Beneficial effects of Cripto-1 for transarterial chemoembolization in hepatocellular carcinoma

Upregulated Cripto-1 expression promoted HCC cell proliferation and invasion

Our previous study had showed that Cripto-1 could be used as a potential prognostic factor for the survival of HCC patients. We further explored the function of Cripto-1 in tumor cell proliferation, migration and invasion in HCC cells. The CCK-8 assay results showed that down-regulated Cripto-1 expression may inhibit tumor cells proliferation in SK-Hep1 cells, while opposite results were showed in up-regulated Cripto-1 expression of SMMC-7721 cells (Figure 1). Transwell invasion assay found that lower invasion and migration ability was observed in sh-Cripto-1-SK-Hep1 cells, compared with those with shControl-SK-Hep1 cells (both P < 0.001, Figure 2A, 2C), while the ability of cell motility and invasion was increased in Cripto-1-SMMC-7721 cells, compared with those with Vector-SMMC-7721 cells (both P < 0.001, Figure 2B, 2D).

Figure 1. Cripto-1 promotes cell proliferation in HCC cells. The cell viability was inhibited in Cripto-1-silenced cells compared with the control cells (A), while the opposite effect of cell viability was observed in Cripto-1-transfected cells (B).

Figure 2. Cripto-1 promotes cell migration and invasion in HCC cell lines. (A, C) Down-regulated Cripto-1 expression in SK-Hep1 cells and suppressed cell migration and invasion. (B, D) Up-regulated Cripto-1 expression in SMMC-7721 cells and accelerated cell migration and invasion. The scale bar represents 50 μm.

As Epithelial-mesenchymal transition (EMT) is a key process in cancer metastasis by which tumor cells acquire migratory characteristics, thereby disassociating from the primary tumor and migrating to distant sites [16, 17], the effect of Cripto-1 on EMT was examined by investigating the expression levels of EMT relevant markers. Western blot and RT-PCR results revealed that increased expression of E-cadherin with decreased expression of N-cadherin, Vimentin, Fibronectin and Snail appeared in sh-Cripto-1-SK-Hep1 cells, compared with the control cells (Figure 3A and 3B). An opposite expression pattern of these genes was observed in Cripto-1-SMMC-7721 cells (Figure 3A and 3B).

Figure 3. Cripto-1 expression was correlated with the expression of epithelial-mesenchymal transition (EMT) relevant markers. Western blot (A) and RT-PCR (B) results showed that transfecting siRNA against Cripto-1 decreased Cripto-1 expression, down-regulated N-cadherin, Vimentin, Fibronectin and Snail and up-regulated E-cadherin. While opposite results were showed in HCC cells with up-regulated Cripto-1 expression.

Prognostic significance of postoperative adjuvant TACE in different Cripto-1 expression

Adjuvant TACE is one of the most common methods to prevent tumor relapse and prolong survival time in HCC. However, the curative effect of adjuvant TACE remains controversial. In this study, our results revealed that adjuvant TACE after hepatectomy could not increase the OS or TTR times in the whole study population (Figure 4A). As Cripto-1 plays an important role in tumor progression and could be an independent prognostic biomarker for HCC patients, we further explored whether the response of patients to adjuvant TACE therapy was different in different Cripto-1 expression of HCC patients. Interestingly, adjuvant TACE may promote tumor relapse and had no influence on OS time in patients with low Cripto-1 expression (Figure 4B). On the contrary, in high Cripto-1 expression patients, those with adjuvant TACE had favorable TTR and OS times (Figure 4C). In patients with high Cripto-1 expression, the median of TTR was 13.0 months, while the median of OS was 26.5 months. The median of TTR and OS times for adjuvant TACE group were 16.5 months and 36.5 months, while there were 10.5 months and 22.5 months in control group. Furthermore, the 5-year TTR and OS rates of the adjuvant TACE group were 36.3% and 40.9%, which were significantly higher than that of the control group (16.7% and 28.1%) (Figure 4C).

Figure 4. Prognostic significance of adjuvant TACE in HCC patients according to Cripto-1 expression. Adjuvant TACE could not increase the OS or TTR times in all HCC patients (A). Kaplan-Meier analysis of the association between adjuvant TACE therapy and OS/TTR in patients with low Cripto-1 expression (B) and high Cripto-1 expression (C).

Cox proportional-hazards regression was used to analyze the correlation of Cripto-1 expression with response to adjuvant TACE in HCC patients. Our data revealed that adjuvant TACE could not be an independent prognostic factor for TTR or OS in HCC patients with low Cripto-1 expression (Table 1). Interestingly, in patients with high Cripto-1 expression, the results showed that without received TACE (P = 0.019), larger tumor size (P = 0.01), multiple tumor (P = 0.001), satellite nodule (P = 0.025) and vascular invasion (P = 0.005) were unfavourable factors for TTR in HCC patients. Moreover, Kaplan-Meier analysis revealed that without received TACE (P = 0.035), high AFP level (P = 0.030), larger tumor size (P = 0.002), multiple tumor (P < 0.001), satellite nodule (P = 0.007) and vascular invasion (P < 0.001) were positively associated with shorter OS in HCC patients (Table 2). Multivariate Cox regression analysis showed that patients who received adjuvant TACE had longer TTR times than those without received TACE therapy (HR = 0.371, 95% CI = 0.229-0.602, P < 0.001). In addition, adjuvant TACE was a potential prognostic biomarker for OS (HR = 0.280, 95% CI = 0.162-0.484, P < 0.001) in patients with high Cripto-1 expression (Table 2).

Cripto-1 expression predicts response of adjuvant TACE in clinical subgroups

The HCC patients with vascular invasion or larger tumor (> 5 cm in diameter) are recommended to receive TACE therapy 1-2 months after hepatectomy [6–8]. It has been known that liver cirrhosis is a significant risk factor for post-operative recurrence [18, 19]. Our findings had similar results with previous study. We found that adjuvant TACE could reduce the recurrence rate and prolong the OS time in HCC patients with vascular invasion (Supplementary Figure 1B) and larger tumor (Supplementary Figure 1D), while adjuvant TACE had no effect on patients with no vascular invasion (Supplementary Figure 1A), small tumor (Supplementary Figure 1C), liver cirrhosis (Supplementary Figure 1F). Interestingly, adjuvant TACE may even promote tumor recurrence in patients with no liver cirrhosis (Supplementary Figure 1E). Furthermore, we wonder whether all the patients with vascular invasion or larger tumor could obtain a benefit from adjuvant TACE after surgery, and whether all the liver cirrhosis patients could not have a benefit from adjuvant TACE. We further assessed the discriminative power of Cripto-1 expression on postoperative adjuvant TACE in patients with vascular invasion, large tumor and liver cirrhosis. In the patients with vascular invasion subgroup (n = 51), low-expressed Cripto-1 patients had no response on adjuvant TACE (Figure 5A), On the contrary, of the patients with high Cripto-1 expression, adjuvant TACE had a better median TTR and OS times than the control group (median TTR for adjuvant TACE [n = 13] versus control group [n = 14]: 12.0 months versus 5.5 months, P = 0.008; median OS for adjuvant TACE [n = 13] versus control group [n = 14]: 20.0 months versus 9.5 months, P = 0.010; Figure 5B). In the patients with larger tumor subgroup (n = 129), patient with low Cripto-1 expression could not have a benefit from adjuvant TACE (Figure 5C). Contrarily, adjuvant TACE could prolong the TTR and OS of patients with high Cripto-1 expression (median TTR for adjuvant TACE [n = 31] versus control group [n = 48]: 13.0 months versus 10.0 months, P = 0.027; median OS for adjuvant TACE [n = 31] versus control group [n = 48]: 34.0 months versus 15.5 months, P = 0.006; Figure 5D). In the patients with liver cirrhosis subgroup (n = 179), adjuvant TACE had no effect on patients with low Cripto-1 expression (Figure 5E). Interestingly, adjuvant TACE was associated with significant improvement in TTR and OS of patients with high Cripto-1 expression (median TTR for adjuvant TACE [n = 39] versus control group [n = 62]: 16.0 months versus 10.0 months, P = 0.017; median OS for adjuvant TACE [n = 39] versus control group [n = 62]: 34.0 months versus 20.5 months, P = 0.049; Figure 5F).

Figure 5. combination of Cripto-1 and tumor features predicts response to postoperative TACE. All patients were divided according to Cripto-1 levels within vascular invasion (A, B), lager tumor size (C, D) or liver cirrhosis (E, F). Kaplan-Meier survival estimates and log-rank tests were used to analyze the correlation of adjuvant TACE therapy and OS/TTR in different subgroups.

Patients and specimens

The informed consents were provided and experiment was approved by the Institutional Review Board and Human Ethics Committee of Affiliated Cancer Hospital & Institute of Guangzhou Medical University.

All the 231 HCC samples were collected from Department of Hepatobiliary Oncology, Sun Yat-sen University (Guangzhou, China) between July 2007 and January 2009 (Supplementary Table 1). Among these patients, 126 patients (54.5%) had undergone curative resections only, and 105 patients (45.5%) received adjuvant TACE 1–2 months after hepatectomy. The patients included in this study following the inclusion: (1) all the tissues were pathological diagnosis; (2) no distant metastases or other malignant diseases (based on enhanced CT and/or MRI); (3) no chemotherapy or radiotherapy before surgery. All patients were enrolled consecutively according to resection tissues and follow-up data. Tumor stage was classified base on the 7^th Edition TNM classification of the American Joint Committee on Cancer Staging and the Barcelona Clinic Liver Cancer (BCLC) staging system.

Establishment of Cripto-1 knockdown HCC cells

In this study, we used two human HCC cell lines: SK-Hep1 and SMCC-7721. The two cell lines were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China) and were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1% penicillin and streptomycin, supplemented with 10% fetal bovine serum (FBS).

Lentiviral containing short hairpin RNAs (shRNA) targeting Cripto-1 was purchased from Hanbio Biotechnology (Hanbio Biotechnology Co., Ltd.) and transfected into SK-Hep1 cell using Lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. Cells transfected with empty vector were used as controls. Stable Cripto-1 knockdown expression clones were selected by Geneticin (Rache Diagnostics, Indianapolis, IN) at the concentration of 500ug/ml.

Plasmid constructs and transfection

Human Cripto-1 cDNA was amplified by PCR and cloned into pcDNA3.1(+) expression vector (Invitrogen, Carlsbad, CA), and then transfected into SMMC-7721 cell using Lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. Cell transfected with empty vector were used as controls. Stable Cripto-1-expressing clones were selected by Geneticin (Rache Diagnostics, Indianapolis, IN) at the concentration of 500ug/ml.

Cell counting kit-8 assay

The HCC cells were detached with trypsin and seeded on a 96-well plate (1x10³ cells/well). After culture in DMEM culture medium supplemented with 10% FBS to allow cells become adherent, cells were observed at different time points (0 h, 24 h, 48 h, 72 h, 96 h and 120 h). Then 10 μl CCK-8 solution was added into each well at different time points and the plate was incubated for additional 2 h. And then, the absorbance at 450 nm was measured using a microplate reader. Wells without addition of CCK-8 solution were taken as the blank. The cell viability was calculated using the formula: Cell viability (100%) = (mean OD of experimental group - mean OD of blank group)/(mean OD of control group - mean OD of blank group) x 100. We performed 3 parallel repeats in each group.

Western blotting

Cell lysates were subjected to 7.5–10% PAGE and transferred to nitrocellulose filter membranes. The membranes were blocked for 1 h in 5 % non-fat dry milk diluted with TBST (10 mM Tris–HCl and 0.05 % Tween 20). The membranes were incubated with the Cripto-1 rabbit polyclonal antibody (working dilution 1:200, Abcam, #ab19917, UK), E-cadherin mouse monoclonal antibody (working dilution 1:300, Abcam, #ab1416, UK), N-cadherin rabbit polyclonal antibody (working dilution 1:200, Abcam, #ab18203, UK), Vimentin mouse monoclonal antibody (working dilution 1:200, Abcam, #ab8978, UK), Fibronectin rabbit polyclonal antibody (working dilution 1:300, Abcam, #ab2413, UK), Snail rabbit polyclonal antibody (working dilution 1:200, Abcam, #ab82846, UK), GAPDH rabbit polyclonal antibody (working dilution 1:5000, Abcam, #ab181602, UK) overnight at 4°C, followed by incubation with appropriate secondary antibodies at room temperature for 2h. The membranes were washed with PBS for three times, and the immunoreactive bands were visualized using an ECL plus Kit, according to the manufacturer’s instructions.

Transwell invasion assays

Invasion assay was performed with BD BioCoat Matrigel Invasion Chambers (Becton Dickinson Labware, Franklin Lakes, NJ) following the manufacturer’s instructions. The matrigel membrane was stained with crystal violet, and migrated cells were counted under a microscope.

Total RNA extraction and qRT-PCR

Total RNA was extracted from HCC cell lines using TRIzol reagent (Invitrogen) following manufacturer’s introductions. The total RNA (2 µg) was reverse transcribed using a PrimeScript RT Kit (Takara, Dalian) for first-strand cDNA synthesis. The primer sequences were: Cripto-1, forward primer, 5′-GGGATACAGCACAGTAAGGAG-3′, reverse primer, 5′-ACGGTGGTAGTTCTGGAGTC-3′. GAPDH, forward primer, 5′-CTCCTCCTGTTCGACAGTCAGC-3′, reverse primer, 5′-CCCAATACGACCAAATCCGTT-3′. E-cadherin, forward primer, 5′-CGAGAGCTACACGTTCACGG-3′, reverse primer 5′-GGGTGTCGAGGGAAAAATAGG-3′. N-cadherin, forward primer, 5′-CACGCCGAGCCCCAGTAT-3′, reverse primer 5′-GCCCCCAGTCGTTCAGGTAA-3′. Vimentin, forward primer, 5′-GACGCCATCAACACCGAGTT-3′, reverse primer, 5′-CTTTGTCGTTGGTTAGCTGGT-3′. Fibronectin, forward primer, 5′-CAGTGGGAGACCTCGAGAAG-3′, reverse primer, 5′-TCCCTCGGAACATCAGAAAC-3′. Snail, forward primer, 5′-TTCTCTAGGCCCTGGCTGC-3′, reverse primer, 5′-TACTTCTGACATCTGAGTGGGTCTG-3′. Expression of Cripto-1 gene and EMT relevant genes were examined by Power SYBR^® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), and the assay was performed on an ABI PRISM 7900 Sequence Detector. The relative expression level (defined as fold change) of Cripto-1 (2^-△△Ct) was normalized to the endogenous 18S rRNA reference (△Ct) and related to the amount of target gene in control sample, which was defined as the calibrator at 1.0.

TACE treatment

All the patients had received adjuvant TACE 1-2 months after surgery. Hepatic arterial angiography was performed firstly, and then preventive chemoembolization was done among patients without tumor stain in the remaining liver. The regimen for preventive adjuvant TACE consisted of lobaplatin 50 mg, Epirubicin (EPI) 50 mg and lipiodol 5 ml. A Contrast-enhanced CT or MRI was performed one month later, and the regimen was finished.

Follow-up

The last follow-up was October 2017. All the patients were followed up every 2 months in the first 2 years after first TACE treatment and every 3-6 months afterwards, with routine monitoring by serum α-fetoprotein (AFP) level, liver function tests, abdominal ultrasonography, and contrast-enhanced computed tomography or magnetic resonance imaging. The main causes of death were HCC recurrence or complicated cirrhosis of the liver.

Statistical analysis

The SPSS software package (version 16.0; Chicago, IL, USA) was used for the statistical analysis. The experimental data between two groups were performed by a two-tailed Student’s t-test. The mRNA level of Cripto-1 and EMT relevant markers in HCC cells were compared using paired Student’s t-test. The Kaplan-Meier analysis with log-rank test was used to calculate the survival time of HCC patients. The Cox proportional hazard regression model was used to analyze the independent prognostic biomarkers for HCC patients. All P values were two-sided and a P value < 0.05 was considered as statistically significant.