Hepatoma cells from mice deficient in glycine N-methyltransferase have increased RAS signaling and activation of liver kinase B1.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain. Electronic address: mlmartinez@cicbiogune.es.
Nuria
N
Martínez-López
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Juan L
JL
García-Rodríguez
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Marta
M
Varela-Rey
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Virginia
V
Gutiérrez
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
David
D
Fernández-Ramos
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Naiara
N
Beraza
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Ana M
AM
Aransay
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
Karin
K
Schlangen
Centro de Investigación Biomédica en red de Enfermedades Hepáticas y Digestivas, Hospital Clinic, Centre Esther Koplovitz, Barcelona, Spain.
Juan Jose
JJ
Lozano
Department of Physiology, University of the Basque Country Medical School, Bilbao, Spain.
Patricia
P
Aspichueta
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee.
Zigmund
Z
Luka
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee.
Conrad
C
Wagner
Institute of Pathology, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
Matthias
M
Evert
Institute of Pathology, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
Diego F
DF
Calvisi
Division of Gastrointestinal and Liver Diseases, USC Research Center for Liver Diseases, Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California.
Shelly C
SC
Lu
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
José M
JM
Mato
CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Technology Park of Bizkaia, Bizkaia, Spain.
María L
ML
Martínez-Chantar
D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001374:Azacitidine / Q000494:pharmacology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019175:DNA Methylation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050938:Glycine N-Methyltransferase / Q000172:deficiency* / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008819:Mice, Nude; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; C413212:RASGRP3 protein, human; D020663:ras Guanine Nucleotide Exchange Factors; D050938:Glycine N-Methyltransferase; C494511:Gnmt protein, mouse; D017346:Protein Serine-Threonine Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C491654:Stk11 protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D019950:Mitogen-Activated Protein Kinase 1; D055372:AMP-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D001374:Azacitidine