Deleted in liver cancer 2 suppresses cell growth via the regulation of the Raf-1-ERK1/2-p70S6K signalling pathway.
BACKGROUND
Deleted in liver cancer 2 (DLC2) gene, a putative tumour suppressor gene, encodes a Rho GTPase-activating protein (RhoGAP) with GAP activity specific for RhoA. It exhibits tumour suppressor functions and inhibits tumour cell proliferation, migration as well as transformation.


OBJECTIVE
In this study, we aimed to investigate the underlying mechanisms of the DLC2 gene in suppressing cell migration and cell growth. HepG2 hepatoma cells were stably transfected with the DLC2γ isoform, which contains the RhoGAP domain.


RESULTS
On performing immunofluorescence staining and Western blot analysis, the expression of the focal adhesion protein paxillin was found to be much reduced in DLC2γ-stable clones. Upon flow cytometric analysis of the cell cycle profiles, the DLC2γ-stable clones were shown to have a higher population of cells arrested at the G1 phase than the EGFP vector-stable clone, suggesting that downregulation of RhoA activity in DLC2γ-stable clones inhibited cell cycle progression. In the DLC2γ-stable clone, the levels of Raf-1 and extracellular signal-regulated kinase (ERK) 1/2 were decreased as compared with those of the parental HepG2, EGFP vector and DLC2γ-GAP defective mutant-stable clones. Furthermore, the ribosomal kinase p70S6K, a downstream target of ERK1/2, was suppressed in the DLC2-stable clones. On the contrary, when DLC2 was knocked down by siRNA in HepG2 cells, the expression levels of phospho-p70S6K and phospho-ERK1/2 were upregulated.


CONCLUSIONS
Our data show that DLC2 inhibits the activity of Raf-1-ERK1/2-p70S6K via its RhoGAP function, resulting in the suppression of cell growth. Further studies on the molecular signalling between DLC2 and p70S6K may provide an insight into its growth suppressor function.
Leung
Thomas Ho-Yin
TH
Department of Pathology, The University of Hong Kong, Hong Kong, China.
Yam
Judy Wai-Ping
JW
Chan
Lo Kong
LK
Ching
Yick-Pang
YP
Ng
Irene Oi-Lin
IO
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051419:Paxillin / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000502:physiology*
D020690:GTPase-Activating Proteins; C496061:PXN protein, human; D051419:Paxillin; D034741:RNA, Small Interfering; C473607:STARD13 protein, human; D025521:Tumor Suppressor Proteins; C085379:rho GTPase-activating protein; D019908:Proto-Oncogene Proteins c-raf; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D048049:Extracellular Signal-Regulated MAP Kinases