LKB1 Signaling Research Articles

Developmental and Cell Cycle Quiescence Is Mediated by the Nuclear Hormone Receptor Coregulator DIN-1S in the Caenorhabditis elegans Dauer Larva

When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-like stage called "dauer" that is specialized for dispersal and survival. The decision to form a dauer larva is controlled by three parallel signaling pathways, whereby a compromise of TGFβ, cyclic guanosine monophosphate, or insulin/IGF-like signaling (ILS) results in dauer formation. Signals from these pathways converge on DAF-12, a nuclear hormone receptor that triggers the changes required to initiate dauer formation. DAF-12 is related to the vitamin D, liver-X, and androstane receptors, and like these human receptors, it responds to lipophilic hormone ligands. When bound to its ligand, DAF-12 acquires transcriptional activity that directs reproductive development, while unliganded DAF-12 forms a dauer-specifying complex with its interacting protein DIN-1S to regulate the transcription of genes required for dauer development. We report here that din-1S is required in parallel to par-4/LKB1 signaling within the gonad to establish cell cycle quiescence during the onset of the dauer stage. We show that din-1S is important for postdauer reproduction when ILS is impaired and is necessary for long-term dauer survival in response to reduced ILS. Our work uncovers several previously uncharacterized functions of DIN-1S in executing and maintaining many of the cellular and physiological processes required for appropriate dauer arrest, while also shedding light on the coordination of nuclear hormone signaling, the LKB1/AMPK signaling cascade, and ILS/TGFβ in the control of cell cycle quiescence and tissue growth: a key feature that is often misregulated in a number of hormone-dependent cancers.

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.

LKB1 and Notch Pathways Interact and Control Biliary Morphogenesis.

BackgroundLKB1 is an evolutionary conserved kinase implicated in a wide range of cellular functions including inhibition of cell proliferation, regulation of cell polarity and metabolism. When Lkb1 is inactivated in the liver, glucose homeostasis is perturbed, cellular polarity is affected and cholestasis develops. Cholestasis occurs as a result from deficient bile duct development, yet how LKB1 impacts on biliary morphogenesis is unknown.Methodology/Principal FindingsWe characterized the phenotype of mice in which deletion of the Lkb1 gene has been specifically targeted to the hepatoblasts. Our results confirmed that lack of LKB1 in the liver results in bile duct paucity leading to cholestasis. Immunostaining analysis at a prenatal stage showed that LKB1 is not required for differentiation of hepatoblasts to cholangiocyte precursors but promotes maturation of the primitive ductal structures to mature bile ducts. This phenotype is similar to that obtained upon inactivation of Notch signaling in the liver. We tested the hypothesis of a functional overlap between the LKB1 and Notch pathways by gene expression profiling of livers deficient in Lkb1 or in the Notch mediator RbpJκ and identified a mutual cross-talk between LKB1 and Notch signaling. In vitro experiments confirmed that Notch activity was deficient upon LKB1 loss.ConclusionLKB1 and Notch share a common genetic program in the liver, and regulate bile duct morphogenesis.

Physiological extracellular electrical signals guide and orient the polarity of gut epithelial cells

Apical-basal polarity in epithelial cells is a fundamental process in the morphogenesis of many tissues. But how epithelial cells become oriented with functionally specialized luminal and serosal facing membranes is not understood fully. Cell-cell and cell-substrate contacts induce the asymmetric distribution of Na+/K+-ATPase pumps on basal membrane and are essential for apical-basal polarity formation. Inhibition of the Na+/K+-ATPase pump abolished apical formation completely. But it is unclear how this pump regulated the apical polarity. We discovered that the transepithelial potential difference (TEP) which is dependent on the basal Na+/K+-ATPase distribution acts as an essential coordinating signal for apical membrane formation through Ror2/ERK1/2/LKB1 signaling. A similar concept applies to all other ion-transporting epithelial and endothelial tissues and this raises the possibility of regulating the TEP as a therapeutic intervention for disorders in which epithelial function is compromised by faulty electrical signaling.

Imiquimod-induced AMPK activation causes translation attenuation and apoptosis but not autophagy

BackgroundAMP-activated protein kinase (AMPK), a principal intracellular energy sensor, plays a crucial role in cell growth, proliferation, apoptosis and autophagy. Imiquimod (IMQ) directly exhibits anti-tumor activity through the induction of apoptosis and autophagic cell death. ObjectiveTo evaluate the role of AMPK in IMQ-induced apoptosis and autophagy. MethodsThe phosphorylation of AMPK and its substrates was detected by immunoblotting. ATP contents were analyzed by an ATP bioluminescence assay. The upstream signaling for AMPK activation was dissected by examination of TLR7/8 expression, over-expression of TLR7/8, the addition of AMPK kinase inhibitors, and the genetic silencing of Myd88 and LKB1. The role of AMPK activation in IMQ-induced autophagy and apoptosis was assessed by inhibiting AMPK, genetically silencing AMPK and over-expressing AMPK dominant-negative mutants. Autophagy and apoptosis were evaluated by a DNA content assay, immunoblotting, EGFP-LC3 puncta detection and acridine orange staining. ResultsIMQ could activate AMPK and autophagy in cancer cells not expressing TLR7/8. IMQ caused ATP depletion and induced LKB1-mediated AMPK activation. The down-regulation of AMPK activity via pharmacological inhibition and genetic silencing resulted in reduced IMQ-induced apoptosis but did not influence autophagy, and this rescue effect was associated with the retention of translation factor activity and anti-apoptotic Bcl-2 family member Mcl-1 protein expression levels. ConclusionIMQ induces AMPK activation independent of TLR7/8 expression, resulting in translation inhibition and subsequent apoptosis through ATP depletion and LKB1 signaling, in skin tumor cells.

Gene Network Analysis of Glucose Linked Signaling Pathways and Their Role in Human Hepatocellular Carcinoma Cell Growth and Survival in HuH7 and HepG2 Cell Lines.

Cancer progression may be affected by metabolism. In this study, we aimed to analyze the effect of glucose on the proliferation and/or survival of human hepatocellular carcinoma (HCC) cells. Human gene datasets regulated by glucose were compared to gene datasets either dysregulated in HCC or regulated by other signaling pathways. Significant numbers of common genes suggested putative involvement in transcriptional regulations by glucose. Real-time proliferation assays using high (4.5 g/L) versus low (1 g/L) glucose on two human HCC cell lines and specific inhibitors of selected pathways were used for experimental validations. High glucose promoted HuH7 cell proliferation but not that of HepG2 cell line. Gene network analyses suggest that gene transcription by glucose could be mediated at 92% through ChREBP in HepG2 cells, compared to 40% in either other human cells or rodent healthy liver, with alteration of LKB1 (serine/threonine kinase 11) and NOX (NADPH oxidases) signaling pathways and loss of transcriptional regulation of PPARGC1A (peroxisome-proliferator activated receptors gamma coactivator 1) target genes by high glucose. Both PPARA and PPARGC1A regulate transcription of genes commonly regulated by glycolysis, by the antidiabetic agent metformin and by NOX, suggesting their major interplay in the control of HCC progression.

Stroke literature synopses: basic science.

Stroke literature synopses: basic science.

Metabolic regulator LKB1 is crucial for Schwann cell-mediated axon maintenance.

Schwann cells (SCs) promote axonal integrity independently of myelination by poorly understood mechanisms. Current models suggest that SC metabolism is critical for this support function and that SC metabolic deficits may lead to axonal demise. The LKB1-AMP-activated protein kinase (AMPK) kinase pathway targets several downstream effectors, including mammalian target of rapamycin (mTOR), and is a key metabolic regulator implicated in metabolic diseases. We found through molecular, structural and behavioral characterization of SC-specific mutant mice that LKB1 activity is central to axon stability, whereas AMPK and mTOR in SCs are largely dispensable. The degeneration of axons in LKB1 mutants was most dramatic in unmyelinated small sensory fibers, whereas motor axons were comparatively spared. LKB1 deletion in SCs led to abnormalities in nerve energy and lipid homeostasis and to increased lactate release. The latter acts in a compensatory manner to support distressed axons. LKB1 signaling is essential for SC-mediated axon support, a function that may be dysregulated in diabetic neuropathy.

LKB1 signalling attenuates early events of adipogenesis and responds to adipogenic cues.

cAMP-response element-binding protein (CREB) is required for the induction of adipogenic transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs). Interestingly, it is known from studies in other tissues that LKB1 and its substrates AMP-activated protein kinase (AMPK) and salt-inducible kinases (SIKs) negatively regulate gene expression by phosphorylating the CREB co-activator CRTC2 and class IIa histone deacetylases (HDACs), which results in their exclusion from the nucleus where they co-activate or inhibit their targets. In this study, we show that AMPK/SIK signalling is acutely attenuated during adipogenic differentiation of 3T3-L1 preadipocytes, which coincides with the dephosphorylation and nuclear translocation of CRTC2 and HDAC4. When subjected to differentiation, 3T3-L1 preadipocytes in which the expression of LKB1 was stably reduced using shRNA (Lkb1-shRNA), as well as Lkb1-knockout mouse embryonic fibroblasts (Lkb1(-/-) MEFs), differentiated more readily into adipocyte-like cells and accumulated more triglycerides compared with scrambled-shRNA-expressing 3T3-L1 cells or Wt MEFs. In addition, the phosphorylation of CRTC2 and HDAC4 was reduced, and the mRNA expression of adipogenic transcription factors Cebpa, peroxisome proliferator-activated receptor γ (Pparg) and adipocyte-specific proteins such as hormone-sensitive lipase (HSL), fatty acid synthase (FAS), aP2, GLUT4 and adiponectin was increased in the absence of LKB1. The mRNA and protein expression of Ddit3/CHOP10, a dominant-negative member of the C/EBP family, was reduced in Lkb1-shRNA-expressing cells, providing a potential mechanism for the up-regulation of Pparg and Cebpa expression. These results support the hypothesis that LKB1 signalling keeps preadipocytes in their non-differentiated form.

Targeting the LKB1 Tumor Suppressor

LKB1 (also known as serine-threonine kinase 11, STK11) is a tumor suppressor, which is mutated or deleted in Peutz-Jeghers syndrome (PJS) and in a variety of cancers. Physiologically, LKB1 possesses multiple cellular functions in the regulation of cell bioenergetics metabolism, cell cycle arrest, embryo development, cell polarity, and apoptosis. New studies demonstrated that LKB1 may also play a role in the maintenance of function and dynamics of hematopoietic stem cells. Over the past years, personalized therapy targeting specific genetic aberrations has attracted intense interests. Within this review, several agents with potential activity against aberrant LKB1 signaling have been discussed. Potential strategies and challenges in targeting LKB1 inactivation are also considered.

Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer

Epithelial ovarian cancer presents mostly with serous, endometrioid or mucinous histology but is treated as a single disease. The development of histotype-specific therapy has been challenging because of the relative lack of studies attributing disrupted pathways to a distinct histotype differentiation. mTOR activation is frequently associated with poor prognosis in serous ovarian cancer, which is the most common and most deadly histotype. However, the mechanisms dysregulating mTOR in the pathogenesis of ovarian cancer are unknown. We detected copy number loss and correlated lower expression levels of LKB1, TSC1, TSC2 and PTEN tumor suppressor genes for upstream regulators of mTOR activity in up to 80% in primary ovarian serous tumor databases, with LKB1 allelic loss-predominant. Reduced LKB1 protein was usually associated with increased mTOR activity in both serous ovarian cancer cell lines and primary tumors. Conditional deletion of Lkb1 in murine ovarian surface epithelial (OSE) cells caused papillary hyperplasia and shedding but not tumors. Simultaneous deletion of Lkb1 and Pten, however, led to development of high-grade ovarian serous histotype tumors with 100% penetrance that expressed WT1, ERα, PAX8, TP53 and cytokeratin 8, typical markers used in the differential diagnosis of serous ovarian cancer. Neither hysterectomy nor salpingectomy interfered with progression of ovarian tumorigenesis, suggesting that neither uterine nor Fallopian tube epithelial cells were contributing to tumorigenesis. These results implicate LKB1 loss in the OSE in the pathogenesis of serous ovarian cancer and provide a compelling rationale for investigating the therapeutic potential of targeting LKB1 signaling in patients with this deadly disease.

Abstract C55: Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency

Abstract Lkb1 has emerged as an important tumor suppressor gene in both sporadic and hereditary cancer. Inactivating germline mutations in the LKB1 gene underlie familial Peutz-Jeghers Syndrome (PJS), a cancer-prone disorder characterized by predisposition to gastrointestinal polyposis. Targeted disruption of Lkb1 in mesenchymal smooth muscle cells was sufficient to recapitulate PJS-type polyposis in mice (Katajisto et al, 2008) and Lkb1 heterozygous mice develop osteoblastic tumors (Robinson et al, 2008). To investigate the mechanism(s) by which Lkb1 loss in mesenchymal tissues drives tumorigenesis, we deleted Lkb1 using two mesenchymal deletors (Fsp1-Cre and Twist2-Cre) expressed in mesenchymal progenitors. Heterozygous deletion of Lkb1 with either Fsp1-Cre (Lkb1flox/+;Fsp1-Cre) or Twist2-Cre (Lkb1flox/+;Twist2-Cre) results in both polyposis and osteoblastic tumor. Homozygous Lkb1 deletion using Fsp1-Cre (Lkb1flox/flox;Fsp1-Cre) leads to rapid polyposis and osteoblastic tumors with complete penetrance, indicating that complete loss of Lkb1 is advantageous for tumorigenesis. AMPK, a key substrate of Lkb1 connected to several other tumor suppressors, has been proposed to mediate Lkb1 tumor suppressive function. However, homozygous deletion of both AMPKα1 and AMPKα2 using Fsp1-Cre does not lead to tumorigenesis. These results implicate other Lkb1 substrate kinases in tumor suppression and suggest Lkb1 loss in a common progenitor underlies mesenchymal tumorigenesis. Katajisto P, et al., (2008) LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis. Nat Genet, 40(4): p. 455-9 Robinson J, et al., (2008) Osteogenic tumours in Lkb1-deficient mice. Exp Mol Pathol, 85(3): p. 223-6 Citation Format: Kaisa Laajanen, Saara Ollila, Iris PL Wong, Kari Vaahtomeri, Gustavo Leone, Benoit Viollet, Makela P. Tomi. Investigating mechanisms of gastrointestinal polyposis and osteoblastic tumor formation induced by mesenchymal Lkb1 deficiency. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C55.

The tumor suppressor LKB1 antagonizes WNT signaling pathway through modulating GSK3β activity in cell growth of esophageal carcinoma

The tumor suppressor LKB1 gene encodes a serine-threonine kinase that regulates cell proliferation and polarity. Inactivation of LKB1 by mutations in LKB1 or loss of its expression is highly correlated with lung, ovarian, and pancreatic cancers, and WNT/β-catenin pathway is also known to be involved in many human malignancies. However, the relationship between LKB1 and WNT signaling pathway in esophageal carcinoma remains unknown. The expression of LKB1 in 62 cases of esophageal cancer patients was determined by quantitative real-time PCR. It was found that LKB1 mRNA level was significantly lower than the adjacent normal epithelium and that the LKB1 downregulation was correlating with TNM stages. Moreover, the expression of WNT target genes such as Cyclin D1, C-MYC, MMP2, and FZD2 was significantly upregulated in esophageal cancer tissues. LKB1 overexpression in TE10 cells inhibited TOPFlash luciferase reporter activity and WNT target gene expression even in the presence of WNT3A. Conversely, LKB1 knockdown enhanced WNT signaling activity in esophageal cancer cells. It was also found that LKB1 antagonized WNT signaling pathway through interaction with GSK3β to downregulate β-catenin expression level. Functional investigation revealed that LKB1 suppressed the promotion effects of WNT3A on the cell growth of TE10 cells. The LKB1 functions in regulating cell growth and WNT target genes expression were impaired by GSK3β inhibition, suggesting that LKB1 antagonized WNT-induced cell proliferation through enhancement of GSK3β activity. Together, the interaction between LKB1 and GSK3β upregulates GSK3β activity to suppress WNT-induced cell proliferation in esophageal carcinoma cells. Loss of LKB1 expression may result in the deregulation of WNT/β-catenin pathway to promote malignant progression of esophageal cancer.

Galangin inhibits proliferation of HepG2 cells by activating AMPK via increasing the AMP/TAN ratio in a LKB1-independent manner

Galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis and autophagy to suppress the proliferation of HepG2 cells. In this study, we demonstrated that galangin induced autophagy by increasing the ratio of AMP/TAN in HepG2 cells. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and LKB1, but inhibited the phosphorylation of AKT and mTOR. Inhibition of AMPK activation suppressed the dephosphorylation of mTOR to block galangin-induced autophagy. AMPK activation by galangin appeared to be independent of the LKB1 signaling pathway because the down-regulation of LKB1 by its siRNA failed to affect galangin-induced autophagy. Collectively, the findings demonstrated a novel mechanism of how galangin induces autophagy via activating AMPK in a LKB1- independent manner. The induction of autophagy can thus reflect the anti-proliferation effect of galangin in HCC cells.

L-4F REVERSES THE EXPERIMENTAL METABOLIC SYNDROME VIA THE HO-1, ADIPONECTIN AND LKB1 SIGNALLING PATHWAY

ObjectivesThe hallmarks of metabolic syndrome such as obesity, insulin resistance, hyperglycemia and associated increase in reactive oxygen species (ROS) are known to decrease hame oxygenase (HO) levels. There are two...

Stromal Liver Kinase B1 [STK11] Signaling Loss Induces Oviductal Adenomas and Endometrial Cancer by Activating Mammalian Target of Rapamycin Complex 1

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

Progress of related targets in LKB1 signaling pathway for non-small cell lung cancer therapy

Progress of Related Targets in LKB1 Signaling Pathway for Non-small Cell Lung Cancer Therapy Jing WANG, Ruili HAN, Diansheng ZHONG Tianjin Lung Cancer Institute; Department of Respriatory Medicine, Tianjin Medical University General Hospital, Tianjin 300052, China Corresponding author: Diansheng ZHONG, E-mail: zhongdsh@hotmail.com This study was supported by the grants from National Natural Science Foundation of China ( to Diansheng ZHONG)(No.30971307, No.81071915) and Tianjin Natural Science Foundation (to Diansheng ZHONG)(No.10JCYBJC13 700). DOI: 10.3779/j.issn.1009-3419.2012.07.08

Altered LKB1/AMPK/TSC1/TSC2/mTOR signaling causes disruption of Sertoli cell polarity and spermatogenesis

Male patients with Peutz-Jeghers syndrome (PJS) have defective spermatogenesis and are at increased risk of developing Sertoli cell tumors. Mutations in the Liver Kinase B1 (LKB1/STK11) gene are associated with the pathogenesis of PJS and have been identified in non-PJS patients with sporadic testicular cancers. The mechanisms controlled by LKB1 signaling in Sertoli cell functions and testicular biology have not been described. We have conditionally deleted the Lkb1 gene (Lkb1(cko)) in somatic testicular cells to define the molecular mechanisms involved in the development of the testicular phenotype observed in PJS patients. Focal vacuolization in some of the seminiferous tubules was observed in 4-week-old mutant testes but germ cell development appeared to be normal. However, similar to PJS patients, we observed progressive germ cell loss and Sertoli cell only tubules in Lkb1(cko) testes from mice older than 10 weeks, accompanied by defects in Sertoli cell polarity and testicular junctional complexes and decreased activation of the MAP/microtubule affinity regulating and focal adhesion kinases. Suppression of AMP kinase and activation of mammalian target of rapamycin (mTOR) signaling were also observed in Lkb1(cko) testes. Loss of Tsc1 or Tsc2 copies the progressive Lkb1(cko) phenotype, suggesting that dysregulated activation of mTOR contributes to the pathogenesis of the Lkb1(cko) testicular phenotype. Pten(cko) mice had a normal testicular phenotype, which could be explained by the comparative lack of mTOR activation detected. These studies describe the importance of LKB1 signaling in testicular biology and the possible molecular mechanisms driving the pathogenesis of the testicular defects observed in PJS patients.

Hepatoma Cells From Mice Deficient in Glycine N-Methyltransferase Have Increased RAS Signaling and Activation of Liver Kinase B1

Patients with cirrhosis are at high risk for developing hepatocellular carcinoma (HCC), and their liver tissues have abnormal levels of S-adenosylmethionine (SAMe). Glycine N-methyltransferase (GNMT) catabolizes SAMe, but its expression is down-regulated in HCC cells. Mice that lack GNMT develop fibrosis and hepatomas and have alterations in signaling pathways involved in carcinogenesis. We investigated the role of GNMT in human HCC cell lines and in liver carcinogenesis in mice. We studied hepatoma cells from GNMT knockout mice and analyzed the roles of liver kinase B1 (LKB1, STK11) signaling via 5'-adenosine monophosphate-activated protein kinase (AMPK) and Ras in regulating proliferation and transformation. Hepatoma cells from GNMT mice had defects in LKB1 signaling to AMPK, making them resistant to induction of apoptosis by adenosine 3',5'-cyclic monophosphate activation of protein kinase A and calcium/calmodulin-dependent protein kinase kinase 2. Ras-mediated hyperactivation of LKB1 promoted proliferation of GNMT-deficient hepatoma cells and required mitogen-activated protein kinase 2 (ERK) and ribosomal protein S6 kinase polypeptide 2 (p90RSK). Ras activation of LKB1 required expression of RAS guanyl releasing protein 3 (RASGRP3). Reduced levels of GNMT and phosphorylation of AMPKα at Thr172 and increased levels of Ras, LKB1, and RASGRP3 in HCC samples from patients were associated with shorter survival times. Reduced expression of GNMT in mouse hepatoma cells and human HCC cells appears to increase activity of LKB1 and RAS; activation of RAS signaling to LKB1 and RASGRP3, via ERK and p90RSK, might be involved in liver carcinogenesis and be used as a prognostic marker. Reagents that disrupt this pathway might be developed to treat patients with HCC.

Tu1946 Osteopontin Prevents Onset of Immune-Mediated Colitis by Inducing Tolerogenic Dendritic Cells

Background We have previously shown that upon formation of immunogenic dendritic cell (DC) T cell contacts, autophagy is induced. This process then functions in a negative feedback loop by destabilization of the interaction. In a subset of Crohn's disease patients, this mechanism is impaired, leading to hyperstable interactions and increased immunogenicity. The correction of autophagy induction after DC-T cell contact in risk allele carriers may form a therapeutic possibilty resulting in normal interaction stability and decreased T cell activation. We therefore sought to determine the signaling events linking DCT cell interactions with the induction of autophagy. Methods Interactions were induced between human monocyte derived DC and allogeneic T cells. Activation of signalling pathways was determined byWestern Blot analysis. Inihibition of signallingmediators was achieved through siRNA and pharmacological inhibitors. Results Upon immunogenic DC-T cell interactions, phosphorylation of AMPKwas increased. AMPK is an inhibitor ofmTOR signaling, and indeed mTOR pathway activity was markedly reduced, as shown by decreased phosphorylation of mTOR itself as well as its downstream target S6. It has been shown previously that inhibition of mTOR is a strong inducer of autophagy. Conversely, HMGB1 and tissue transglutaminase 2, which are also known to influence autophagic activity, did not appear to be involved in autophagy induction under these circumstances. Interestingly, AMPK activation wasmediated through activation of LKB1, a molecule which has been shown to be involved in mediating the polarity of intestinal epithelial cells. We show that LKB1 is recruited to the site of DCT cell contact, and that inhibition of LKB1 signalling results in impaired induction of autophagy after cell-cell contact. Consequently, cell contacts formed by LKB1 deficient DC were hyperstable and resulted in increased immunogenicity, similar to the effects of autophagy deficiency. Conclusion This data shows that upon DC-T cell interaction, autophagy is induced as a regulatory mechanism and this induction is mediated through the LKB1-AMPKmTOR signaling pathway. In subsets of CD patients this regulatory mechanism is disturbed, leading to hyperstable interactions and excessive immunity. Boosting the mTOR pathway may be an interesting therapeutic option in CD patients carrying risk alleles in autophagyrelated genes.