Abnormal expression of paxillin correlates with tumor progression and poor survival in patients with gastric cancer

This study was approved by the ethics committee of Sun Yat -sen University Cancer Center and written informed consents for using the samples for research purpose were obtained from all the patients before surgery. We collected 239 paraffin-embedded, archived tissue samples from patients who underwent surgery in Sun Yat-sen University Cancer Center (Guangzhou, China) from 2004 to 2008. Moreover, we obtained paired fresh gastric cancer tissues and adjacent nontumorous tissues from 30 of the 239 patients and kept in liquid nitrogen until use. All the patients had a histologically confirmed diagnosis of gastric cancer after resection. Tumor stage was determined according to the 7th edition of the International Union Against Cancer (UICC) on Tumor-Node-Metastasis (TNM) staging system. All the patients were followed-up regularly every three months after surgery with a median follow-up time of 32.5 months (range from 4 to 75 months). All the patients did not receive any pre-operative treatment. The patients who received adjuvant chemotherapy after surgery were based on 5-FU, platinum or taxol regimens. Relevant clinicopathological information including age, gender, tumor size, tumor depth, lymph node invasion, distant metastasis, differentiation status, TNM stage and treatment strategies were obtained from patients’ medical files.

The human gastric cancer cell lines HGC27, SGC7901, BGC823, AGS and MKN28 were obtained from either the RIKEN Cell Bank or the American Type Culture Collection and were cultured with RPMI 1640 medium (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, invitrogen, Carlsbad, CA, USA) in a humidified chamber with 5% CO₂ at 37°C. Human gastric epithelial cell line GES-1 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and was cultured with Dulbecco’s Modified Eagle Medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml).

Total RNA was extracted from the tissues or cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and complementary DNA (cDNA) was synthesized with 2 μg of total RNA by using M-MLV transcriptase (Promega, Madison, WI). Real-time PCR was performed with an ABI PRISM® 7500 Fast Real-time PCR System (Applied Biosystems, CA, USA) and a SYBR Premix Ex Taq™ kit (Takara, Japan); β-actin expression was used as a reference. The following temperature profiles were used: initial heating at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 65°C for 10 s. The primers used were:

PXN forward: 5′-ACGTCTACAGCTTCCCCAACAA-3′;

PXN reverse: 5′-AGCAGGCGGTCGAGTTCA-3′;

β-actin forward: 5′-TGGATCAGCAAGCAGGAGTA-3′;

β-actin reverse: 5′-TCGGCCACATTGTGAACTTT-3′.Data were analyzed using the 2^-△△ct method.

Total cellular proteins were extracted from tissues or cells and separated by SDS-PAGE gels, Western blot analysis was performed according to a standard method as previously described [22]. For immunoblotting of PXN, a rabbit PXN antibody (1:1000, Cell Signaling Technology, USA) was used, and an anti-GAPDH antibody (1:2000; Santa Cruz Biotechnology, USA) was used as loading control.

IHC analysis of PXN was performed according to a previously described method [23]. Briefly, the formalin-fixed, paraffin-embedded tissue samples were cut into 4 μm slides, dewaxed in xylene, rehydrated with graded of alcohols, and then treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The slides were boiled in 0.01 mol/L sodium citrate buffer (pH 6.0) in a microwave oven to retrieve tissue antigens. Tissue samples were pretreated with 10% normal goat serum to inhibit non-specific staining and incubated at 4°C with a primary antibody (ab#32084, abcam) over night. Tissue sections were then washed with PBST, treated with an anti-rabbit secondary antibody, incubated with streptavidin horseradish peroxidase complex, and finally developed using diaminobenzidine tetrahydrochloride (DAB).

To assess the expression of PXN, we qualified and scored both the extent and intensity of immunoreactivity. In this study, the scores of the extent of staining were evaluated according to the percentage of cells that had positive immunoreactivity in every microscopic field of view: 0, <25%; 1, 25%-50%; 2, 50%-75%; 3, 75%-100%. The scores of IHC intensity ranging from 0 to 3 were determined as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. By multiplying the scores for extent and intensity, a total score (range, 0 to 9) was achieved. PXN expression level was considered high with scores of ≥ 4 and low with scores of < 4.

For overexpression of endogenous PXN, the coding sequence of PXN was amplified and subcloned into the pcDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’ instructions. AGS cells were then transfected with a negative control vector or a PXN expressing plasmid using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). To knockdown endogenous PXN expression in cells, small interfering RNA (siRNA) duplex oligonucleotides targeting human PXN mRNA (si-PXN) was obtained from Ribobio (Guangzhou, China). The targeting sequences were: si-PXN#1: GCAGCAACCTTTCTGAACT; si-PXN#2: GTGTGGAGCCTTCTTTGGT. In the present study, we used si-PXN#1 as it could effectively reduced endogenous PXN expression in our preliminary experiments. The target sequence for scrambled siRNA was 5′-GTCTCCACGCGCAGTACATTT-3′. SGC7901 cells were transfected with si-PXN or scramble siRNA according to the manufacturer’s instructions.

The 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide (MTT) assay was performed to test cell proliferation following a method as previously described [24]. Cells were seeded in a 96-well plate at 1 × 10³ cells/well, the spectrophotometric absorbance was measured for each sample at 490 nm, all the experiments were performed in triplicate and repeated for 3 times, and the average was calculated.

For the colony formation assay, cells (500/well) were seeded in a six-well plate and cultured for 14 days at 37°C with 5% CO₂ humidified air. Colonies were stained with 0.1% crystal violet (1 mg/ml) and the numbers of colonies containing more than 50 cells were counted. The experiment was performed in triplicate and repeated for three times.

The cell migratory capacity was determined using transwell chambers (BD Biosciences) according to a method previously described [25]. Briefly, cells (1 × 10⁵/well) were suspended in 100 μl serum-free medium and then added to the upper chamber of the inserts, RPMI 1640 medium (GIBCO) containing 10% FBS (500 μl) was added to the lower chamber as the chemotactic factor. After culture for 22 hours, non-migrated cells on the upper surface were removed gently with a cotton swab and cells that migrated to the lower side of the department were fixed and dyed with 0.1% crystal violet. The numbers of migrated cells were calculated by counting five different views under the microscopy. The experiment was performed in triplicate and repeated for three times.

All the data were presented as mean ± SD, a Student t-test or Chi-square test was employed to compare the differences as appropriate. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Multivariate analysis with Cox proportional hazards model was used to investigate independent prognostic factors. All P-values were two-sided, and a P-value of < 0.05 was considered statistically significant. Statistical analysis was performed by the SPSS software package (version 16.0, SPSS Inc) or GraphPad prism 5.

The protein and mRNA level of PXN was evaluated in gastric cancer tissues and cell lines. In paired primary gastric cancer tissues and adjacent nontumorous tissues, Western blot analysis revealed overexpression of PXN in cancer tissues compared with adjacent nontumorous tissues (Figure 1A). The level of PXN mRNA was increased in 27 of 30 (P < 0.001) cancer tissues in comparison with that of nontumorous tissues (Figure 1B). Moreover, Western blot analysis showed the expression levels of PXN were higher in gastric cancer cell lines than that of normal gastric epithelial cell line GES-1 (Figure 1C). Consistent with the level of protein, real-time PCR analysis confirmed PXN mRNA level was elevated in all five gastric cancer cell lines (Figure 1D).

We further investigated the expression of PXN in 239 paraffin-embedded gastric cancer tissues by IHC. As shown in Figure 2, PXN protein was mainly located in the cytoplasm of tumor cells. Positive staining was detected in 186 of 239 (77.8%) patients. One hundred and fifty-two patients were possessed with low PXN expression while the other eighty-seven with high PXN expression based on the IHC scores. The associations between PXN expression and clinical and pathologic parameters were summarized in Table 1. High PXN expression was significantly correlated with distant metastasis (P = 0.001), TNM stage (P = 0.021) and survival status (P = 0.004). However, there was no association between PXN expression level and age, gender, tumor depth, differentiation status and treatment. Kaplan-Meier analysis with log-rank test was employed to investigate the prognostic role of PXN for gastric cancer patients. Patients with high PXN expression had significantly worse overall survival than patient with low PXN expression (Figure 3). In addition, Univariate analysis revealed high PXN expression is a risk factor of death in gastric cancer patients (HR = 2.05, 95%CI, 1.47-2.86; P < 0.001, Table 2). Besides, other factors including lymph node invasion, distant metastasis and TNM stage were also correlated with overall survival as demonstrated by univariate analysis (Table 2). However, age, gender, differentiation status, treatment and tumor depth were not prognostic indicators in this study. Multivariate analysis indicated only distant metastasis and PXN expression were independent prognostic factors for gastric cancer patients (P < 0.001 and P = 0.020, respectively, Table 2).

We further investigated the role of PXN on the tumor cell growth and proliferation through gain-of-function and loss-of-function analysis. AGS cells were transfected with pcDNA3.1(+)-PXN to overexpress PXN (Figure 4A). The effects of PXN on cell growth and proliferation were then evaluated by MTT and colony formation assays. The results showed that ectopic expression of PXN significantly promoted the viability of AGS cells (Figure 4B, P = 0.017). The colony formation assay also indicated the number of colonies was markedly increased in PXN overexpressing cells compared with control cells (Figure 4D and E, P = 0.026). The impact of PXN on cellular proliferation was further confirmed by performing MTT and colony formation assays after PXN knockdown in SGC7901 cells (Figure 4A). As indicated in Figure 4C, F and G, compared with that of control cells, knockdown of PXN significantly inhibited the growth rate and colony formation ability of SGC7901 cells (P = 0.019 and P = 0.011, respectively).

As PXN expression is associated with distant metastasis in gastric cancer patients, we then evaluated the potential role of PXN on cellular migration by transwell assays. AGS cells were transfected with PXN overexpressing or control plasmid and seeded in the chamber, and their migratory abilities were determined 24 hours later. The results showed ectopic expression of PXN significantly increased the migratory capacity of AGS cells (Figure 5A and B, P = 0.013). On the contrary, knockdown of PXN dramatically reduced the migrated cell number of SGC7901 (Figure 5C and D, P = 0.032).