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  current news   Press   selected story    
     
  24 June 2015  
 
ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in hepatocellular carcinoma
 
 




Authors
Jianxiang Chen1,2, Hongping Xia1, Xiaoqian Zhang2, Sekar Karthik1, Seshachalam Veerabrahma Pratap1, Wanjin Hong2 and Kam M. Hui1,2,4

1 Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of   Cancer Research, National Cancer Center, Singapore, Singapore;
2 Institute of Molecular and Cell Biology, A*STAR, Biopolis Drive Proteos, Singapore, Singapore;
3 Cancer and Stem Cell Biology Program, Duke-National University of Singapore Graduate Medical   School, Singapore, Singapore;
4 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore,   Singapore.

Published in Journal of Hepatology, 2015 vol. 62 j 1287–1295

Abstract
Early HCC recurrence is mainly responsible for the poor overall survival of patients with HCC and is the major obstacle to improving prognosis. Therefore, identifying molecules that contribute to HCC recurrence could provide potential targets for novel therapeutic strategies for the clinical management of HCC. Gene and protein expression profiles of ECT2 were analyzed by microarrays, immnoblotting and immunohistochemistry in human HCC samples. siRNA and lentivirus-based gene knockdown were employed to dissect the molecular functions of ECT2 in vitro and in vivo. Upregulation of ECT2 is significantly associated with early HCC tumour recurrence and poor survival. Expression and function of ECT2 was negatively regulated by TP53. Knockdown of ECT2 induced apoptosis, attenuated oncogenicity and the ability of HCC cells to metastasis by markedly suppressed the activation of Rho GTPases. Knockdown ECT2 or Rho also suppressed ERK activation and silencing of Rho or ERK markedly reduced cell migration. Stable ECT2 knockdown in HCC patient-derived xenografts resulted in significant retardation of tumour growth and suppressed ERK activation. High expression of ECT2 correlated with high ERK phosphorylation and poor survival of HCC patients. Furthermore, ECT2 regulate the expression and stability of RacGAP1 to accelerate ECT2-mediated Rho activation to promote metastasis. Conclusions: ECT2 is closely associated with the activation of the Rho/ERK signalling axis to promote early HCC recurrence. ECT2 crosstalks with RacGAP1 to promote GTP exchange ability of Rho signalling in HCC.

Figure:

Figure legend: A Microarray analysis of ECT2 gene expression in histologically normal liver tissues from patients with colorectal metastases (NN), matched normal liver of HCC patients (MN-HCC), recurrent tumours (R-HCC), and non-recurrent tumours (NR-HCC). B, The subcutaneous xenograft mice model using ECT2 stable knockdown HCCLM3 cells was used to investigate the tumorigenesis. After 5 weeks, the mice and tumours were photographed and the changes in tumour volume were analysed. C, Human Phospho-Kinase antibody analysis of the HCCLM3 cells transfected with si RNAs targeting Scramble control and ECT2 genes. The p-ERK1/2 was significantly decreased while HSP60 was upregulated markedly after ECT2 gene knockdown, and they were shown in black frames. The “R” indicates reference control dots. D, Diagram depicting upstream and downstream signals of ECT2 that might contribute to HCC recurrence. → indicates direct activation, ┤indicates direct inhibition, and dotted arrow indicates possible indirect activation. E, Representative confocal images of Duolink assay of ECT2 and RacGAP1 interaction. The rabbit and mouse IgG antibodies were used for the control group. Duolink-positive dots were calculated per cell both in interphase and mitosis (n=60). F, Endogenous co-IP was used to determine interaction between ECT2 and RacGAP1 in HCCLM3 cells. 1% Immunoprecipitation lysate was used for input detection. G, RhoA GTP pull-down assay was analyzed by Western blotting 48 h after siRNA treatment. Scram: scramble siRNAs. Relative protein expression was calculated as RhoA-GTP/total RhoA by Image-J (NIH, USA). Relative expression of the scramble siRNA group was taken as 1. GAPDH and HSP70 were used as loading controls. H, Transwell migration assay was analyzed and the migrated and stained cells were calculated.


For more information on Wanjin HONG 's laboratory, please click here.