Tuberous sclerosis gene products in proliferation control

Two genes, TSC1 and TSC2, have been shown to be responsible for tuberous sclerosis (TSC). The detection of loss of heterozygosity of TSC1 or TSC2 in hamartomas, the growths characteristically occurring in TSC patients, suggested a tumor suppressor function for their gene products hamartin and tuberin. Studies analyzing ectopically modulated expression of TSC2 in human and rodent cells together with the finding that a homolog of TSC2 regulates the Drosophila cell cycle suggest that TSC is a disease of proliferation/cell cycle control. We discuss this question including very recent data obtained from analyzing mice expressing a modulated TSC2 transgene, and from studying the effects of deregulated TSC1 expression. Elucidation of the cellular functions of these proteins will form the basis of a better understanding of how mutations in these genes cause the disease and for the development of new therapeutic strategies.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2^Y1571H cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2^R611Q, which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.     

Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner

Serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a key metabolic stress-responsive factor that promotes the adaptation of cells to their microenvironment. Elevated concentrations of intracellular AMP, caused by metabolic stress, are known to activate AMPK by phosphorylation of the catalytic subunit. Recently, the tumor suppressor serine/threonine protein kinase LKB1 was identified as an upstream kinases, AMPKKs. In the current study, we found that stimulation with growth factors also caused AMPK-alpha subunit phosphorylation. Interestingly, even an LKB1-nonexpressing cancer cell line, HeLa, exhibited growth factor-stimulated AMPK-alpha subunit phosphorylation, suggesting the presence of an LKB1-independent pathway for AMPK-alpha subunit phosphorylation. In the human pancreatic cancer cell line PANC-1, AMPK-alpha subunit phosphorylation promoted by IGF-1 was suppressed by antisense ataxia telangiectasia mutated (ATM) expression. We found that IGF-1 also induced AMPK-alpha subunit phosphorylation in the human normal fibroblast TIG103 cell line, but failed to do so in a human fibroblast AT2-KY cell line lacking ATM. Immunoprecipitates of ATM collected from IGF-1-stimulated cells also caused the phosphorylation of the AMPK-alpha subunit in vitro. IGF-1-stimulated ATM phosphorylation at both threonine and tyrosine residues, and our results demonstrated that the phosphorylation of tyrosine in the ATM molecule is important for AMPK-alpha subunit phosphorylation during IGF-1 signaling. These results suggest that IGF-1 induces AMPK-alpha subunit phosphorylation via an ATM-dependent and LKB1-independent pathway.     

Oncogenic H-ras induces cyclin B1 expression in a p53-independent manner

Alcohol abuse is a major risk factor for cancer of the upper alimentary tract, the upper respiratory tract, and liver. Chromosome damage is used as early effect biomarker in the surveillance of human exposure to genotoxic carcinogens. In the present study, two genetic markers, namely chromosome aberrations (CAs) and micronuclei (MN), were used to evaluate genetic damage in peripheral lymphocytes from 20 alcoholics, 20 abstinent alcoholics, and 20 controls. Composition of the three groups was fairly similar as regards sex, age and smoking habits. A highly significant increase was observed in the frequencies of CA and MN in lymphocytes of alcoholics as compared both with controls and abstinent alcoholics. However, no correlation was found between the length of alcohol abuse and the frequencies of either biomarkers in alcoholics. CA and MN frequencies in abstinent alcoholics were similar than those in controls.Our data indicate that CA and MN can be two useful biomarkers to assess genetic damage associated with alcohol abuse. They could be included in programs for cancer prevention in alcoholics. Abstinence appears to normalize the frequency of both MN and CA. This could offer therapists another tool to help alcoholics change their lifestyle.     

Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner

Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.     

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apop-tosis protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.     

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by exogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mitochondrial membrane potential (△Ψm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived activator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 for 24 h. The results showed that △Ψm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P＜0.05) and cyclosporin A (CsA) could inhibit the collapse of △Ψm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P＜0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the release of Smac was caspase-dependent.     

Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner

Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations.     

A novel Apaf-1-independent putative caspase-2 activation complex

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.     

Kremen1 and Dickkopf1 control cell survival in a Wnt-independent manner

In multicellular organisms, a tight control of cell death is required to ensure normal development and tissue homeostasis. Improper function of apoptotic or survival pathways can not only affect developmental programs but also favor cancer progression. Here we describe a novel apoptotic signaling pathway involving the transmembrane receptor Kremen1 and its ligand, the Wnt-antagonist Dickkopf1. Using a whole embryo culture system, we first show that Dickkopf1 treatment promotes cell survival in a mouse model exhibiting increased apoptosis in the developing neural plate. Remarkably, this effect was not recapitulated by chemical Wnt inhibition. We then show that Dickkopf1 receptor Kremen1 is a bona fide dependence receptor, triggering cell death unless bound to its ligand. We performed Wnt-activity assays to demonstrate that the pro-apoptotic and anti-Wnt functions mediated by Kremen1 are strictly independent. Furthermore, we combined phylogenetic and mutagenesis approaches to identify a specific motif in the cytoplasmic tail of Kremen1, which is (i) specifically conserved in the lineage of placental mammals and (ii) strictly required for apoptosis induction. Finally, we show that somatic mutations of kremen1 found in human cancers can affect its pro-apoptotic activity, supporting a tumor suppressor function. Our findings thus reveal a new Wnt-independent function for Kremen1 and Dickkopf1 in the regulation of cell survival with potential implications in cancer therapies.In multicellular organisms, long-distance communication between cells is typically achieved by secreted ligands that diffuse through the extracellular medium and bind transmembrane receptors on target cells. Signal propagation through the plasma membrane is then achieved by receptor conformational changes upon ligand binding and classically involves modulation of enzymatic activity, interaction with intracellular partners or ion permeability. The classical view that transmembrane receptors only signal when bound to their ligand is now outdated, especially since the emergence of the dependence receptor concept. Dependence receptors do not form a family per se, but rather regroup a variety of receptors (transmembrane but also nuclear) that share the ability to trigger cell death unless bound to their respective ligand(s).¹ Dependence receptors therefore display two signaling activities: a ‘positive'' signaling in the presence of a ligand that can modulate various cellular processes (proliferation, differentiation, migration, etc.), and a ‘negative'' signaling in the absence of ligand consisting in the activation of a pro-apoptotic cascade downstream the receptor. To date, more than a dozen dependence receptors have been identified among which, deleted in colorectal cancer (DCC), Unc5A-D, Patched, TrkA/C, PlexinD1 and others,^{1, 2, 3, 4, 5, 6, 7} all of which play major roles during embryonic development, especially in the nervous system. Dependence receptors are also involved in cancer: as their apoptotic activity may allow to restrain cells within a ligand-rich environment and eliminate those that migrate away (such as metastatic cells), they were proposed to act as conditional tumor suppressors.¹ Accordingly, loss of function of a dependence receptor or overexpression of a ligand can confer a selective advantage to tumor cells.Many signaling pathways involved during embryonic development are misregulated in cancers. Among them, the wingless-related integration site (Wnt)-signaling pathway has been extensively studied in both developmental and cancer paradigms.^{8, 9} The Wnt1 ligand itself was first identified as a proto-oncogene¹⁰ and later shown to be required for mammalian brain patterning.¹¹ Although Wnt signaling was reported to elicit multiple cellular responses, most of the physiological effects described so far were shown to be mediated by the so-called canonical pathway: extracellular Wnt ligands bind transmembrane receptors of the Frizzled family associated with LDL receptor-related protein (LRP) co-receptors, leading to the activation of an intracellular signaling cascade whose final effector, β-catenin, modulates the expression of target genes.¹² Critical regulations of the Wnt pathway occur at the extracellular level by means of secreted antagonists.¹³ Dickkopf-1 (Dkk1) is one of such secreted inhibitors. It was shown to bind Kremen1/2 (Krm1/2) as well as LRP5/6 transmembrane receptors and subsequently inhibit Wnt downstream signaling.^{14, 15, 16, 17} During vertebrate embryonic development, Dkk1-mediated Wnt antagonism in anterior regions is strictly required for head induction.^{18, 19} In cancers, Dkk1 function remains a matter of debate as it was proposed to act as either a positive or a negative factor depending on the context.^{20, 21, 22} Such a discrepancy could reflect a yet unknown function for Dkk1 in addition to its well-characterized Wnt-inhibiting activity.In this study, we unravel a new Wnt-independent anti-apoptotic function for Dkk1 and find it is mediated by its transmembrane receptor Krm1, which behaves as a dependence receptor.     

Mdm2-mediated pRB downregulation is involved in carcinogenesis in a p53-independent manner

Mdm2 promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Although a p53-independent role of Mdm2 has been reported, the underlying mechanism remains unclear. In the present study, we indicated that Mdm2 is involved in p53-independent carcinogenesis via downregulation of pRB. Expression of pRB showed an apparent inverse correlation with Mdm2 expression in 30 patients with non-small cell lung cancer. There were some cases with the p53 mutations in which a high level of Mdm2 and a low level of pRB were expressed. Mdm2 promoted ubiquitination of pRB in cells without wild-type p53. Furthermore, pRB-mediated G1 arrest in a p53-deficient cell line, SRB1, was significantly enhanced by a mutant Mdm2 that lacks pRB ubiquitination activity. Soft-agar colony formation activity of p53-knockout MEF was increased by wild-type Mdm2 but not mutant Mdm2. These findings suggest that overexpression of Mdm2 can perturb a RB pathway regardless of the p53 gene status, promoting carcinogenesis.