ERK signalling axis to promote early recurrence in human hepatocellular carcinoma

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

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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

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ECT2 mRNA expression (log2)

ECT2 mRNA expression (log2)

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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-

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Fig. 1. Upregulation of ECT2 in HCC tissues is associated with early tumour recurrence. (A) Microarray analysis of ECT2 gene expression in histologically normal liver tissues from patients with colorectal metastases (NN), matched normal livers of HCC patients (MN-HCC), recurrent tumours (R-HCC), and non-recurrent tumours (NR HCC). (B) The expression of ECT2 and GAPDH was compared by Western blotting in 14 pairs of HCC patient tissues (T, tumour; N, matched normal liver) and in 7 R and 7 NR tumours. (C) ECT2 expression in HCC cell lines by Western blotting. (D) Representative images of ECT2 expression in HCC patients’ tissues by IHC. (E) Expression of ECT2 was associated with disease-free survival (DFS) in HCC patients.

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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

JOURNAL OF HEPATOLOGY HCC patients (MN) and 70 HCC tumour tissues including 34 nonrecurrent (NR)-HCC (with no recurrent disease observed in P5 years) and 36 recurrent (R) HCC (with recurrent disease detected in 62 years) samples [19]. ECT2 expression was significantly upregulated in tumour samples compared to both NN and MN tissues (p <0.001; Fig. 1A). Furthermore, ECT2 expression was markedly upregulated in early recurrent tissues compared to NRHCC tissues (p <0.001; Fig. 1A). Similarly, ECT2 protein expression was investigated in 14 pairs of HCC tissues (Supplementary Table 1A) and ECT2 was found significantly increased in HCC tumour samples (T) compared to matched normal liver tissues (N) (Fig. 1B). ECT2 protein expression was also markedly elevated in early recurrent HCC samples compared to NR samples (Fig. 1B). ECT2 expression was markedly upregulated in 10 out of the 11 HCC cell lines that we studied (Fig. 1C). Immunohistochemical (IHC) staining confirmed that ECT2 expression was elevated in HCC compared to histologically normal liver tissues, and it was especially higher in recurrent HCC than in NR-HCC. (Fig. 1D). Importantly, it was demonstrated that patients with high ECT2 expression (expression level above the median expression of all the HCC tissues studied) had a significantly shorter disease-free duration compared to patients with low ECT2 expression (expression level below the median expression of all the HCC tissues studied) (p <0.001; Fig. 1E; Supplementary Table 1B). Corroborating these results, univariate and multivariate analyses showed that ECT2 could be an independent predictor of poor prognosis for patients with HCC (Table 1). Taken together, these data identify ECT2 as a novel prognostic factor in HCC. ECT2 promotes proliferation and tumourigenesis of HCC cells To investigate the mechanistic role of ECT2 in HCC, we employed the siRNA or shRNA targeting ECT2 for functional studies. Firstly, knockdown of ECT2 expression by siRNA significantly attenuated the growth of Hep3B and HCCLM3 cells (Fig. 2A), indicating that ECT2 promotes HCC growth. Moreover, ECT2 knockdown increased the level of apoptosisinduced cleaved caspase-3 and DNA damage marker p-Ser139

H2AX in Hep3B, HuH7 and HCCLM3 cells, while it decreased the levels of pro-caspase-9 and PARP-1, compared to siScramble or siLuciferase treatment (Fig. 2B). The percentage of TUNEL-positive cells was also markedly increased after silencing ECT2 (p <0.001) (Fig. 2C). Taken together, these data demonstrate that ECT2 promotes proliferation and resistance to apoptosis in HCC cells. To evaluate whether ECT2 is oncogenic or tumorigenic in HCC, we generated ECT2 stable knockdown HuH7, Hep3B, and HCCLM3 cell lines (Supplementary Fig. 1A). Soft agar colony formation assay was employed to evaluate the oncogenic potential of these cells. ECT2 stable knockdown clones showed significantly attenuated colony formation ability compared to scramble knockdown clones (Fig. 2D). In addition, ECT2 stable knockdown cells also exhibited delayed cell growth kinetics compared to control cells (Supplementary Fig. 1B–D). Subcutaneous xenograft HCCLM3 tumour model was employed to study the tumorigenicity of ECT2. ECT2 stable knockdown HCCLM3 tumours (shECT2) were significantly smaller than the stable scramble knockdown ones (shScramble) (Fig. 2E). Taken together, these data show that ECT2 contributes to anchorage-independent growth and confer a tumorigenic potency in vivo. Loss of ECT2 leads to reduced cell migration and invasion of HCC cells in vitro ECT2 has previously been demonstrated to regulate cell migration in Caenorhabditis elegans [17]. To further investigate the role of ECT2 in HCC progression, we tested the effect of ECT2 on metastasis. Silencing ECT2 in both Hep3B and HCCLM3 cells significantly suppressed their migrative and invasive abilities (p <0.001; Fig. 2F). In addition, a wound healing assay also showed that the migrative ability of ECT2 stable knockdown HCCLM3 cells was significantly attenuated compared to scramble stable knockdown cells (p <0.05; Fig. 2G). The tumour suppressor p53 regulates cell migration and negatively regulates ECT2 expression [13]. High frequency of p53 mutations was associated with poorly or moderately

Table 1. Univariate and multivariate analyses showing that ECT2 expression could serve as an independent prognostic factor for early recurrent HCC disease.

Variables

Univariate analysis RR (95% CI) p value

Age ≤60 years (n = 32) vs. >60 years (n = 35) Gender Male (n = 59) vs. female (n = 8) Cirrhosis Yes (n = 32) vs. no (n = 32) AJCC stage I (n = 42) vs. II and III (n = 25) Tumour venous infiltration VI (n = 24) vs. NI (n = 43) Child-Pugh's grade A (n = 51) vs. B (n = 16) Tumour size ≤5 cm (n = 41) vs. >5 cm (n = 26) ECT2 expression High (n = 33) vs. low (n = 34)

Multivariate analysis RR (95% CI) p value

0.55 (0.27-1.09)

0.088

n.s.

2.57 (0.61-10.7)

0.196

n.s.

1.95 (0.97-3.91)

0.058

n.s.

2.15 (1.07-4.29)

0.030*

n.s.

0.38 (0.19-0.77)

0.007*

n.s.

0.45 (0.22-0.91)

0.029*

2.21 (1.06-4.59)

0.034*

2.15 (1.09-4.23)

0.026*

2.35 (1.19-4.65)

0.014*

4.33 (2.0-9.41)

0.000**

4.16 (1.89-9.18)

0.000** ⁄

Univariate and multivariate Cox regression analysis was performed as described in Supplementary Materials and methods. n.s., not significant; p <0.05;

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⁄⁄

p <0.01.

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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

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Fig. 2. Expression of ECT2 is associated with cell growth, oncogenesis, tumourigenesis and the metastatic ability of HCC cells. (A) The knockdown effect of ECT2 by siRNA and live cells observed after trypan blue staining were counted on different days post-treatment. (B) Caspase-related apoptosis was analysed at 72 h post-treatment by immunoblotting. (C) TUNEL assays were performed 72 h after ECT2-siRNA transfection. TUNEL-positive cells (green) indicated cells with DNA damage and/or apoptotic cells. 7-AAD (red) indicated the nucleus. Scale bar: 20 lm. The percentage of TUNEL-positive cells was calculated (200 cells were monitored). (D) The oncogenic activity of ECT2 stable knockdown cells was analysed by soft agar colony formation assay. (E) ECT2 stable knockdown HCCLM3 cells were inoculated subcutaneously to monitor tumour development. At 5 weeks after inoculation, the mice and tumours were photographed and the changes in tumour volume were analysed. (F) Representative images showing the effect of siRNA treatment on cell migration and invasion 1 day after seeding in vitro. (G) Representative images of wound healing assay to demonstrate the effect of ECT2 stable knockdown on HCC cell migration. Three independent experiments were performed for the in vitro cell migration and invasion assay and the wound healing assay, respectively.

differentiated tumours with a high rate of recurrence [20]. To investigate the potential role of p53 in ECT2-induced migration in HCC, we studied the HCC cell lines SK-Hep1 and HepG2 which endogenously express wild-type p53 [21,22]. Silencing of p53 in 4

these cells significantly elevated the expression and promoter activity of ECT2 (Supplementary Fig. 2A–D). Enhancement in the migratory and invasive abilities of these cells could subsequently be observed (Supplementary Fig. 2E and F).

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

JOURNAL OF HEPATOLOGY Furthermore, the observed enhancement in the migratory and invasive abilities of these cells was impaired by double-knockdown of ECT2 and p53 (Supplementary Fig. 2E and F), suggesting that p53 could promote early HCC recurrence partially through ECT2. Knockdown of ECT2 impairs the activation of Rho/ERK signals ECT2 is a Rho GEF that can promote Rho signals [17]. To investigate the effect of ECT2 on small Rho GTPase activities in HCC, we knocked down ECT2 by siRNA. ECT2 knockdown impaired the activity of Cdc42, Rac1, and Rho compared to siScramble- and GTPcS-treated siScramble controls (Fig. 3A). To identify potential downstream signalling targets of ECT2 and Rho, we employed the phospho-kinase screening arrays in HCCLM3 cells treated with scramble or ECT2 siRNAs. We found that p-ERK1/2 was markedly suppressed compared to the reference control (R) after silencing ECT2 (Fig. 3B), suggesting that ERK kinase might be an important downstream target of ECT2 signalling in HCC. To confirm this result, we silenced ECT2 expression and investigated the Rho/ERK, FAK and AKT signals by Western blot. Silencing ECT2 suppressed the activation and/ or phosphorylation of RhoA, ERK, FAK, and AKT signals (Fig. 3C and D). Furthermore, the migrative ability of HCCLM3 cells was significantly attenuated after ERK1/2, RhoA or ECT2 siRNA treatment (Fig. 3E). These results corroborated well with the results obtained on treating HCC cells with the ERK1/2 inhibitor (VX-11e, ChemieTek) or RhoA inhibitor (Rho inhibitor I, Cytoskeleton, Inc). VX-11e or the RhoA inhibitor I attenuated the migration of HCCLM3 cells in a dose-dependent manner (Fig. 3F). Having demonstrated that ECT2 contributes to apoptosis resistance in HCC cells, we then test whether the ERK signalling pathway is involved in this process. Knockdown of ECT2 or ERK induced mitochondria-dependent apoptosis by upregulating the pro-apoptotic factors such as Bax, Bad, Bak and Puma and conversely, while by decreasing anti-apoptotic factors such as BclxL, Bcl-2 and Mcl-1 in HCCLM3 cells (Supplementary Fig. 3A). Cell cycle analysis demonstrated that attenuation of the ECT2/ ERK signal by siRNAs could induce G1 phase cell cycle arrest of HCCLM3 cells (Supplementary Fig. 3B). Furthermore, the attenuation of ECT2/ERK signal decreased the Cyclin E/CDK2/Rb signal and activated the p53 pathway in HCCLM3 cells (Supplementary Fig. 3C). These data were consistent with previous reports demonstrating that the ERK activity duration affects the promotion of G1 phase progression to S phase [23,24]. Together, these observations support that ECT2 signalling contributes to the oncogenic phenotypes through the ECT2/ERK pathway in HCC (Fig. 3D). ECT2 expression correlates with ERK activation in vivo The observed role of ERK signalling in ECT2-induced oncogenesis prompted us to evaluate whether the attenuated tumorigenicity observed in ECT2 stable knockdown xenograft was caused by the attenuation of ECT2/ERK signal, we investigated the ECT2/ ERK signal in tumours at 5 weeks after tumour inoculation by Western blot and IHC analysis. ECT2 stable knockdown was validated in the shECT2 xenograft tumours (Fig. 3G and H) and it was observed that depletion of ECT2 resulted in the reduction of pERK1/2 compared to the shScramble controls (Fig. 3G and H),

demonstrating that ERK signalling could contribute to ECT2-induced tumorigenicity in vivo. To demonstrate the clinical correlation of ECT2 and p-ERK1/ 2, a duplicate set of 43 tumour sections obtained from HCC patients (including 17 with NR disease and 26 with early recurrent disease) were stained for both ECT2 and p-ERK1/2 (Supplementary Table 1C). ECT2 was highly expressed in 41.9% (18/43) of the sections studied, including 94.4% (17/18) of the patients with early recurrent disease, representative images are shown (Fig. 3I and J). The p-ERK1/2 was highly expressed in 41.9% (18/43) of these tumour sections, including 77.8% (14/18) of the patients with early recurrent disease (Fig. 3I and J). In addition, high ECT2 expression was significantly associated with poor overall survival (p <0.0001) and disease-free survival (p <0.0001; Fig. 3K), and high p-ERK1/2 expression was also significantly associated with poor overall survival (p = 0.0049) and disease-free survival (p <0.0272; Fig. 3L). These data suggest that ECT2 could promote early HCC recurrence via ERK pathway. RacGAP1 promotes ECT2-mediated RhoA activation and metastasis of HCC cells To further understand the molecular mechanism of how ECT2 may promote metastasis by regulating RhoA/ERK pathway and to identify its potential interacting co-factors, we performed ingenuity pathway analysis (IPA). As a consequence, RACGAP1 was identified as a potential cofactor that interacted with ECT2 (Fig. 4A) and it was demonstrated that ECT2 and RACGAP1 could be precipitated together as one complex in HCCLM3 cells (Fig. 4B). While silencing ECT2 expression alone suppresses the GTPbinding activity of RhoA (p <0.01; Fig. 4C), silencing RACGAP1 alone did not suppress the GTP-binding activity of RhoA (Fig. 4C). However, knockdown of both RACGAP1 and ECT2 resulted in significantly better suppression of RhoA activity than silencing ECT2 alone (Fig. 4C), indicating the possibility that RACGAP1 could promote ECT2-mediated RhoA-GTP exchange in HCC cells. Furthermore, silencing both RACGAP1 and ECT2 suppressed the metastatic potential of HCC cells more strongly compared to that observed with silencing ECT2 (p <0.01) or RACGAP1 individually (p <0.001, Fig. 4D). ECT2 interacts and colocalizes with RACGAP1 in HCC To validate whether ECT2 and RACGAP1 exist in one complex spatially in HCC, we did immunofluorescence co-staining or Duolink in situ assay of ECT2 and RacGAP1 in HCCLM3 and Hep3B cells. ECT2 and RACGAP1 could be colocalized at the spindle and midbody of mitotic HCC cells (Fig. 4E). During interphase, ECT2 and RACGAP1 were mainly localized in the nucleus (Fig. 4E). It was observed that the expression of ECT2 colocalized with RACGAP1 during both interphase and mitosis (Fig. 4F). The DuolinkÒ assay provides precise detection and quantification of protein-protein complex interactions. In our study, DuolinkÒ complexes could be largely detected in interphase and mitotic cells, confirming the physical interaction of ECT2 and RACGAP1 (Fig. 4G). Moreover, the detection of more DuolinkÒ complexes in mitotic cells than in interphase cells (p <0.001; Fig. 4G) would suggest that ECT2 and RACGAP1 function not only in mitosis but also in interphase cells.

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Research Article

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p-ERK1/2 high (≥2) 100 p-ERK1/2 low (<2) 80 60 R = 12, NR = 13 40 R = 14, NR = 4 20 p = 0.0271 0 2 4 6 8 10 0 Years

DF survival %

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Low scored tumor tissues Patient #4 Patient #5

High scored tumor tissues Patient #1 Patient #2 Patient #3

Fig. 3. ECT2 regulates the migration and tumorigenesis of HCC cells via the RhoA/ERK signalling axis. (A) Pull-down and immunoblotting assays of the GTPase activity of Cdc42, Rac1, and Rho after different treatments. GTPcS added to siScramble lysate acted as a positive control. (B) Human Phospho-Kinase antibody analysis of HCCLM3 cells lysates obtained after ECT2 and Scramble control siRNAs treatment. p-ERK1/2 was significantly decreased while HSP60 was upregulated markedly after ECT2 gene knockdown (indicated by black boxes). The ‘‘R’’ indicates reference control. (C) Pull-down assays for RhoA activity were prepared in scrambled or ECT2 siRNA-treated HCCLM3 cells and the activity of downstream signalling molecules was analysed by immunoblotting. GAPDH was used as a loading control. (D) Diagram depicting upstream and downstream signals of ECT2 that might be contributing to HCC recurrence. ? Indicates direct activation, a indicates direct inhibition and a dotted arrow indicates possible indirect activation. (E) RhoA expression level and ERK activation were analysed by immunoblotting at day 2 after ERK- or RhoA-siRNA treatment. In vitro cell migration of cells was studied. (F) Rho inhibitor I and ERK inhibitor (VX-11e) were added to HCCLM3 cells at the indicated dose. After 24 h, the active forms of RhoA and ERK were detected by immunoblotting and the ability of the cells to migrate was also studied. (G) ERK activation and ECT2 level were detected by immunoblotting in two tumours generated after subcutaneous inoculation of ECT2 stable knockdown cells in mice. (H) Representative images of IHC staining showing the expression of ECT2 and p-ERK1/2. Representative images of three HCC patients’ tumours with high (I) or low (J) ECT2 and p-ERK1/2 expression are shown. Twenty-eight of 43 samples showed high ECT2 or p-ERK1/2 levels. Among the tumours highly expressing ECT2, there were 17 R and 1 NR HCC samples. For the high p-ERK1/2 expressing tumours, there were 14 R and 4 NR HCC samples. High expression of ECT2 (K) or p-ERK1/2 (L) was related to poor overall survival and disease-free survival (DF).

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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

JOURNAL OF HEPATOLOGY

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Fig. 4. ECT2 functionally and physically interacts with RACGAP1 to promote its RhoA-GTP exchange, cell migration and the protein stability of RACGAP1. (A) IPA analysis to identify potential molecules that can interact with ECT2. The interaction between ECT2 and RACGAP1 is highlighted in blue. The red coloured nodes indicate the upregulated genes in early recurrent HCC. (B) Endogenous co-IP was used to determine the interaction between ECT2 and RACGAP1 in HCCLM3 cells. 1% of the lysate for immunoprecipitation was used for input detection. (C) RhoA-GTP pull-down assay was analysed 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 scrambled siRNA group was taken as 1. GAPDH and HSP70 were used as loading controls. (D) In vitro Transwell migration assays. The cells that migrated through the membrane were stained and counted. (E) Representative confocal images showing the localization of ECT2 and RacGAP1 (red) at different phases in the cell cycle. Images were analysed at 63 magnification following immunostaining. a-Tubulin (a-T) staining (green) indicates the microtubules; Hoechst (blue) was used to stain DNA. Scale bar; 5 lm. (F) Colocalization of ECT2 and RACGAP1 was investigated during interphase (I) and mitosis (M) by confocal microscopy. Cells showing colocalization at the spindle (M: in mitosis) or in the nucleus (I: in Interphase) were counted. n = 200. (G) Representative confocal images of results obtained to study ECT2 and RacGAP1 interaction by immunoprecipitation and DuolinkÒ protein–protein interaction assays. The rabbit and mouse IgG antibodies were used for the controls. DuolinkÒ-positive dots were calculated per cell both in interphase and mitosis (n = 60). (H) Correlation analysis of the expression of ECT2 and RACGAP1 using our previously established microarray database consisting of 70 HCC patients [19]. R >0.5 indicates a significant correlation. (I) Western blot assay of ECT2 and RACGAP1 in cells at 72 h post-treatment. The protein density was determined by Image-J software (NIH, USA) and the protein density ratio of ECT2 and RacGAP1 to GAPDH was calculated. The protein density of the siScamble treatment group was regarded as 1. (J) A CHX chase experiment was used to analyse the stability of RACGAP1 protein after siRNA treatment in cells. The relative protein density ratio of RACGAP1 to b-actin was calculated at 0, 1, and 2 h post-CHX treatment (100 lg/ml). p <0.05 was considered statistically significant. Representative data from three independent experiments were employed for quantitation.

Journal of Hepatology 2015 vol. xxx j xxx–xxx

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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 ECT2 functionally interacts with RACGAP1 and regulates the stability of RACGAP1 To probe the functional role of the ECT2 and RACGAP1 interaction in HCC, we examined their expression in HCC patients’ samples. In HCC tumour tissues, the expression of ECT2 correlated well with RACGAP1 expression (r = 0.77, p <0.0001; Fig. 4H). Silencing ECT2 expression decreased RACGAP1 protein expression by 53–61% and reciprocally, silencing RACGAP1 expression reduced ECT2 protein expression by 53–65% (Fig. 4I), indicating ECT2 and RacGAP1 interacted and the expression and/or protein stability could be by synchronous regulation. To study protein stability, a cycloheximide (CHX) chase assay was performed. It was demonstrated that two hours post-CHX treatment, the expression of RACGAP1 was reduced to 68% in Hep3B cells and 75% in HCCLM3 cells in the scramble knockdown group. In comparison, for the ECT2 knockdown group, the expression of RACGAP1 was only 10% in Hep3B cells and 39% in HCCLM3 cells relative to their expression at starting point (Fig. 4J), strongly suggesting that ECT2 could protect against the degradation of RACGAP1 in HCC cells.

Although the oncogenic function of ECT2 has been demonstrated in multiple pathways, studies thus far have been focused towards understanding its function in cell division and cell transformation. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant-negative Rho-N19 in NIH3T3 cells, suggesting the involvement of Rho signal in ECT2 transformation [9]. In NSCLC, ECT2 has been linked in the PKCiotaPar6alpha/Rac1 pathway activation and transformation [11]. In ovarian cancer, ECT2 interacts with PKCiota to activate a MEK/ ERK signalling axis that drives tumorigenesis [32]. In situ hybridization analysis also showed that high ECT2 expression was associated with cells undergoing mitosis in regenerating mouse liver, indicating a potential regulatory role of ECT2 in the cell cycle [33]. It is likely that ECT2 is a tissue-dependent and multi-functional oncogenic driver in human cancers and interplays with multiple pathways to promote malignant transformation in different tissues. Here, we demonstrated that in human HCC, ECT2 promotes tumorigenesis, metastasis and early recurrence primarily through Rho/ERK signalling pathway. ECT2 can interact with different cellular molecules to promote early recurrent HCC disease and could therefore serve as a potential candidate for designing novel therapeutic strategies in the clinical management of HCC.

Discussion The prognosis of HCC remains dismal and our knowledge of the underlying cellular molecular pathways driving its pathogenesis remains limited. In this study, we showed that ECT2 was significantly upregulated in HCC tumours and was strongly associated with early recurrent HCC disease. We further demonstrated that ECT2 played critical regulatory roles in the cell proliferation, oncogenic transformation, tumourigenesis and metastasis of HCC cells and ECT2/Rho/ERK signal partially mediated ECT2 oncogenic function and to promote HCC recurrence. Moreover, ECT2/Rho/ERK oncogenic signal could be potentiated by RacGAP1 though interacting with ECT2 and accelerating the GTP exchange of ECT2. Cancer recurrence is a complex phenomenon and is believed to be mediated through a number of mechanisms, including the concept of cancer stem cells [2,5,25,26]. The data presented here suggested that ECT2 may potentially affect multiple cellular processes that drive HCC progression and recurrence through ERK activity. This could therefore suggest a role of ECT2 in the induction of a cancer stem cell phenotype or a similar role of ECT2 contributing to ERK-dependent sorafenib resistance [27]. The upregulation of ECT2 has also been reported in several types of human cancer [10–12]. ECT2 is amplified as part of the 3q26 amplicon, a region frequently targeted for chromosomal alterations in human tumours, has been detected in lung squamous cell carcinomas (LSCC) [11] and cervical cancer [28]. However, no amplification was reported in HCC in a recent study [29], indicating the upregulation of ECT2 in HCC could reside on the dysregulation of RNA and/or protein. Alternatively, it is also possible that ECT2 is activated in HCC through other molecular mechanisms such as by the inactivation of p53 [13] or Rb [30], both are tumour suppressors that are frequently mutated in HCC [31]. Furthermore, we have previously demonstrated that RACGAP1 can be an independent predictor for early recurrent HCC disease [19]. It was therefore interesting to observe, in this study, that the expression of ECT2 significantly correlated with RACGAP1 expression.

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Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements We thank the Tissue Repository of the National Cancer Centre Singapore for tissue samples. This work was supported by grants from the National Medical Research Council of Singapore (to L.L.O. and to K.M.H.) and by the SingHealth Foundation (to J.C.).

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2015.01. 014.

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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