ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma

Cancer Letters 337 (2013) 96–106

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Insulin receptor tyrosine kinase substrate activates EGFR/ERK signalling pathway and promotes cell proliferation of hepatocellular carcinoma Yu-Ping Wang a,b,c, Li-Yu Huang a,b, Wei-Ming Sun d,e, Zhuang-Zhuang Zhang a,b, Jia-Zhu Fang a,b, Bao-Feng Wei a,b, Bing-Hao Wu a,b, Ze-Guang Han a,b,f,⇑ a

Key Laboratory of Systems Biomedicine (Ministry of Education) of Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Rui-Jin Road II, Shanghai 200025, China Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China Institute of Medical Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, 199 Dong-Gang West Road, Lanzhou 730000, Gansu, China d Institute of Combined Traditional Chinese and Western Medicine, School of Basic Medical Sciences, Lanzhou University, 199 Dong-Gang West Road, Lanzhou 730000, Gansu, China e Department of Endocrinology, The First Hospital of Lanzhou University, 1 Dong-Gang West Road, Lanzhou 730000, Gansu, China f Shanghai Center of Systems Biomedicine, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China b c

a r t i c l e

i n f o

Article history: Received 24 February 2013 Received in revised form 7 May 2013 Accepted 14 May 2013

Keywords: IRTKS Hepatocellular carcinoma Proliferation EGFR/ERK signalling

a b s t r a c t Insulin receptor tyrosine kinase substrate (IRTKS) is closely associated with actin remodelling and membrane protrusion, but its role in the pathogenesis of malignant tumours, including hepatocellular carcinoma (HCC), is still unknown. In this study, we showed that IRTKS was frequently upregulated in HCC samples, and its expression level was significantly associated with tumour size. Enforced expression of IRTKS in human HCC cell lines significantly promoted their proliferation and colony formation in vitro, and their capacity to develop tumour xenografts in vivo, whereas knockdown of IRTKS resulted in the opposite effects. Furthermore, the bromodeoxyuridine (BrdU) incorporation analyses and propidium iodide staining indicated that IRTKS can promote the entry into S phase of cell cycle progression. Significantly, IRTKS can interact with epidermal growth factor receptor (EGFR), results in the phosphorylation of extracellular signal-regulated kinase (ERK). By contrast, inhibition of ERK activation can attenuate the effects of IRTKS overexpression on cellular proliferation. Taken together, these data demonstrate that IRTKS promotes the proliferation of HCC cells by enhancing EGFR–ERK signalling pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Although liver cancer currently ranks worldwide as the fifth most common malignant disease in men and seventh in women, it is the second and sixth leading cause of cancer-related death, respectively, with half of the incidence and deaths occurring in China [1]. In primary liver cancers, hepatocellular carcinoma (HCC) is the major histological subtype, contributing 70–85% of total liver cancer worldwide [2]. Despite the remarkable achievements that have been attained in the treatment of primary hepatocellular carcinoma, the long-term survival rate for HCC is still quite low [3]. Although a large number of molecules and signalling pathways that are related to the development of HCC have been identified [4–7], the molecular mechanisms underlying the ⇑ Corresponding author at: Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China. Tel.: +86 21 50801325; fax: +86 21 50800402. E-mail addresses: [email protected] (Y.-P. Wang), [email protected] (L.-Y. Huang), [email protected] (W.-M. Sun), [email protected] (Z.-Z. Zhang), [email protected] (J.-Z. Fang), [email protected] (B.-F. Wei), [email protected] (B.-H. Wu), [email protected] (Z.-G. Han). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.05.019

tumourigenesis and proliferation of HCC are still poorly understood. Our group previously obtained insulin receptor tyrosine kinase substrate (IRTKS) by cloning the full-length cDNA from the human hypothalamus–pituitary–adrenal axis [8]. IRTKS belongs to the IRSp53 protein family of which the main family members include IRSp53 (BAIAP2), IRTKS (BAIAP2 L1), FLJ22582 (BAIAP2 L2), MIM, and ABBA [9]. Each of these proteins contains a conserved IRSp53/MIM domain (IMD) at the N-terminus and a canonical SH3 domain near the C-terminus [9]. The IMD structure belongs to the larger family of Bin-amphyipysin-Rvs67 (BAR) domains. IRSp53 is a typical representative of the family that functions in the regulation of membrane ruffling and cell shape [10,11] and is related to the formation of filopodia and lamellipodia [12]. IRTKS has been reported to be a substrate of insulin receptor tyrosine kinase and has been observed to induce bundle actin filaments [13]. To date, most of the reports concerning IRTKS pertain to its involvement in actin remodelling and membrane protrusion [14– 18]. However, the biological function of IRTKS in HCC proliferation remains unknown. In this study, we first demonstrate that IRTKS is frequently upregulated in human HCC and is significantly associated with tu-

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mour size in an independent HCC cohort. Functional research indicated that high levels of IRTKS promoted both in vitro and in vivo cell proliferation by enhancing ERK activity, and IRTKS interacted with EGFR and regulated its phosphorylation status. These data suggest that IRTKS might balance EGFR activity as a new adaptor. 2. Materials and methods 2.1. Tissue specimens All liver cancer specimens were acquired from patients who underwent surgical resection with informed consent. Specimens of both the tumour and adjacent normal tissue were collected from each patient, and the diagnosis of HCC was validated by pathological examination. The use of human and animal tissues in this investigation was approved by the ethics committee of the Chinese National Human Genome Centre at Shanghai. 2.2. Liver cancer cell lines Fourteen cell lines derived from liver tumours (QGY-7703, Focus, Hep3B, HepG2, HepG2.2.15, Huh7, LM3, LM6, MHCC-H, MHCC-L, PLC/PRF/5, SK-Hep-1, SNU398, and YY-8103) and the L02 cell line derived from foetal liver tissue were used in this study. These cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, USA) supplemented with 10% foetal bovine serum (GIBCO, USA) at 37 °C in a humidified 5% CO2 incubator. 2.3. Antibodies and reagents

97

containing IRTKS, the plasmid pShuttle-IRTKS-IRES-hrGFP was constructed, and the IRTKS ORF was subcloned into the AdEasy™ XL Adenoviral Vector System (Stratagene, USA). 2.8. RNA interference (RNAi) Two siRNAs against IRTKS were chemically synthesised (Shanghai GenePharma Co.) to target the coding region and 30 -untranslated region (UTR) of IRTKS: siRNA-1 (50 -CCAGUCCCUUGAUCGAUAUTT-30 and 50 -AUAUCGAUCAAGGGACUGGTA-30 ) and siRNA-2 (50 -GCUUAAGCAAAUCAUGCUUTT-30 and 50 -AAGCAUGAUUUGCUUAAGCAG-30 ). In addition, siRNA-NC (50 -UUCUCCGAACGUGUCACGUTT-30 and 50 ACGUGACACGUUCGGAGAATT-30 ) was also synthesised for use as a control. The synthesised DNA fragments encoding the short hairpin RNA (shRNA) used for the knockdown of endogenous IRTKS were inserted into the pGCsi-H1Neo-GFP plasmid derived from pSUPER (Oligoengine, USA). The sequences of the oligonucleotides for RNAi IRTKS were as follows: shRNA-1 (forward, 5-GATCCCCCCAGTCCCTTGATCGATATTTCAAGAGAATATCGATCAAGGGACTGGTTTTTGGAAA-3, and reverse, 5-AGCTTTTCCAAAAACCAGTCCCTTGATCGATATTCTCTTGAAATATCGATCAAGGGACTGGGGG-3) and shRNA-2 (forward, 5-GATCCCCGCTTAAGCAAATCATGCTTTTCAAGAGAAAGCATGATTTGCTTAAGCTTTTTGGAAA-3, and reverse, 5-AGCTTTTCCAAAAAGCTTAAGCAAATCATGCTTTCTCTTGAAAAGCATGATTTGCTTAAGCGGG-3). pSUPER shRNA-NC contained irrelevant nucleotides and served as a negative control. 2.9. Cell transfection Cell transfection was performed with Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s protocols. 2.10. Cell proliferation

An anti-IRTKS rabbit polyclonal antibody was raised against the GST-IRTKS fusion protein [19]. Antibodies directed against IRTKS (mouse), EGFR (total), EGFR (phosphorylated Tyr1173), b-catenin, b-actin, BrdU, and Ki67 were obtained from Santa Cruz Biotechnology (USA). Akt (total), Akt (phosphorylated Ser473), ERK (total), ERK (phosphorylated Thr202/Tyr204), phosphorylated tyrosine, and PD98059 were obtained from Cell Signaling Technology (USA).

Transiently transfected cells were cultured in a 96-well plate for 6 days, and cell viability was tested using the Cell Counting Kit-8 (Dojindo Laboratories, Japan), according to the instructions of the manufacturer. The optical density measured at 450 nm was used as an indicator of cell viability. 2.11. Colony formation

2.4. RNA extraction Ò

Total RNA was extracted with the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. To avoid possible genomic DNA contamination, RNAase-free DNAase I (Takara, Japan) was used. The concentration and quantity of RNA were assessed using a Nanodrop Spectrophotometer (ND-1000, Wilmington, USA). 2.5. Semi-quantitative and real-time PCR Reverse transcription was performed in a 25-ll reaction volume using M-MLV Reverse Transcriptase (Promega, USA) with a total of 2 lg of RNA. Semi-quantitative RT-PCR and real-time PCR were performed using a Thermal Cycler Dice Detection System and SYBR green dye (TaKaRa, Japan), according to the manufacturer’s protocol. The following primers were used to amplify a 205-bp PCR product for IRTKS: forward, 50 -GAAGGATGGCTGGCTCTATG-30 , and reverse, 50 -GCATTCCAAGTAGTCGGGTG-30 . The housekeeping gene b-actin was used as an endogenous control with the following primers: forward, 50 -AGAGCCTCGCCTTTGCCGATCC-30 , and reverse, 50 -CTGGGCCTCGTCGCCCACATA-30 . 2.6. Immunoblotting analysis Cell extracts were collected in 2 loading lysis buffer (50 mM Tris–HCl [pH 6.8], 2% SDS, 10% 2-mercaptoethanol, 10% glycerol, and protease inhibitor cocktail, Sigma, USA). The total cellular protein was separated using 8% SDS–PAGE and transferred to Hybond-C nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK). After blocking with PBS containing 5% BSA or nonfat milk, the membrane was incubated with the appropriate primary antibody (1:1000) at room temperature for 2 h or at 4 °C overnight, followed by incubation with IRDye 800CW or 680RD secondary antibodies (1:10,000, Li-COR Biosciences, USA). The protein bands were detected using the Odyssey Infrared Imaging System (Li-COR Biosciences, USA). b-actin was used as a loading control. 2.7. Construction of recombinant plasmids and adenoviral vectors The full-length IRTKS ORF (1536 bp, GenBank accession number NM_018842) was amplified from the pFLAG-CMV2-IRTKS plasmid [20]. The primers were as follows: forward, 50 -TACTCGAGATGTCCCGGGGGCCCGAGGAG-30 , and reverse, 50 -GAGGATCCTCGAATGATGGGTGCCGAGCGATCATTCG-30 . The PCR product was inserted into the expression vector pcDNA3.1/myc-His(-)B-3  FLAG-IRES-hrGFP, derived from pcDNA™3.1/myc-His(-)B (Invitrogen, USA). To construct the adenoviral vector

HCC cells transfected with the vectors containing IRTKS (or the empty vector as a control) or IRTKS shRNA (or shRNA-NC as a control) were cultured in 100-mm dishes for colony formation; G418 (Life Technologies, USA) was added to the culture medium at a final concentration of 0.6 to 1 mg/ml. After 3–4 weeks, proliferating colonies were dyed with crystal violet and counted. For the soft agar colony formation assay, the transfected cells were cultured and grown on 24-well plates containing 0.5% top agar and 1% base agar. The plated cells were cultured for 3–4 weeks, and the colonies were counted using a dissecting microscope. 2.12. Tumour xenografts in vivo Male BALB/c nude mice (5–6 weeks old) were purchased from Shanghai Experimental Animal Center. The kinetics of tumour formation were assessed by measuring the tumour sizes at 3 or 4-day intervals. Tumour size was measured with digital callipers, and the tumour volume was calculated using the following formula: volume = 0.5  width2  length. 2.13. Immunohistochemistry (IHC) assays Five micrometre thick paraffin-embedded tumour sections were pretreated with methanol to inactivated endogenous peroxidase. The sections were incubated with anti-IRTKS or anti-Ki-67 antibody (1:100) at 4 °C overnight, then incubated with the horseradish peroxidase (HRP)-conjugated antibodies (DACO, Kyoto, Japan) at 37 °C for 1 h. The signals were detected by Diaminobenzidine (DAB) Substrate Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. 2.14. Cell cycle analysis Flow cytometry was performed to analyse the cell cycle distribution. For DNA content detection, the cells were fixed in 70% ethanol, resuspended in PBS, and treated with RNase A (10 mg/mL) and propidium iodide (10 lg/mL) for 30 min each. Samples were measured using a FACSCalibur flow cytometer, CellQuest (BD Biosciences, USA). 2.15. Bromodeoxyuridine (BrdU) incorporation assay Two methods were used to determine the population of cells in S phase of the cell cycle. For immunofluorescence assays, the transfected cells were treated with BrdU (Sigma–Aldrich, USA) for 2 h and incubated with an anti-BrdU antibody

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Fig. 1. IRTKS is upregulated in HCC and liver cancer cell lines. (A) Expression of IRTKS in 68 pairs of human HCC and their corresponding non-tumourous samples. The IRTKS expression level was determined by real-time PCR and normalised to an endogenous control (b-actin). The statistical analysis was performed using paired t-tests. (B) Paired comparison of IRTKS expression levels between primary HCC samples and the corresponding noncancerous tissue samples. (C) Immunoblotting analysis results of 8 pairs of HCC (C) and the adjacent non-HCC liver tissue (N). (D) Expression levels of IRTKS determined by immunoblotting analysis in 8 HCC cell lines. b-actin was used as an internal control. All the above experiments were repeated at least 3 times to confirm the reproducibility of the results. The data are presented as the mean ± SD. P < 0.001 versus the control. (1:100), followed by incubation with Alexa FluorÒ Dyes (1:200, Invitrogen, USA). Confocal microscopy (Carl Zeiss, Germany) was performed to analyse the cellular incorporation of BrdU. In flow cytometric detection, the cells treated with BrdU were incubated with an FITC-conjugated mouse anti-BrdU monoclonal antibody and an mouse IgG1 isotype served as a control (20 ll, BD Pharmingen™, USA). The samples were measured using a FACSCalibur flow cytometer.

Table 1 The correlations of IRTKS expression with various clinicopathological features of HCC.

2.16. Immunofluorescence assays An immunofluorescence assay was used to detect the colocalisation of IRTKS and EGFR. The cells were incubated with anti-IRTKS and -EGFR antibodies (1:200), with Alexa FluorÒ 488 (green) and Alexa FluorÒ 546 (red)-coupled secondary antibodies (1:200, Invitrogen, USA), respectively. The stained cells were observed by Zeiss confocal microscopy and a ZEISS LSM Image Browser (Carl Zeiss, Germany). 2.17. Co-immunoprecipitation (Co-IP) HCC cells were resuspended in 1 ml of lysis buffer (20 mM Tris, [pH 7.5], 150 mM NaCl, 1.0% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail). The cell lysates were immunoprecipitated by incubation with 1 lg anti-IRTKS or anti-EGFR antibody, followed by immunoblotting with antibodies (1: 1000) against p-Y, IRTKS, EGFR, and p-EGFR (Tyr 1173). The cell lysates incubated with 1 lg IgG served as a control. 2.18. Statistics Significant differences and variances were evaluated using Student’s t tests. Averaged data are presented as means ± standard deviation (SD). P < 0.05 was considered to be significant.

Clinicopathological feature

Number of cases

Expression of IRTKS (mean ± SEM)

Pvalue

Gender Male Female

49 7

0.008 ± 0.001 0.007 ± 0.001

0.516

Age (years) P60 <60

23 33

0.009 ± 0.001 0.007 ± 0.001

0.044*

HBsAg P0.5 <0.5

44 12

0.007 ± 0.001 0.009 ± 0.001

0.129

HCV Positive Negative

1 55

0.013 0.007 ± 0.001

–

Diameter (cm) P5 <5

28 28

0.009 ± 0.001 0.006 ± 0.001

0.002**

Degree of differentiation Well 4 Moderately and 52 poorly

0.005 ± 0.001 0.008 ± 0.001

0.158

0.007 ± 0.001 0.008 ± 0.001

0.521

TNM stage I + II III + IV * **

7 49

P < 0.05. P < 0.01 between the two groups.

3. Results 3.1. IRTKS is upregulated in HCC and is associated with tumour size To observe whether IRTKS contributes to HCC progression, we analysed IRTKS mRNA expression in 68 HCC patients and 14 HCC

cell lines by real-time RT-PCR and western blotting. We found that IRTKS mRNA expression was significantly upregulated in the HCC tissue samples compared to the corresponding adjacent nontumourous liver samples (Fig. 1A, P < 0.001). In these 68 samples,

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Fig. 2. Overexpression of IRTKS promotes HCC cellular proliferation and colony formation in vitro. (A) Ectopic IRTKS promoted the proliferation of 4 HCC cell lines. (B) Overexpressed IRTKS enhanced the colony formation of the HCC cell lines, as shown by representative plates of cells transfected with the IRTKS expression vector and empty control vector. The histograms represent the numbers of colonies, and the data are shown as the mean ± SD. (C) Forced IRTKS expression promoted colony formation in soft agar. The histograms represent the numbers of colonies, and the data are shown as the mean ± SD. All the above experiments were repeated at least 3 times to confirm the reproducibility of the results. P < 0.05, P < 0.01 versus the control.

27 (39.7%) HCC cases exhibited an IRTKS mRNA level at least 2-fold higher than the corresponding nontumourous liver tissue (Fig. 1B). Eight pairs of typical cases are illustrated (Fig. 1C Supplementary Fig. S1A and S1D). Moreover, the expression of IRTKS in most of the examined human HCC cell lines was determined (Fig. 1D, Supplementary Fig. S1B and S1C) to exist at a higher basal level when compared to normal adult liver and foetal liver tissue. In addition, we evaluated the expression of IRTKS in 56 HCC patients, and the correlations between the IRTKS expression level and the clinicopathological characteristics of HCC are summarised in Table 1. The expression of IRTKS in HCC patients did not significantly correlate with gender, HBsAg, differentiation, and TNM stage. In contrast, IRTKS expression was significantly associated with tumour size, suggesting that IRTKS might have a stimulatory role in the progression of HCC. Furthermore, IRTKS expression was associated with the age of the patient, implying that IRTKS is perhaps connected with other factors.

3.2. Overexpression of IRTKS promotes HCC cell proliferation and colony formation in vitro To determine the effect of IRTKS on HCC cells, recombinant pcDNA3.1-IRTKS was transiently transfected into 2 HCC cell lines with high expression levels (Huh7 and YY-8103) and 2 HCC cell lines with relatively low expression levels (Hep3B and SK-hep-1), as based on their expression profiles (Fig. 1D). After evaluating the detectability of the recombinant IRTKS plasmid (Supplementary Fig. S2A and S2B), we observed that cell growth and colony formation were significantly promoted by IRTKS overexpression when compared to that of the cells transfected with the empty vector (Fig. 2A and B). Moreover, in soft agar, the ectopic IRTKS-transfected HCC cell lines were endowed with abundant energy, displaying more invasive growth that was accompanied by increases in colony quantity and size, relative to the cells transfected with the empty vector (Fig. 2C). This strong ability for anchorage-

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Fig. 3. Knockdown of IRTKS inhibits HCC cellular proliferation and colony formation in vitro. (A) IRTKS knockdown suppressed the proliferation of 4 HCC cell lines. (B) IRTKS RNAi limited colony formation in the HCC cell lines, as shown by representative plates of cells transfected with the IRTKS shRNA constructs and shRNA-NC control. The histograms represent the number of colonies, and the data are shown as the mean ± SD. (C) IRTKS RNAi suppressed colony formation in soft agar. The histograms represent the number of colonies, and the data are shown as the mean ± SD. All the above experiments were repeated at least 3 times to confirm the reproducibility of the results.  P < 0.05, P < 0.01 versus the control.

dependent and -independent growth suggested a specific requirement for IRTKS in maintaining normal cell proliferation. 3.3. Knockdown of IRTKS inhibits HCC cell proliferation and colony formation in vitro To further evaluate the effects of IRTKS on cell proliferation and colony formation, we used chemically synthesised siRNAs and constructed an shRNA derived from the recombinant pSUPER plasmid to knockdown endogenous IRTKS in the 4 HCC cell lines. As expected, both siRNAs significantly knocked down endogenous IRTKS (Supplementary Fig. S3A) and inhibited the growth of the HCC cells when compared to the si-NC-transfected cells (Fig. 3A). To investigate the effect of IRTKS on colony formation, two recombinant pSUPER-producing shRNAs were constructed and transfected into the HCC cell lines Hep3B, SK-hep-1, Huh-7, and YY-8103 (Supplementary Fig. S3B). The resulting data showed that both shRNAs significantly inhibited the colony formation of these cell lines compared to the control shRNA-NC-infected cells (Fig. 3B). Furthermore, the downregulation of IRTKS reduced the anchorageindependent growth of these HCC cell lines in soft agar and

significantly decreased the number of larger colonies compared to the cells transfected with the negative control shRNA (Fig. 3C). These collective data implied that endogenous IRTKS might be essential for maintaining cell proliferation and colony formation in HCC cells. 3.4. IRTKS enhances tumourigenicity in vivo To determine whether IRTKS upregulation contributes to HCC oncogenesis and progression, we constructed a recombinant adenoviral vector producing IRTKS. As expected, these adenoviruses efficiently infected SK-hep-1 and YY-8103 cells, as shown by Green Fluorescent Protein (GFP, Supplementary Fig. S4A). The SK-hep-1 and YY-8103 cells infected with Ad-GFP and Ad-IRTKS were injected subcutaneously into athymic mice, and tumourigenicity was assessed in the xenograft model. The results showed that IRTKS overexpression facilitated tumour growth in both HCC cell lines (Fig. 4A). In our experiments, the size and weight of the tumours formed from cells overexpressing IRTKS were significantly higher than those of xenografts formed from cells infected with the control Ad-GFP (Fig. 4B and C, and Supplementary Fig. S4B).

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Fig. 4. IRTKS enhances tumourigenicity in vivo. (A) Xenograft tumour growth of SK-hep-1 and YY-8103 cells was promoted by infection with a recombinant adenovirus carrying IRTKS; cells carrying the empty vector were used as the controls (n = 7). Xenograft tumour growth was monitored every 3 days by tumour diameter measurement (mean ± SD). (B and C) All the xenograft tumours were removed from the experimental mice and weighed (mean ± SD). (D) Xenograft tumour growth of YY-8103 stable cell lines was delayed by stable knockdown of endogenous IRTKS; cell subclone line with a stable shRNA-NC was used as the controls (n = 5). Xenograft tumour growth was monitored every 4 days by tumour diameter measurement (mean ± SD). (E and F) All the xenograft tumours were removed from the experimental mice and weighed (mean ± SD). P < 0.05, P < 0.01, P < 0.001 versus the control.

The upregulation of the recombinant IRTKS protein was detected using an anti-IRTKS antibody in the excised xenograft tumours (Supplementary Fig. S4C), and tumour sections were measured by Ki67 to observe proliferation (Supplementary Fig. S4D). To further identify whether IRTKS downregulation restrains HCC oncogenesis, we used two YY-8103 stable cell lines that containing stable knockdown of endogenous IRTKS by transfecting pSUPER vector containing shRNA-1 and shRNA-2. As expected, the 2 stable knockdown subclones, shRNA-1-4 and shRNA-2-9 (Supplementary Fig. S5A), showed significantly slower cell growth than the subclone line with a stable shRNA-NC (Fig. 4D, and Supplementary Fig. S5B). Inversely, the size and weight of the tumours formed from cells IRTKS knockdown were significantly lower than the control shRNA-NC (Fig. 4E and F). Finally, the downregulation of the IRTKS protein and Ki67 level were detected in the excised xenograft tumours (Supplementary Fig. S5C and S5D), suggesting that IRTKS plays an important role in boosting cell overgrowth in vivo.

3.5. IRTKS influences the G1-S cell cycle transition Changes in cell proliferation are usually associated with alterations in the cell cycle. To evaluate the function of IRTKS in cell cycle progression, a flow cytometric analysis was used following the staining of the transfected cells with propidium iodide (PI). There were significant increases in the S-phase cell fraction of the IRTKS-transfected SK-hep-1 and YY-8103 cells; in contrast, siRNA reduced the S-phase fraction of these cells compared to the control si-NC cells (Fig. 5A and Supplementary Fig. S5A). In addition, de novo DNA synthesis was identified by BrdU incorporation in the transfected cells. Immunofluorescence assays with an anti-BrdU antibody showed that the IRTKS-transfected SK-hep-1 and YY8103 cells exhibited significant increases in BrdU incorporation, whereas siRNA reduced the BrdU-incorporated proportion of SKhep-1 and YY-8103 cells (Fig. 5B). Moreover, a flow cytometric analysis with an FITC-conjugated anti-BrdU antibody also supported the observation of ectopic IRTKS expression in the SK-

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Fig. 5. IRTKS promotes the G1-S transition of the cell cycle. (A) The cell cycle distributions were analysed after SK-hep-1 and YY-8103 cells were infected with plasmids or siRNA. The histogram columns represent the means. (B) The immunofluorescence assays depict the cells that incorporated BrdU (red), and the cell nucleus was dyed with DAPI (blue). The histograms represent the percentage of BrdU incorporation, and the data are shown as the mean ± SD. All the above experiments were repeated 3 times.  P < 0.05, P < 0.01 versus the control (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

hep-1 cells (Supplementary Fig. S5B). These data indicated that IRTKS could enhance the proliferation of HCC cells by promoting the entry into S phase of cell cycle progression. 3.6. IRTKS promotes cell proliferation by enhancing ERK signalling The common cell signalling pathways involved in cell proliferation include EGFR/ERK [21], PI3K/Akt/mTOR [22], and Wnt/b-catenin [23]. To determine which pathway is related to IRTKS, we used western blot analysis to detect changes in p-ERK, p-Akt, and bcatenin. After the upregulation of IRTKS in the SK-hep-1 cells, we did not observe a difference in b-catenin and p-Akt between those cells and the vector control. Interestingly, forced IRTKS expression did increase the level of p-ERK when compared with that of the cells transfected with the empty vector. In contrast, the opposite results were obtained with the downregulation of IRTKS in SKhep-1 cells (Fig. 6A and B). To further confirm the association between the effects of the proliferation promoted by IRTKS and ERK, we used PD98059, an inhibitor of ERK. Serum-starved SK-hep-1 and YY-8103 cells overexpressed IRTKS and stimulated with EGF (20 ng/ml for 10 min), exhibited substantial increases in ERK phosphorylation compared to the cells containing the empty vector. Furthermore, EGF plus PD98059 (10 lM for 1 h) rescued the change in ERK phosphorylation caused by the overexpression of IRTKS (Fig. 6C and Supple-

mentary Fig. S6A); downregulation of IRTKS also impaired the status of ERK phosphorylation (Fig. 6D and Supplementary Fig. S6B). In addition, we observed an effect of ectopic IRTKS expression and PD98059 on HCC proliferation: the result showed that inhibiting ERK phosphorylation could rescue the proliferation-promoting role of IRTKS in the SK-hep-1 and YY-8103 cell lines (Fig. 6E). Therefore, we inferred that the characteristic promotion of proliferation by IRTKS was achieved by enhancing ERK signalling.

3.7. IRTKS interacts with EGFR and affects EGFR activation EGFR/ERK signalling is an important pathway in the regulation of cellular proliferation [24,25]. To elucidate whether the enhancement of ERK signalling by IRTKS occurs through an interaction with EGFR, we used an immunofluorescence assay to observe the location of the two molecules in cell. In serum-starved YY-8103 cells treated with EGF (20 ng/ml for 0, 10, 30, 60, 90, and 120 min), we noted that IRTKS was localised near the cell membrane at 10–60 min after EGF stimulation; the co-localisation of endogenous IRTKS and EGFR was enhanced at 30 min after EGF stimulation (Fig. 7A). This finding suggested that IRTKS might interact with EGFR and then modulate EGFR activation in response to EGF stimulation.

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Fig. 6. IRTKS promotes cellular proliferation by enhancing ERK signalling. (A) After overexpression and knockdown of IRTKS, canonical signalling pathway nodes were screened using immunoblotting. (B) The relative quantification of bands was performed by the optical density scanning of (A). (C) ERK phosphorylation was detected by immunoblotting after the cells overexpressing IRTKS and treated with PD98059 (an ERK inhibitor). (D) Phosphorylation of ERK was detected by immunoblotting after IRTKS knockdown. (E) Cell proliferation was detected after the cells overexpressing IRTKS and treated with PD98059 (mean ± SD). The results of the above analyses were from 3 independent experiments. P < 0.05, P < 0.01, P < 0.001 versus the control; #P < 0.05, ##P < 0.01, ###P < 0.001 IRTKS plus PD98059 versus IRTKS.

To test this hypothesis, we used reciprocally endogenous coimmunoprecipitation (Co-IP) experiments in SK-hep-1 and YY8103 cells with anti-IRTKS and EGFR antibodies. As expected, EGFR was immunoprecipitated by the anti-IRTKS antibody in both cell lines, and the interaction was strengthened with EGF stimulation. We also observed that IRTKS tyrosine phosphorylation was enhanced after EGF treatment (Fig. 7B). Similarly, IRTKS was immunoprecipitated by the anti-EGFR antibody, and EGF stimulation could also enhance this association. The phosphorylation levels of EGFR at Tyr 1173 immunoprecipitated by IRTKS, in addition to the site involved in MAP kinase signalling activation [26], were increased after EGF stimulation (Fig. 7C). In conclusion, these collective data indicate that IRTKS function may be an adaptor of EGFR that positively regulates ERK signalling.

4. Discussion The abnormal expression of genes is very common in tumours. Indeed, certain ‘‘driver genes’’ can affect tumour proliferation [27,28], metastasis [29,30], and differentiation [31,32]. In addition, some genes are ‘‘passenger genes’’ that might be used as clinical

markers [33,34]. Mounting evidence suggests an important role for IRTKS in the regulation of actin filaments, including pedestal formation [14,15,18], actin assembly and polymerisation [13,16,35,36], and lamellipodia formation [9]. However, the functional role of IRTKS in HCC proliferation remains unknown. In this study, we showed that IRTKS is frequently upregulated in human HCC and that this upregulation is significantly associated with tumour size. These data suggest that IRTKS might play a specific functional role in HCC. Clearly, the most elementary characteristic of cancer cells is their ability to maintain chronic proliferation [37]. Accordingly, the development of an effective intervention targeting proliferous disease should improve the mortality rate and surgical opportunity of cancer patients. In this study, the forced expression of IRTKS promoted the proliferation, colony formation, and anchorage-independent growth of HCC cells both in vitro and in vivo. In contrast, the downregulation of IRTKS produced the opposite results, further confirming that IRTKS can facilitate HCC cell proliferation. ERK is a well-documented, important signalling node that regulates cell growth and differentiation [38,39]. In addition, the activities of Akt [40,41] and b-catenin [42,43] are also related to cell proliferation. After preliminary screening, we found that IRTKS

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Fig. 7. IRTKS interacts with EGFR and affects EGFR phosphorylation. (A) Dynamic images of YY-8103 cells are shown with EGF stimulation for the indicated times. (B and C) Reciprocal Co-IP experiments were performed using cell extracts from SK-hep-1 and YY-8103 cells with anti-IRTKS (B) and -EGFR (C) antibodies. The asterisk () indicates the IgG heavy chain. (D) Schematic representation of the regulation of HCC proliferation involving IRTKS that is formed following EGF stimulation.

can affect the phosphorylation status of ERK. We further identified EGFR, an important proliferation-associated receptor and upstream molecular of ERK signalling [44,45], in this response. Strikingly, the results of this study showed that IRTKS regulates cell proliferation by associating with EGFR and adjusting the tyrosine-specific protein kinase activity intrinsic to the EGF receptor. To our knowledge, this is the first report to explore the role of IRTKS in the tumourigenesis of HCC, and our results indicate that IRTKS functions in promoting the proliferation of HCC cells by interacting with EGFR. Interestingly, the data provided in Table 1 exhibit that IRTKS expression is associated with patient age. Because a previous study

revealed that IRTKS is a substrate of the insulin receptor tyrosine kinase [13], it is possible that IRTKS is involved in the insulin signalling pathway, as the older age group is the population at high risk for diabetes. In summary, we identified the promotion of proliferation as another important function of IRTKS. IRTKS is frequently upregulated and associated with tumour size in HCC. The ectopic expression of IRTKS facilitates HCC cell proliferation, colony formation, and anchorage-independent growth by strengthening the interaction with EGFR and via the positive regulation of ERK phosphorylation. Using these data, we derived a simple signalling flow chart

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(Fig. 7D). Future investigations will assess the precise interaction between IRTKS and EGFR in relation with the EGFR/Shc/GRB2 complex [46]. Expanding on the role of IRTKS in HCC cell proliferation will promote our understanding of the sophisticated mechanisms underlying liver cancer progression and might improve the development of new treatment regimens for curing liver cancer.

[13]

[14]

[15]

Funding [16]

This work was supported by Chinese National Key Program on Basic Research (2010CB529200), China National Key Projects for Infectious Disease (2012ZX10002012-008 and 2013ZX10002010006), and National Natural Science Foundation (81071722 and 81272271).

[17]

[18]

Conflict of Interest We declare that there are no potential conflicts of interest. Acknowledgements We gratefully acknowledge Qing Deng and Xiao Xu for help in the cell cycle analysis, Da-Li Zheng, Bing-Bing Wan, Yan-Dong Li, Rui-Fang Liu, Na Cheng, and Hui Chen for experimental assistance, and Fei Chen, Qian-Lan Fei, and Jin-Shan Li for help in cell culturing.

[19] [20]

[21]

[22]

[23]

Appendix A. Supplementary material [24]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.canlet.2013. 05.019.

[25]

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