisms underlying this phenomenon are still poorly understood. The production of yolk protein precursors (YPPs), a central event in egg maturation, is called vitellogenesis. YPPs are synthesized in the fat body, the insect analogue of the vertebrate liver. Mosquito vitellogenesis is regulated by the steroid hormone 20 hydroxyecdysone (20E). However, 20E alone is not capable of activating vitellogenesis in vivo. Here, we report that amino acid signaling through the nutrient-sensitive target of rapamycin (TOR) pathway is essential for the activation of YPP gene expression. An increase in extracellular amino acid levels, similar to the increase observed after a blood meal, is critical for 20E stimulation of YPP gene expression. Treatment with the TOR kinase inhibitor rapamycin significantly inhibits YPP expression. We used RNA interference to knockdown the expression of two key proteins of the TOR signaling pathway, TOR, and tuberous sclerosis complex 2. Knockdown of TOR inhibited amino acid stimulation while knockdown of tuberous sclerosis complex 2, a negative regulator of TOR signaling, resulted in enhanced YPP expression. Thus, amino acid-based TOR signaling regulates the activation of egg development after a blood meal, an adaptation to the unique life style of mosquitoes.     

SGT1 is essential for Nod1 activation

The Nod-like receptor family in man contains proteins that recognize invasive bacteria. Nod1, a member of this family, is activated by specific peptidoglycan-derived muropeptides that contain meso-diaminopimelic acid. Plants contain a large family of proteins known as resistance (R) proteins that have common structural features with the Nod-like receptors and are essential for protection against a variety of plant pathogens. Extensive genetic studies have shown that the R protein function is determined by multiple proteins including SGT1, Rar1, and HSP90. Here we show that SGT1 positively regulates Nod1 activation. Depletion of SGT1 with siRNA did not affect stability of Nod1 protein or of downstream signaling molecules but did prevent multiple cellular responses associated with Nod1 activation. In contrast, depletion of the mammalian orthologue of Rar1, Chp1, had no effect on Nod1-dependent cellular activation. Finally, depletion of HSP90 or addition of a pharmacologic inhibitor of HSP90 resulted in loss of Nod1 protein. Thus, we show common regulatory pathways in plant R protein and human Nod1-dependent pathways and provide the basis for understanding the Nod1 pathway.     

From the Cover: Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.     

Small molecule inhibitors of Smoothened ciliary localization and ciliogenesis

Vertebrate Hedgehog (Hh) signals involved in development and some forms of cancer, such as basal cell carcinoma, are transduced by the primary cilium, a microtubular projection found on many cells. A critical step in vertebrate Hh signal transduction is the regulated movement of Smoothened (Smo), a seven-transmembrane protein, to the primary cilium. To identify small molecules that interfere with either the ciliary localization of Smo or ciliogenesis, we undertook a high-throughput, microscopy-based screen for compounds that alter the ciliary localization of YFP-tagged Smo. This screen identified 10 compounds that inhibit Hh pathway activity. Nine of these Smo antagonists (SA1-9) bind Smo, and one (SA10) does not. We also identified two compounds that inhibit ciliary biogenesis, which block microtubule polymerization or alter centrosome composition. Differential labeling of cell surface and intracellular Smo pools indicates that SA1-7 and 10 specifically inhibit trafficking of intracellular Smo to cilia. In contrast, SA8 and 9 recruit endogenous Smo to the cilium in some cell types. Despite these different mechanisms of action, all of the SAs inhibit activation of the Hh pathway by an oncogenic form of Smo, and abrogate the proliferation of basal cell carcinoma-like cancer cells. The SA compounds may provide alternative means of inhibiting pathogenic Hh signaling, and our study reveals that different pools of Smo move into cilia through distinct mechanisms.     

The kinase domain of mitochondrial PINK1 faces the cytoplasm

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD.     

Nuclear accumulation of the AT1 receptor in a rat vascular smooth muscle cell line: effects upon signal transduction and cellular proliferation

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II-AT(1) receptor interactions in vascular cells. Rat vascular smooth muscle cell line A10 was transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII, encodes AII fused downstream of enhanced cyan fluorescent protein) and the rat AT(1a) receptor (pAT(1)R/EYFP, encodes the rat AT(1a) receptor fused upstream of enhanced yellow fluorescent protein). The AII fluorescent fusion protein possesses no secretory signal peptide and deconvolution microscopy established that is maintained within these cells predominantly in the nucleus. AT(1)R/EYFP was absent from the nucleus when expressed exclusively or in untreated cells but accumulated in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, expression of ECFP/AII stimulated proliferation of A10 vascular smooth muscle cells (VSMCs) 1.6-fold (P < 0.05). Transfection of a control, pECFP/AII(C) (which encodes a scrambled AII peptide fused to ECFP) had no growth effect. In light of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of A10 cells activated cAMP response element-binding protein (CREB) as determined by one-hybrid assays and immunoblots. Expression of intracellular ECFP/AII similarly activated CREB. However, intracellular and extracellular AII activated CREB through different phosphorylation pathways. Exogenous AII treatment of A10 cells activated p38MAPK and ERK1/2 phosphorylation as determined by Western blot analyses and one-hybrid assays. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059 each partially inhibited exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation in this system. In contrast, expression of ECFP/AII (intracellular AII) in A10 VSMCs activated p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibited intracellular AII-induced CREB phosphorylation. In summary, extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor-ligand complex. Extracellular AII also stimulates a unique pathway, apparently reflecting interaction with plasma membrane-associated AT(1)R.     

Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of Parkinson's disease

Increasing evidence suggests that apoptosis may be the underlying cell death mechanism in the selective loss of dopaminergic neurons in Parkinson's disease. Because the inhibition of caspases provides only partial protection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP(+)) model of Parkinson's disease, we investigated the role of the proapoptotic c-Jun N-terminal kinase (JNK) signaling cascade in SH-SY5Y human neuroblastoma cells in vitro and in mice in vivo. MPTP/MPP(+) led to the sequential phosphorylation and activation of JNK kinase (MKK4), JNK, and c-Jun, the activation of caspases, and apoptosis. In mice, adenoviral gene transfer of the JNK binding domain of JNK-interacting protein-1 (a scaffold protein and inhibitor of JNK) inhibited this cascade downstream of MKK4 phosphorylation, blocked JNK, c-Jun, and caspase activation, the death of dopaminergic neurons, and the loss of catecholamines in the striatum. Furthermore, the gene transfer resulted in behavioral benefit. Therefore, inhibition of the JNK pathway offers a new treatment strategy for Parkinson's disease that blocks the death signaling pathway upstream of the execution of apoptosis in dopaminergic neurons, providing a therapeutic advantage over the direct inhibition of caspases.     

Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner

Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking the angiogenesis inhibitor thrombospondin-1 (TSP1) exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several proangiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated endothelial cell responses are inhibited by recombinant type 1 repeats of TSP1 and a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and proangiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis.     

Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes

IGF-I induces skeletal muscle hypertrophy by stimulating protein synthesis and suppressing the protein degradation pathway; the downstream signaling pathways Akt-mammalian target of rapamycin (mTOR)-p70-kDA-S6-kinase (p70S6K), and Forkhead box O1 (FoxO1) play essential roles in this regulation. Reactive oxygen species (ROS) modulate the signaling of various growth factors via redox regulation. However, the role of ROS in IGF-I signaling is not fully understood. In this study, we investigated whether ROS regulate the signaling and biological action of IGF-I in C2C12 myocytes. We found that IGF-I induces ROS in C2C12 myocytes. While treatment with H(2)O(2) significantly enhanced IGF-I-induced phosphorylation of the IGF-I receptor (IGF-IR), IGF-IR phosphorylation was markedly attenuated when cells were treated with antioxidants. The downstream signaling pathway, Akt-mTOR-p70S6K was subsequently down-regulated. Furthermore, the phosphorylation of FoxO1 by IGF-I decreased concomitantly with the restoration of the expression of its target genes, Atrogin-1 and muscle RING finger 1, which are related to muscle atrophy. Nox4 knockdown, which is reportedly to produce ROS in insulin signaling, attenuated IGF-I-induced IGF-IR phosphorylation, indicating that Nox4 is involved in the regulation of IGF-I signaling. Importantly, antioxidant treatments inhibited IGF-I-induced myocyte hypertrophy, demonstrating that ROS are necessary for IGF-I-induced myocyte hypertrophy in vitro. These results indicate that ROS play an essential role in the signaling and biological action of IGF-I in C2C12 myocytes.