A total of 178 primary HCC samples were randomly selected from patients who received curative resection for HCC at the Liver Cancer Institute and Zhongshan Hospital, Fudan University (Shanghai, China) from February 2006 to November 2006. This study was approved by the Zhongshan Hospital Research Ethics Committee. Follow-up procedures have been described previously (23). Each patient was followed-up until March 2012, with a median follow-up of 56 months (range, 2–69 months). The clinical characteristics of HCC patients are presented in Table I.

Preparation of tissue microarray and immunohistochemistry procedures were performed as described (23). Antibodies used were anti-human MST4 (rabbit, polyclonal, 1:100; Proteintech) and anti-human p-ERK (rabbit, polyclonal, 1:200; Cell Signaling Technology). Two independent pathologists who did not have information regarding the patient characteristics assessed the immunohistochemistry staining. The immunohistochemistry staining intensity was graded on a scale from 0 to 3 (0, no staining; 1, weak staining; 2, mild staining; 3, strong staining). The staining extent was graded on a scale from 0 to 3 based on the percentage of immunoreactive tumor cells (0%; 1–25%; 26–50%; 51–100%). The final score was obtained by multiplying the staining extent score by the staining intensity score. If the final score was <3, then it was defined as low expression. If the final score was >3, then it was defined as high expression.

Flag-tagged and HA-tagged full-length MST4 cDNA were cloned in pcDNA3 (Invitrogen) to create pFlag-MST4 and pHA-MST4, respectively. Lentiviral vectors pLKO.1 TRC (Addgene plasmid 10879) and pWPI.1 (Addgene plasmid 12254) were used for producing recombinant lentiviruses. For RNA interference of MST4, DNA fragments encoding hairpin precursors for shMST4 group 1 (5′-CCTCTTGGTGTAT AGTATTTA-3′) and shMST4 group 2 (5′-CCAGATTGCTA CCATGCTAAA-3′) were inserted into pLKO.1 TRC, respectively. A scrambled siRNA precursor (Scr) of similar GC content as shMST4 group 1 and shMST4 group 2, but with no sequence identity with MST4, was used as a negative control. For overexpression of MST4, flag-tagged MST4 cDNA was cloned in pWPI.1.

The human hepatic non-tumor cell line LO2, the embryonic kidney cell line HEK293T, and HCC cell lines SMMC7721, PLC, and Hep3B were obtained from Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). MHCC-97H and MHCC-LM3 cell lines were established at the Liver Cancer Institute and Zhongshan Hospital, Fudan University. All cells were cultured in DMEM high-glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO₂.

The expression plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Helper plasmids pSPAX2 (Addgene plasmid 12260) and pMD2.G (Addgene plasmid 12259) were cotransfected with pLKO.1-based or pWPI.1-based plasmids in HEK293T cells to package recombinant lentiviruses. Supernatants from cotransfections were used for infection of cultured cells.

The detected cells were harvested in 6-well plates. The total RNA of each group was isolated using TRIzol (Gibco Laboratories). Reverse-transcription was performed with a PrimeScript reverse-transcription reagent kit (Takara Biotechnology, Dalian, China). Gene-specific primers for human MST4 and GAPDH were generated by Sangon Biotech (Shanghai, China). The following primer sequences were used: MST4: F-5′-TTCGAGCTGGTCCATTTGATG-3′, R-5′-TGA ATGCAGATAGTCCAGACCT-3′; and GAPDH: F-5′-TGTG GGCATCAATGGATTTGG-3′, R-5′-ACACCATGTATTCCG GGTCAAT-3′. The iCycler and iQ real-time polymerase chain reaction (Bio-Rad, Hercules, CA, USA) and SYBR-Green PCR Master Mix (Toyobo, Osaka, Japan) were used for amplification and detection. The steps of amplification were 95°C for 30 sec, 60°C for 30 se and 72°C for 30 sec for 40 cycles. The data were analyzed by using the real-time polymerase chain reaction analysis software Bio-Rad iQ5. All experiments were performed in triplicate.

Cell proliferation was measured by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). According to the instructions, cultured cells were seeded onto 96-well plates at 5×10³ per well (n=5 for each time-point) in a final volume of 100 μl. At indicated time-points, CCK-8 solution (10 μl) was added to each well and the absorbance at 450 nm was monitored with Tecan Infinite M200 NanoQuant Daily (Mannedorf, Switzerland).

To detect clonogenesis ability, the cells (1×10³ cells) were suspended with DMEM containing 10% FBS and cultured in a 6-well plate at 37°C with 5% CO₂. After 14 days, colonies were photographed and counted. All experiments were performed in triplicate.

The invasive ability of HCC cells was detected by 24-well transwell chambers with 8-μm pores of polycarbonate membranes (Costar, Cambridge, MA, USA) coated with Matrigel (BD Pharmingen, San Jose, CA, USA). The upper chamber was filled with FBS-free DMEM medium and the bottom chamber was filled with DMEM containing 10% FBS as a chemoattractant. Upper chambers containing cells (5×10⁴) were filled and incubated at 37°C. After 48 h, cells migrating to the bottom of the membrane were stained with Giemsa (Sigma Chemical, St. Louis, MO, USA). Then, the cells were imaged and counted with a microscope (Leica, UK). All experiments were performed in triplicate.

Cells on the cover slips were fixed with 4% paraformaldehyde for 30 min and permeabilized in 1% Triton X-100 for 15 min at room temperature. After blocking with 1% bovine serum albumin for 30 min, the cells were incubated with primary antibodies for 4 h. The following antibodies were used: human anti-E-cadherin (mouse monoclonal antibody, 1:50; Santa Cruz), Vimentin (mouse monoclonal antibody, 1:50; Santa Cruz), and human anti-MST4 (rabbit, polyclonal, 1:100; Proteintech). Then, samples were incubated with Alexa 488-conjugated and 594-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. Slides were mounted in medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed under a fluorescence microscope.

The proteins were extracted with RIPA buffer containing protease inhibitors, separated on a denaturing 10% SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride membrane. After blocking with 5% non-fat milk in phosphate-buffered saline containing 0.05% Tween-20, the membrane was incubated with primary antibodies overnight at 4°C, followed by incubating with peroxidase secondary antibodies for 1 h at 37°C. The following antibodies were used: anti-human MST4 (rabbit, polyclonal, 1:500; Proteintech); anti-human GAPDH (rabbit, polyclonal, 1:1000; Cell Signaling Technology); anti-human p-ERK, ERK, E-cadherin, N-cadherin, Vimentin, Snail, and Slug, and the secondary antibody belonging to the EMT Antibody Sampler kit (rabbit, polyclonal, 1:1,000; Cell Signaling Technology). Western blotting was detected and analyzed with Image Acquisition using Image Quant LAS 4000 (GE Healthcare Life Sciences, MI, USA) and Quantity One software (Bio-Rad Laboratories), respectively.

Statistical analysis was performed with SPSS 13.0 (SPSS, Chicago, IL, USA). All data are presented as the mean ± standard deviation. The Student’s t-test and one-way analysis of variance were applied to analyze the difference of two groups or more than two groups, respectively. Kaplan-Meier analysis was used for survival analysis and log-rank test was used to compare patient survival between subgroups. A Cox proportional hazards model was adopted for multivariate analysis. Receiver-operating characteristic (ROC) curve analysis was applied to assess the predictive values of variables. P<0.05 was considered statistically significant for all tests.

Our previous cDNA array study showed that MST4 had higher expression level in HCC with metastasis than those without metastasis (22), indicating the correlation between MST4 level and metastatic potential of HCC. In this study, we first examined the protein expression of MST4 in the normal liver cell line and HCC cell lines with different metastatic abilities. Western blot analysis showed that MST4 was detected in higher levels in HCC cell lines with high metastatic potential (MHCC97H and MHCCLM3) than those cell lines with low-metastatic potential (Hep3B, PLC, and SMMC7721) and normal liver cell line L02 (Fig. 1A)

We then tested the mRNA and protein levels of MST4 in HCC samples with vascular invasion (VIHCC) and with non-vascular invasion (non-VIHCC), and found that they were much higher in VIHCC than that in non-VIHCC (P<0.05) (Fig. 1B and C), which is in agreement with our previous cDNA array results. Immunohistochemistry staining showed that MST4 expression was negative in normal and peritumoral liver tissue, relatively positive in non-VIHCC, and strongly positive in VIHCC (Fig. 1D). These results indicate that high expression of MST4 is closely associated with aggressive metastasis in HCC.

We then evaluated the association of MST4 expression and the outcomes of 178 HCC patients with 5-year follow-up after operation using immunostaining and tissue microarrays. The clinicopathologic characteristics of the patients are shown in Table I. MST4 staining was mainly found in the cytoplasm of tumor cells and around the nucleus but was absent in most stromal cells (Fig. 2A). According to the HCC staining scores, all the samples were stratified into two grades: strong and mild MST4-positive staining were defined as high MST4 expression (n=61), and low MST4-positive staining and MST4-negative staining were defined as low MST4 expression (n=117).

Statistical analysis showed that the high MST4 expression group displayed significantly worse overall survival (OS) (median OS, 58 months versus >63 months; log-rank=8.12; P=0.004) (Fig. 2B) and shortened time to tumor recurrence (TTR) (median TTR, 43.0 months versus >63 months; log-rank 10.31; P=0.001) (Fig. 2C) compared to the low MST4 expression group. Large tumor size, microvascular invasion, multiple tumors, and advanced TNM classification of malignant tumors stage were found to be associated with worse OS and shortened TTR in univariate analysis. Multivariate analysis results revealed that MST4 intensity in tumors is an independent prognosticator for both OS (relative risk, 1.863; P=0.012) and TTR (relative risk, 2.348; P=0.001) (Table II), suggesting that MST4 is a valuable predictor of clinical outcomes in HCC patients.

Considering the close association of high MST4 expression and high recurrence rate of HCC, we examined whether high MST4 level plays a key role in HCC proliferation and invasion. To verify this inference, lentivirus-mediated knockdown in highly invasive MHCC-97H cells was performed to assess the functional involvement of MST4 in HCC cell proliferation, colony formation, and invasion in vitro. We generated three MST4-specific shRNAs (shMST4) and infected MHCC-97H cells with a lentivirus expressing shMST4 precursor, and found the second group of shMST4 induced >80% reduction which was used for further study (Fig. 3A, left panel). Compared to the scrambled shRNA group (SCR group) and wild-type group (WT group), the proliferation and the clone formation in the MST4 knockdown group (KD group) were restrained significantly (P<0.01) (Fig. 3B, left panel and Fig. 3C, upper panel). Furthermore, depletion of MST4 also led to dramatic decline in invasiveness potential (transwell assay, P<0.01) (Fig. 3D, lower panel). We generated the SMCC7721 clone with stable lentivirus-mediated overexpression of MST4 (MST4 group) and the SMCC7721 empty vector as the control group (MOCK group) and found that exogenous expression of MST4 in the MST4 group significantly enhanced HCC cell proliferation, colony formation, and invasion compared with wild-type and mock groups(Fig. 3). Taken together, these results suggest that MST4 plays an important role in HCC proliferation and invasion.

EMT is an important process in tumor proliferation and invasion. To investigate whether MST4 altered the EMT process in HCC metastatic progression, we examined the expression level of E-cadherin, N-cadherin, and Vimentin, the cell markers of EMT, in HCC cells. As shown in Fig. 4A and C, relative to the scrambled shRNA group (SCR group), decreased expression of MST4 (KD group) resulted in upregulation of E-cadherin and downregulation of N-cadherin and Vimentin, whereas overexpression of MST4 (MST4 group) led to downregulation of E-cadherin and upregulation of N-cadherin and Vimentin, compared to the control group (MOCK group). Immunofluorescence staining also confirmed these results in Fig. 4B and D. The expression of E-cadherin is negatively regulated by transcription factors, such as Snail and Slug (24). As illustrated in Fig. 4A and C, the levels of Snail and Slug were dramatically downregulated in the MHCC-97H knockdown group and significantly upregulated after MST4 overexpression in SMCC7721. These results indicate that MST4 may promote malignant progression of HCC by inducing the EMT process.

Mounting evidence indicates that Snail and Slug play crucial roles in initiating EMT process (25,26), and they are both important downstream targets of the ERK signaling pathway (27,28). To further investigate the mechanisms of MST4 in promoting EMT process in HCC cells, we examined the influence of MST4 overexpression on the ERK pathway.

HCC cell lines SMMC7721 and Hep3B with MST4 overexpression were generated with stable lentivirus-mediated MST4 (MST4 group) and the empty vector (control MOCK group). As shown in Fig. 5A and B, the ratio of p-ERK to ERK was upregulated in the MST4 group compared to the mock group. This trend was similar to that of the mesenchymal markers such as Snail, Slug, and Vimentin, but was contrary to that of E-cadherin.

To further confirm the key role of the p-ERK/ERK pathway in the promotion of EMT with MST4, we used the p-ERK inhibitor (U0126, 20 μM; Sigma-Aldrich) to decrease the activation of ERK. As shown in Fig. 5, U0126 treatment partially reversed the MST4-induced increase in the ratio of p-ERK to ERK and the mesenchymal markers but did not bring it back down to control levels. In addition, U0126 treatment also suppressed the migratory and invasive behavior of the SMMC7721 and Hep3B cells with MST4 overexpression (Fig. 5B and D). These data revealed that overexpression of MST4 promote the EMT process, at least in part, through upregulation of the ERK signaling pathway.

On the basis of the in vitro finding that MST4 regulated phosphorylation of ERK in HCC, we further investigated the relationship between MST4 and p-ERK in HCC tissue microarray. Positive p-ERK staining was partly detected in cytoplasmic and nuclear HCC cells (Fig. 6A), and p-ERK expression was significantly correlated with MST4 expression (data not shown). To further confirm the prognostic value of MST4 and p-ERK, HCC tissues were classified into the following three groups according to the positive staining value of MST4 and p-ERK: group 1 (n=67), low MST4 and low p-ERK expression; group 2 (n=81), either high MST4 or high p-ERK expression; and group 3 (n=30), high MST4 and high p-ERK expression. There were significant differences in OS (P=0.001) (Fig. 6B) and TTR (P<0.001) (Fig. 6C) among the three groups.

Multivariate survival analysis results showed that MST4 alone could predict death and recurrence precisely with the areas under the receiver-operating characteristic curve of 0.584 [95% confidence interval (CI), 0.503–0.672] and 0.579 (95% CI, 0.502–0.668), respectively. The combination of MST4 and p-ERK further elevated the area under the curve and was better for predicting death (0.622; 95% CI, 0.532–0.707) (Fig. 6D) and recurrence (0.623; 95% CI, 0.537–0.708) (Fig. 6E) for HCC.