Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer

Diego F. Calvisi, Sara Ladu, Elizabeth A. Conner, Daekwan Seo, Jer Tsong Hsieh, Valentina M. Factor, Snorri S. Thorgeirsson

Research output: Contribution to journalArticle

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Abstract

Background & Aims: 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.

Original languageEnglish (US)
Pages (from-to)311-319
Number of pages9
JournalJournal of Hepatology
Volume54
Issue number2
DOIs
StatePublished - Feb 2011

Fingerprint

ras GTPase-Activating Proteins
Liver Neoplasms
Hepatocellular Carcinoma
GTPase-Activating Proteins
Mutation
Apoptosis
Immunoblotting
Microsatellite Repeats
Methylation
Small Interfering RNA
Transfection
Real-Time Polymerase Chain Reaction
Down-Regulation
Cell Line
Survival

Keywords

  • HCC
  • Methylation
  • Ras GAPs

ASJC Scopus subject areas

  • Hepatology

Cite this

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. / Calvisi, Diego F.; Ladu, Sara; Conner, Elizabeth A.; Seo, Daekwan; Hsieh, Jer Tsong; Factor, Valentina M.; Thorgeirsson, Snorri S.

In: Journal of Hepatology, Vol. 54, No. 2, 02.2011, p. 311-319.

Research output: Contribution to journalArticle

Calvisi, Diego F. ; Ladu, Sara ; Conner, Elizabeth A. ; Seo, Daekwan ; Hsieh, Jer Tsong ; Factor, Valentina M. ; Thorgeirsson, Snorri S. / Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer. In: Journal of Hepatology. 2011 ; Vol. 54, No. 2. pp. 311-319.
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AU - Calvisi, Diego F.

AU - Ladu, Sara

AU - Conner, Elizabeth A.

AU - Seo, Daekwan

AU - Hsieh, Jer Tsong

AU - Factor, Valentina M.

AU - Thorgeirsson, Snorri S.

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N2 - Background & Aims: 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.

AB - Background & Aims: 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.

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