Research Article

ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in human hepatocellular carcinoma Jianxiang Chen1, Hongping Xia1, Xiaoqian Zhang2, Sekar Karthik1, Seshachalam Veerabrahma Pratap1, London Lucien Ooi3, Wanjin Hong2, Kam M. Hui1,2,4,5,⇑ 1 Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore; 2Institute of Molecular and Cell Biology, A⁄STAR, Biopolis Drive Proteos, Singapore, Singapore; 3Division of Surgical Oncology, National Cancer Centre, Singapore 169610, Singapore; 4Cancer & Stem Cell Biology Program, Duke-National University of Singapore Graduate Medical School, Singapore, Singapore; 5Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Background & Aims: Early recurrence is the major obstacle for improving the outcome of patients with hepatocellular carcinoma (HCC). Therefore, identifying key molecules contributing to early HCC recurrence can enable the development of novel therapeutic strategies for the clinical management of HCC. Epithelial cell transforming sequence 2 (ECT2) has been implicated in human cancers, but its function in HCC is largely unknown. Methods: ECT2 expression was studied by microarrays, immunoblotting and immunohistochemistry in human HCC samples. siRNA- and lentiviral vector-mediated knockdown were employed to decipher the molecular functions of ECT2. Results: The upregulation of ECT2 is significantly associated with early recurrent HCC disease and poor survival. Knockdown of ECT2 markedly suppressed Rho GTPases activities, enhanced apoptosis, attenuated oncogenicity and reduced the metastatic ability of HCC cells. Moreover, knockdown of ECT2 or Rho also suppressed ERK activation, while the silencing of Rho or ERK led to a marked reduction in cell migration. Stable knockdown of ECT2 in vivo resulted in significant retardation of tumour growth and the suppression of ERK activation. High expression of ECT2 correlates with high ERK phosphorylation and poor survival of HCC patients. Furthermore, ECT2 enhances the expression and stability of RACGAP1, accelerating 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. In addition, ECT2 can crosstalk with RACGAP1 to catalyse the GTP exchange involved in Rho signalling to further regulate tumour initiation and metastasis. Ó 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Hepatocellular carcinoma (HCC); Early recurrence; ERK activation; GTP exchange; Rho GTPases; RACGAP1. Received 22 July 2014; received in revised form 9 December 2014; accepted 8 January 2015 ⇑ Corresponding author. Address: Division of Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore. Tel.: +65 6436 8338; fax: +65 6226 3843. E-mail address: [email protected] (K.M. Hu).

Introduction Hepatocellular carcinoma (HCC) is a highly lethal malignancy [1– 4]. Early HCC recurrence is implicated in poor patient survival and is the major obstacle to improving prognosis [2,3]. Therefore, identifying molecules that contribute to early HCC recurrence could provide potential targets for developing novel therapeutic strategies to clinically manage HCC [5]. Epithelial cell transforming sequence 2 (ECT2) interacts with several members of the Rho GTPase family and is one of the most important factors catalysing guanine nucleotide exchange (GEF) on the small Rho GTPases [6–8]. ECT2 was originally reported as an oncogene that transformed NIH-3T3 cells, and ECT2 lacking the N-terminal was reported to lead to malignant transformation of NIH-3T3 cells and MAPK activation including JNK, p38 and ERK [9]. The overexpression of ECT2 and its potential downstream molecules have been described in several human cancers including lung, oesophageal and glioblastoma [6,8,10–12]. ECT2 has also been shown to be negatively regulated by wild-type p53 expression via protein methyltransferases in human non-small cell lung carcinoma H1299 and breast cancer MCF7 cells [13]. In non-small-cell lung cancer (NSCLC), ECT2 was shown to be associated with Rac1 activation leading to ECT2-dependent NSCLC anchorage-independent growth and invasion in vitro [11]. In addition, ECT2 has also been reported to interact with RacGAP1 in cytokinesis [14,15] and to be involved in epithelial cell polarity and migration [8,16,17]. However, little is known about the molecular role of ECT2 in HCC. This study demonstrated that ECT2 is highly expressed in early recurrent HCC tumours and is closely associated with activation of the Rho/ERK signalling axis to promote early HCC recurrence. In addition, ECT2 can work in conjunction with RACGAP1 to catalyse the GTP exchange involved in Rho signalling to further regulate tumour initiation and metastasis.

Journal of Hepatology 2015 vol. xxx j xxx–xxx

Please cite this article in press as: Chen J et al. ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in human hepatocellular carcinoma. J Hepatol (2015), http://dx.doi.org/10.1016/j.jhep.2015.01.014

Research Article Materials and methods

old male BALB/c nude mice by subcutaneous injection. Student’s t test was used to evaluate differences between the tumour sizes in the shScramble- and shECT2transfected groups. Detailed information on the other materials and methods is in the Supplementary Materials and methods.

Tissues The collection of tumour and adjacent normal liver tissue from HCC patients was approved by our Institutional Review Board (IRB) and all tissues studied were provided by the Tissue Repository of the National Cancer Centre Singapore (NCCS). Written informed consent was obtained from participating patients and relevant clinical and histopathological data provided to the researchers in an anonymised manner.

Results ECT2 is significantly upregulated in HCC and correlates with early tumour recurrence and patient survival

Animal studies

2 C C

H

C C H

#2

1

0

#2

#2

0

C

#1

C

C C H

H

T N T N T N

H

#9 C

C H

C

C

#8

#7 C

HCC tissues

T N

T N T N T N 100 kDa 36 kDa

Recurrence

Non-recurrence

ETC2

-H

-H

100 kDa 36 kDa

GAPDH

N # H 2 uH H 7 ep H 3b ep PL G2 C M /PR ah F /5 H lavu LE SK SN He U p1 H 44 C 9 C M LM H 3 C M C9 H 7 C H C 97 L

R

R N

100 kDa 36 kDa

D

Patients’ tissues

Normal

M

#1

T N T N T N

ETC2 GAPDH

n = 36

C

C C

C C

N

-H N M N M

HCC cell lines

C

n = 10

4

H

N

n =10 n = 41 n = 70

6

C

2

T N T N T N

C

4

8

C

6

T N

ETC2 GAPDH

10

8

0

***

C

10

12

H

***

ECT2 mRNA expression (log2)

ECT2 mRNA expression (log2)

12

H C C #3 H C C #4 H C C #5 H C C #1 4 H C C #1 5 H C C #1 6

B

A

H C C #2

To identify genes associated with early recurrent HCC disease, we have previously analysed and compared the gene expression profiles of 121 liver tissues: 10 histologically normal liver tissues from patients with colorectal cancer that metastasized to the liver (NN), 41 histologically normal adjacent liver tissues from

All experiments on mice were approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). Tumour volumes (V) were monitored every week and calculated according to the formula V = 0.52  length  width2, as previously described [18]. Stably transfected HCCLM3 cells were resuspended in PBS and implanted into the left and right flanks (5  106 cells per flank) of 6-weeks-

ETC2 GAPDH

ETC2 low (R = 11, NR = 23)

0.8 0.6

ETC2 low ETC2 high ETC2 low-censored ETC2 high-censored

0.4

ETC2 high (R = 25, NR = 8) 0.2 0.0

Log-rank, p