Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer.
OBJECTIVE
Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis.


METHODS
The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines.


RESULTS
In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis.


CONCLUSIONS
Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.
Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4262, USA.
Diego F
DF
Calvisi
Sara
S
Ladu
Elizabeth A
EA
Conner
Daekwan
D
Seo
Jer-Tsong
JT
Hsieh
Valentina M
VM
Factor
Snorri S
SS
Thorgeirsson
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000209:etiology* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation; D019175:DNA Methylation; D006801:Humans; D008113:Liver Neoplasms / Q000209:etiology* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019656:Loss of Heterozygosity; D048848:MAP Kinase Kinase Kinase 5 / Q000502:physiology; D051761:Paired Box Transcription Factors / Q000037:antagonists & inhibitors; D051966:Phospholipase C gamma / Q000502:physiology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology; D020703:ras GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018631:ras Proteins / Q000235:genetics / Q000502:physiology*
C462952:DAB2IP protein, human; D051761:Paired Box Transcription Factors; C101485:homeobox protein PITX1; D020703:ras GTPase-Activating Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D051966:Phospholipase C gamma; D018631:ras Proteins