SSX2IP promotes metastasis and chemotherapeutic resistance of hepatocellular carcinoma

Background Synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP), which has been identified as an acute myeloid leukemia associated antigen, is a potential target for leukemia immunotherapy. In rodents, its homologous gene, ADIP, plays an important role in the regulation of cell adhesion and migration, underlying its potential role in promoting metastasis of other cancers. Methods To investigate the correlation between the expression level of SSX2IP and the clinicopathologic factors of hepatocellular carcinoma (HCC), 53 cases were studied by qPCR and statisted. To directly testing SSX2IP’s contribution to HCC in animal models, 45 nude mice were enrolled in peritoneal spreading and liver metastasis models. For the migration and invasion assays, cell culture experiments were performed using QCMTM 24-Well Colorimetric Migration Assay Kit and Cell Invasion Assay Kit (Millipore). Moreover we examined the influence of SSX2IP overexpression on the chemosensitivity of hepatocellular carcinoma cells to two most common chemotherapy drugs (5-Fu and CDDP) using Cell counting kit-8 (CCK-8). The chemotherapeutic drugs sensitivity was evaluated by IC50 parameter. Results Statistical analysis of clinical cases revealed that the SSX2IP high expression group had inclinations towards larger tumor size, more tumor thrombus and shorter survival period, implying a strong correlation between the expression level of SSX2IP and HCC tumorigenesis. Consistently in abdominal cavity metastasis and liver metastasis models of immune-deficient mice, SSX2IP was able to promote the metastasis of hepatoma cells. At the cytological level, SSX2IP stimulates the wound healing, metastasis and invasion of hepatoma cells, and reduces the sensitivity of hepatoma cells to 5-Fu and CDDP. Conclusions Our results showed that SSX2IP promotes the development and metastasis of hepatocellular carcinoma and contributes to the drug resistance of hepatoma cells, suggesting that SSX2IP is expected to become a new diagnostic and prognostic marker and a new target of the treatment of hepatocellular carcinoma.


Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, ranking the fifth of the global malignant tumor incidence and the third of cancer related cause of death [1,2]. In China, hepatocellular carcinoma incidence ranks second only to lung cancer and counts for about half of the global number, thus people's health and life quality have been seriously affected. As the disease symptom is not obvious, early diagnosis is difficult; most of the patients have advanced cancer at the time of diagnosis, and the disease is easy to relapse even if the lesions were resected. Although chemotherapy is an important method in combined treatment of hepatocellular carcinoma, its effect becomes less unsatisfactory as hepatoma cells from advanced cancer often develop resistance to chemotherapeutic drugs. Therefore, identifying molecular markers suitable for early diagnosis and new therapeutic targets for treatment will greatly benefit the many HCC patients around the world.
The SSX2IP gene is located on chromosome 1p22.3, spanning 46 kb region and containing 14 exons [3]. The locus has a higher frequency of deletions, amplifications and translocations [4][5][6]. SSX2IP was originally identified through its interaction with SSX2, a transcriptional repressor [7], and was suggested to regulate the role of SSX2 in testicular tissue and malignant cells [8]. Previous studies indicated that SSX2IP is an acute myeloid leukemia-associated antigen, as analysis of clinical cases showed that the expression of SSX2IP was identified in the serum of 33% patients with acute myeloid leukaemia (AML), but not in healthy volunteers [9]. SSX2IP expression was most apparent on the surface of myeloid leukaemia cells in mitosis [10,11]. Several studies further strengthened the argument that SSX2IP was an acute myeloid leukemia-associated antigen and a potential immunotherapy target for leukemia [12][13][14]. So far, the role of SSX2IP in other human tumors remains elusive.
Afadin DIL domain-interacting protein (ADIP) is the homologous gene of SSX2IP in rodents, and the amino acid sequences of ADIP in mouse and rat show 88% and 87% identity with SSX2IP respectively. Cytologically, ADIP localizes at cell-cell adherens junctions and promotes cell migration by activating Rac protein through Vav2 [15,16]. All of the above characteristics are closely related to the adhesion capacity and metastatic characteristics. Given the functionality prompts of SSX2IP and its homologue gene ADIP, we decided to explore the role of SSX2IP in the development and progression of hepatocellular carcinoma.

Cell culture and transfection
Human hepatocellular carcinoma cell lines SMMC-7721 and BEL-7402 were preserved in our institute. Briefly, cells were grown in DMEM supplemented with 10% fetal calf serum. Cells were maintained at 37°C with 5% CO 2 . SSX2IP was constructed into pEGFP-C1 eukaryotic expression vector. We obtained stably transfected clones by G418 selection (Promega, 800 μg/ml). A stable transfectant of the pEGFP-C1 empty vector was used as a control.

Migration, invasion and scratch wound healing assays
For the cell migration assays, cell culture was performed using QCM TM 24-Well Colorimetric Migration Assay Kit (Millipore) according to the manufacturer's instructions. For the invasion assay, Cell Invasion Assay Kit (Millipore) was used according to the manufacture's instructions. Cells (1 × 10 5 ) in 300 μl serum-free medium were added to the upper chambers and cultured for 48 h. Non-migrating or non-invading cells were removed with cottons swabs, Cells that migrated or invaded to the bottom of the membrane were stained with the cell stain buffer provided in the assay kit and counted under microscope and photographed. Three independent experiments were performed for the same conditions. For the scratch wound healing assay, cells were cultured in serum-free medium for 24 h and wounded with pipette tips. Fresh medium was replaced. The wound closing procedure was observed for 48 h, and photographs were taken every 24 h.

Nude mice study
Both peritoneal spreading and liver metastasis models were observed. 4-week-old male BALB/C nude mice were purchased from the Institute of Zoology, Chinese Academy of Sciences of Shanghai. All experiments were performed in accordance with the official recommendations of the Chinese animal community. In peritoneal spreading experiment, 5 mice were enrolled in each group. 1 × 10 6 cells of BEL-7402, BEL-7402 Vector and BEL-7402 SSX2IP were injected into each mouse. In liver metastasis study, 10 mice were enrolled in each group. 1 × 10 6 cells of BEL-7402, BEL-7402 Vector and BEL-7402 SSX2IP were injected into caudal vena. 6 weeks after intraperitoneal injection, mice were killed by cervical decapitation, the abdominal masses were excised and fixed with buffered formalin for further morphological analysis. 10 weeks after caudal vena injection, mice were killed by cervical decapitation. The liver of mice was examined carefully and the metastatic masses were counted.
Chemotherapeutic drugs sensitivity analysis by cell counting kit-8 Hepatocellular carcinoma cells (3 × 10 3 cells/well) were seeded in 96-well plates and treated with different chemotherapeutics 5-fluorouracil (5-Fu) and Cis-diamminedichloroplatinum (CDDP) in different concentrations for 72 h. Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was added into each well. After 4 h incubation at 37°C, the coloring reaction was quantified by an automatic plate reader (BIO.TEK, USA). Growth inhibitory effects were expressed as cell viability curve (OriginPro 8.5.1, USA). The chemotherapeutic drugs sensitivity was evaluated by IC50 parameter (inhibitory concentration of 50% cells).
Patients HCC tissues were collected from patients at Zhongshan Hospital, Fudan University, after obtaining the subjects' informed consent and with institutional review board approval of the hospital.

mRNA expression analysis
Total RNA was extracted using Trizol solution (Invitrogen). Reverse transcription was performed according to the manufacturer's instructions (Promega). qPCR was performed in triplicate using the SYBR Green Mastermix on the ABI Prism 7900 Sequence Detection System (Applied Biosystems). qPCR primers used as follows: SSX2IP:

Statistics
Data were shown as mean ± SD. Statistically significant differences were analyzed using Student t test, when there were only two groups, multiple comparisons were performed by one-way analysis of variance. For survival analysis, Kaplan-Meier method was used. Log rank test was used to analyze differences between statistical significances. All statistical analyses were performed using the SPSS 13.0 software (SPSS Inc, Chicago, IL, USA). P < 0.05 was considered statistically significant.

Results
The expression levels of SSX2IP in HCC patients were significantly correlated with tumor size, tumor thrombosis and survival time To examine the correlation between the expression level of SSX2IP and clinicopathologic factors in HCC, 53 cases were statistically analysed. The clinical and pathologic characteristics of these cases were shown in Table 1. Among them, 32 samples showed a~2-fold up-regulation of SSX2IP compared with the matched non-tumor controls. These samples were classified as the high expression group, and the other 21 samples were classified as the low expression group. Importantly, the SSX2IP high expression group had inclinations towards larger tumor size and more tumor thrombus (P < 0.05). However, there were no significant relationships with other clinicopathologic factors. Notably, 51 out of 53 patients were re-interviewed with a re-interview rate of 96%, Kaplan-Meier survival analysis showed the patient survival time in SSX2IP high expression group (n = 31) was significantly shorter than in the low expression group (n = 20) (P = 0.004, Figure 1). Taken together, our patient cases studies suggest that SSX2IP may have a previously unrecognized role in promoting hepatocellular carcinoma.

SSX2IP promotes peritoneal spreading and liver metastasis of hepatocellular carcinoma cells in nude mice
To directly test the contribution of SSX2IP in HCC, we carried out peritoneal spreading and liver metastatic assays in mice models. A total of 45 nude mice were enrolled in these experiments, among which, 15 mice were used for peritoneal spreading assay and 30 for liver metastatic assay. The results were shown in Figure 2 and summarized in Table 2 and Table 3. Strikingly, the peritoneal nodules were significantly enforced in SSX2IP   over expression group compared with the parental and empty vector-transfected groups (6.2 ± 0.84 vs 2.6 ± 0.89, 2.4 ± 1.14, **P < 0.001, Figure 2A-B and Table 2). The tumorigenicity of liver metastasis was also observed. In SSX2IP overexpression group, 7 out of 10 mice showed tumorigenicity in liver, which is significantly higher than that in the parental group (2/10) and the empty vectortransfected group (1/10) (P = 0.023, Figure 2C-D and  Figure 3).

SSX2IP promotes migration and invasion of hepatocellular carcinoma cells
In order to further assess the influence of SSX2IP on HCC cells, we employed cell migration and invasion assays to determine these two key factors of malignant progression and metastasis upon SSX2IP overexpression. As shown in result, overexpression of SSX2IP led to significantly enhanced migration and invasion of both SMMC-7721 and BEL-7402 ( Figure 4). Number of migratory SMMC-7721 SSX2IP cell line is significantly higher than the other two control groups (123.33 ± 9.45 vs 59.67 ± 4.73 and 51.33 ± 6.03), and number of invasive SMMC-7721 SSX2IP cell line is significantly higher compared to the control groups (92.00 ± 7.00 vs 43.67 ± 4.16 and 38.00 ± 3.61, **P < 0.001, Figure 4A-B). Similarly, number of migratory BEL-7402 SSX2IP cell line is significantly higher than the other two control groups (154.67 ± 14.05 vs 103.67 ± 10.70 and 109.00 ±7.55), and number of invasive BEL-7402 SSX2IP cell line is significantly higher compared to the control groups (113.00 ± 6.56 vs 63.33 ± 11.50 and 60.67 ± 11.02, *P < 0.01, Figure 4C-D). These results displayed an enhanced effect of SSX2IP on hepatocellular carcinoma cells migration and invasion.

SSX2IP reduces the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs
The positive effect of SSX2IP on HCC cells' metastatic status prompted us to speculate that it may also contribute to the drug resistance of HCC cells. To test this idea, we assayed chemosensitivity to the two most common chemotherapy drugs  Figure 5A-B). The IC50s of SMMC-7721 SSX2IP and BEL-7402 SSX2IP for CDDP were 11.38 ± 1.42 μM and 9.86 ± 1.24 μM, respectively, while the IC50s of SMMC-7721, SMMC-7721 Vector , BEL-7402 and BEL-7402 Vector for CDDP were 5.72 ± 0.75 μM, 6.18 ± 0.81 μM, 6.13 ± 0.24 μM and 5.90 ± 0.47 μM, respectively (**P < 0.001, Figure 5C-D). We could concluded that SSX2IP contributes to reduce the sensitivity of HCC cells to chemotherapeutic drugs.  Table 3 Animal experiments for liver metastasis   migration assay have been widely used for studying the motility of tumor cells to reflect their ability to penetrate extracellular matrix and to migrate. Hepatoma cells with high expression of SSX2IP were shown a very strong mobility, and the number of cells which had penetrate the filter membrane in this group was significantly higher than in the parental and empty vector-transfected groups. These results suggest that SSX2IP could promote the migratory and invasive ability of hepatoma cells.
The dynamic changes of cell adhesion characteristics and the reorganization of cytoskeletal play important roles in the process of tumor invasion and metastasis [17,18]. ADIP, the homologous gene of SXX2IP in rodents, localizes at cell-cell adherent junctions and binds to αactinin, a kind of F-actin-bundling protein. ADIP may play a key role in the organization of the cytoskeleton at cellcell adherent junctions and may connect the nectin-afadin and E-cadherin-catenin systems [15]. These systems have been shown to maintain the epithelial to mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), and studies of a variety of tumors have revealed that this conversion characteristics of the tumor cells is an important reason for the tumor metastasis and recurrence [19][20][21]. Therefore, it is suggested that SSX2IP may regulate the EMT-MET by affecting E-cadherin-catenin systems and thereby promote the invasion and metastasis of tumor cells.
On the other hand, ADIP has been shown to promote cell movement by regulating the activation of Rac1 [16]. Rac1 belongs to the Rho family and the Ras superfamily of small GTP-binding proteins, and it is an important regulator which connects membrane surface receptors and actin cytoskeleton and plays a role of molecular switch. Members of Rho family regulate the actin assembly and affect cell movement, tumor invasion and migration [22]. The guanine nucleotide exchange factor specific to Rac1 can regulate cytoskeleton reorganization, affect cell polarity and promote cell motility and migration by promoting the conversion of Rac-GDP (inactivation) to Rac1-GTP (activation) [23]. As our study indicated a role of SSX2IP in promoting the mobility of HCC cells, we speculated that SSX2IP may do so via activating Rac1. We are actively investigating the influence of SSX2IP on Rac1 activation.
Chemotherapy plays an irreplaceable role in the clinical treatment of HCC [24]. However, resistance of hepatoma cells to chemotherapy drugs often makes chemotherapy unsatisfactory [25]. High expression of some resistance related genes in tumor cells is the underlying cause of drug resistance that eventually leads to ineffective treatment. Our data showed SSX2IP reduced the sensitivity of hepatoma cells to 5-Fu and CDDP and significantly increased IC50, which indicated that SSX2IP may be a resistance related gene with the ability to promote the resistance of hepatoma cells to chemotherapeutic drug. Although the exact function of SSX2IP in drug resistance is still unknown, clues can be taken from its rodent homologue ADIP, which directly binds to β'-COP to participate in the vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum [26]. As is well known, many resistance genes encode transporter proteins, such as ABCG2 in the ABC protein family [27,28]. We speculate that SSX2IP is likely to be a player in the intracellular transportation system.
Studies of SSX2IP in tumor-related fields have been focused on leukemia in the past, while little is known about its role in oncology of other types of cancers. Our results suggest that SSX2IP can significantly promote the motility, invasion and migration of hepatoma cells and improve their resistance to chemotherapeutic drugs. Importantly, since SSX2IP has been identified as an acute myeloid leukaemia-associated antigen and a potential immunotherapy target for leukemia, this study has expanded the usage of those SSX2IP related applications to HCC. Further in-depth exploration of the molecular mechanisms of SSX2IP in promoting the migration, invasion and drug resistance of HCC will be needed to clarify the role of SSX2IP in HCC development and to provide theoretical basis for future clinical applications.

Conclusions
Our studies showed that SSX2IP can promote the development and metastasis of hepatocellular carcinoma. It reduces the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs and could be a resistanceassociated gene. All the above, our findings suggest that SSX2IP may serve as a new target for clinical diagnosis and treatment of hepatocellular carcinoma.