Introduction

Lung cancer is the leading cause of death among malignant tumors worldwide [1–3]. Although targeted therapies have been established, mutations such as EGFR and KRAS causing activation of these gene products are identified only in a limited number of patients. Many complex genetic, epigenetic, and microenvironmental factors play important roles in growth and invasion of tumor cells [4–7]. Thus, identification of these oncogenes/tumor suppressors and elucidation of their mechanism is an important mission for development of new treatment.

RASSF4 is a RASSF family protein. To date, RASSF family proteins such as RASSF1 and RASSF2 have been shown to directly interact with Ras proteins with the characteristics of effectors [8–11]. These proteins can induce cell death in a Ras-dependent manner. Moreover, these proteins are all frequently downregulated during tumor development by promoter methylation [12]. Several studies have shown that RASSF4 is downregulated in human cancers, suggesting involvement of RASSF4 in carcinogenesis [13]. However, there was no report concerning protein expression pattern and clinical significance of RASSF4 in human nonsmall cell lung cancer. Its biological roles in lung cancer cells also remain unexplored.

Here, we investigated RASSF4 protein expression in NSCLCs and analyzed its correlation with clinicopathological factors. We also examined the function of RASSF4 as a tumor suppressor in lung cancer cell lines and provide evidence that RASSF4 inhibits lung cancer cell proliferation and invasion.

Materials and methods

Tissue samples

The study was approved by the ethical committee of China Medical University. A total of 89 cases of NSCLC were retrieved from the Pathology Archive of the First Affiliated Hospital of China Medical University from 2007 to 2010. Clinicopathological information about the patients was obtained from patient records. All of the enrolled patients underwent curative surgical resection without having prior chemotherapy or radiation therapy.

Immunohistochemistry

Tumor specimens were fixed with neutral formalin, embedded in paraffin, and 4-μm thick sections were prepared. The sections were deparaffinized in xylene, rehydrated in graded alcohol series. Antigen retrieval was performed using 0.01-M citrate buffer for 2-min boil. Hydrogen peroxide was applied to block peroxidase, and then the slides were incubated with normal goat serum. The primary antibody for RASSF4 (1:400, Proteintech, USA) was incubated overnight at 4 °C. Sections were stained in parallel with nonimmune IgG to provide a negative control. The expression of RASSF4 was then detected by the EliVision plus kit (Maixin, Fuzhou, China). DAB visualization was then performed, and the slides were counterstained with hematoxylin.

Two independent investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at ×400 magnification. Immunostaining of RASSF4 was scored following a semiquantitative scale by evaluating in representative tumor areas, the intensity, and percentage of cells. Cytoplasmic staining was considered as positive. The intensity of RASSF4 staining score was indicated as 0 (none), 1 (weak), and 2 (strong). Percentage scores were indicated as 1 (1–25 %), 2 (26–50 %), 3 (51–75 %), and 4 (76–100 %). The scores of each tumor sample were multiplied to give a final score of 0 to 8, and the total expression of RASSF4 was determined as low expression with score ≤4 and high expression (+) with score >4.

Cell culture and transfection

Human NSCLC cell lines NHBE, H460, A549, H1299, H157, H3255, and small cell lung cancer (SCLC) cell line H446 were purchased from ATCC. The cells were cultured using RPMI-1640 (Gibco, Invitrogen, NY, USA) with 10 % fetal bovine serum in an incubator at 37 °C with 5 % CO2. For transfections, pCMV6-RASSF4 plasmid and the control empty vector pCMV6 were purchased from Origene (Origene, Rockville, MD, USA). Cells were transfected using Attractene reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RASSF4 small interfering RNA (siRNA) and negative control siRNA were purchased from Dharmacon (GE healthcare, USA). siRNA transfection was performed using Dharmafect1 reagent (GE healthcare, USA).

Western blotting

Total proteins from cells were extracted in lysis buffer and quantified using the Bradford method. Thirty-microgram protein was separated by SDS-PAGE. Samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4 °C with antibody against RASSF4 (1:800, Sigma, USA), cyclin D1, MMP2, MMP9 (1:1000, Cell Signaling, USA), and GAPDH (1:500, Santa Cruz, USA). After incubation with HRP-coupled anti-mouse or rabbit IgG antibody (1:1000 dilution, Cell Signaling Technology, USA) at 37 °C for 2 h, target proteins on PVDF membrane were visualized using Pierce ECL kit and captured using a DNR BioImaging System (DNR, Jerusalem, Israel).

Colony formation and MTT assays

Colony formation assay

Forty-eight hours after transfection, cells were plated into 6-cm culture dishes (about 1000 per dish). After 2 weeks, plates were washed with PBS, and Giemsa staining was performed to visualize colony. The colonies with more than 50 cells were counted using a microscope.

MTT assay

Cells were plated in 96-well plates (approximately 3000 cells per well) and cultured for 5 days. Twenty microliters of 5 mg/ml MTT solution was added to the well. After incubation for 4 h, the medium was removed, and remaining MTT formazan was dissolved in 150 μl of DMSO. Solution was measured at 490 nm.

Matrigel invasion assay

Cell invasion assay was performed using a 24-well Transwell chamber. The inserts were coated with 18-μl Matrigel (1:4 dilution, BD Bioscience, USA). After the transfection, cells were trypsinized and re-suspended in 100 μl of serum-free medium, which were transferred to the upper chamber. Total 600-μl medium containing 10 % FBS was added to lower chamber. After 18-h incubation, noninvaded cells on the upper chamber were removed using cotton tips, and the invaded cells were stained using hematoxylin. Invading cell number was counted in five high power fields under the microscope.

Statistical analysis

SPSS version 16 for Windows was used for all statistical analyses. A χ 2 test was used to examine possible correlations between RASSF4 expression and clinicopathologic factors. Student’s t test was used to compare densitometry data on focus numbers between control and SIX1-transfected cells. All p values are based on a two-sided statistical analysis, and p < 0.05 was considered to indicate statistical significance.

Results

Expression pattern and clinical significance of RASSF4 in NSCLC

We investigated RASSF4 expression in 89 NSCLC tissue specimens and 20 normal lung tissue specimnes by immunohistochemistry. In normal lung tissues, positive cytoplasmic staining was observed in normal bronchial epithelial cells and alveolar cells (Fig. 1a). As for lung cancer tissues, 53 cases showed strong cytoplasmic RASSF4 staining and 36 cases (40.5 %) showed downregulated RASSF4 staining compared with normal tissue (Fig. 1c, d). RASSF4 protein was mainly located in the cytoplasmic compartment of tumor cells. We examined the correlation between RASSF4 status and clinical factors. We found that RASSF4 downregulation significantly associated with advanced TNM stage (Table 1, p = 0.0073) and positive nodal status (p = 0.0167). No difference was observed in the RASSF4 status according to the age, gender, differentiation, and tumor size (Table 1).

Fig. 1
figure 1

Expression of RASSF4 in nonsmall cell lung cancers. a Positive cytoplasmic staining of RASSF4 in normal alveolar cells. b Positive staining of RASSF4 in a case of lung adenocarcinoma. c Negative staining of RASSF4 in lung squamous cell carcinoma. d Negative RASSF4 staining in a case of lung adenocarcinoma (magnification ×200). e Survival analyses of patients with RASSF4 expression and those without. The overall survival was significantly lower in patients with RASSF4-negative NSCLCs than in patients with RASSF4-positive NSCLCs

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Table 1 Distribution of RASSF4 status in NSCLC according to clinicopathological characteristics
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Furthermore, Kaplan-Meier survival analysis showed a significantly lower overall survival in patients with negative RASSF4 compared with those with positive expression (p < 0.05, log rank test; Fig. 1e). In addition, univariate analysis showed that TNM stage and RASSF4 status were both significant prognostic factors (TNM stage: hazard ratio 2.138, p < 0.001; RASSF4 status: hazard ratio 0.505, p = 0.0214), and multivariate analysis using a Cox regression model indicated that TNM stage was an independent, unfavorable prognostic factor (Table 2).

Table 2 Univariate and multivariate analysis for predictive factors in patients with NSCLC
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RASSF4 is downregulated in lung cancer cell lines and inhibits cell proliferation and invasion

Relative expression level of RASSF4 was analyzed by western blot in a panel of lung cancer cell lines. In accordance with tissue samples, the RASSF4 protein expression was remarkably decreased in NSCLC cell lines, especially in H460, A549, H1299, and H446, compared with normal HBE cell line (Fig. 2a). A549 and H460 cell lines were selected for RASSF4 transfection. We upregulated RASSF4 expression using RASSF4 plasmid, and transfection efficiency was confirmed by western blot analysis (Fig. 2b). RASSF4 overexpression in A549 and H460 cells greatly inhibited the proliferation rate (day 5, p < 0.05) and the potential of colony formation (p < 0.05) (Fig. 3a, b). To characterize the effect of RASSF4 on cell invasion, matrigel invasion assay was performed in A549 and H460 cells. As shown in Fig. 3c, significant reduced invading ability was observed in cells with RASSF4 transfection compared with empty controls. In addition, we performed siRNA depletion in H157 cell line with relatively high RASSF4 expression. As shown in supplementary Fig. 1, RASSF4 depletion significantly upregulated cancer cell proliferation rate and invading ability.

Fig. 2
figure 2

RASSF4 expression in lung cancer cell lines and its transfection efficiency. a Endogenous expression of RASSF4 was examined in HBE and lung cancer cell lines by western blot. Lung cancer cell lines have significant downregulated RASSF4 expression. b Western blot analysis showed that pCMV6-RASSF4 plasmid markedly increases its levels in H460 and A549 cells compared with control

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Fig. 3
figure 3

RASSF4 restoration inhibits cell proliferation and invasion. a MTT assay in H460 and A549 cells transfected with RASSF4 plasmid. Time-dependent decrease in cell proliferation after RASSF4 transfection compared with control. b Colony formation assay was performed in cells transfected with RASSF4 siRNA and cells transfected with control. A marked decrease in colony formation is seen in the groups with RASSF4 restoration. c Matrigel invasion assay showed that RASSF4 transfection decreased cell invasion in A549 and H460 cell lines. *p < 0.05

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RASSF4 inhibits cell cycle and regulates cyclin D1, and MMP2 and MMP9 expression

The aforementioned results indicate that RASSF4 leads to decreased cellular proliferation and invasion. We further checked the role of RASSF4 on cell cycle progression. As shown in Fig. 4a, RASSF4 overexpression inhibited G1-S transition in H460 and A549 cell lines. To underline the possible mechanisms, we examined a panel of growth and invasion-related proteins. As shown in Fig. 4b, RASSF4 transfection significantly inhibited cell cycle protein cyclin D1 and invasion-related protein MMP2 and MMP9.

Fig. 4
figure 4

RASSF4 transfection inhibits cell cycle progression, with cyclin D1 and MMP9 donwregulation. a Cell cycle analysis showed that RASSF4 transfection decreased cell percentage in S phase and increased the cell percentage in G1 phase. b Western blot analysis showed that RASSF4 restoration could decrease the protein expression of cyclin D1, MMP2, and MMP9. *p < 0.05

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Discussion

Epigenetically inactivation of RASSF4, which is caused by methylation and/or deletions, has been investigated in a number of cancers. Reduced RASSF4 mRNA expression and hypermethylated promoter region of RASSF4 was found in head and neck squamous cell carcinoma and nasopharyngeal carcinoma [14, 15]. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines [16]. However, there was no study concerning protein expression of RASSF4 in lung cancer tissues and its correlation with clinicopathological parameters. In addition, biological roles of RASSF4 in human nonsmall cell lung cancer cells remain elusive. In this study, we demonstrated that RASSF4 protein expression was significantly decreased in 38.6 % NSCLC tissues, which was significantly correlated with lymph node metastasis and TNM stage, suggesting RASSF4 as a putative tumor suppressor in NSCLC. Importantly, we showed that loss of RASSF4 correlated poor patient prognosis. To data, this is the first report concerning protein expression pattern and clinical significance of RASSF4 in human cancers. We also examined RASSF4 expression in several lung cancer cell lines. RASSF4 expression was significantly lower in four of seven cancer cell lines compared with HBE, which was consistent with immunohistochemical findings showing RASSF4 as a tumor suppressor.

Using RASSF4 plasmid, we demonstrated that its restoration in NSCLC cell lines with low endogenous expression significantly inhibited proliferation and cell cycle progression. Previous reports showed evidence supporting RASSF4 as a tumor suppressor. It has been reported that RASSF4 overexpression induced cell death and inhibited survival in MCF-7 cell line [16]. Our result was in accord with these studies, suggesting the role of RASSF4 as a tumor suppressor as in lung cancer cells. To data, the effect of RASSF4 on cell cycle and cell invasion has not been investigated. The mechanism of RASSF4 as a tumor suppressor also has not been elucidated. Thus, we checked cell cycle progression and related proteins and found that RASSF4 overexpression inhibited S phase percentage and cyclin D1 expression. Cyclin D1 play a critical role during cell cycle progression at G1-S checkpoint. Many studies have demonstrated that cyclin D1 was overexpressed in nonsmall cell lung cancer and correlated with malignant progression and poor prognosis [17, 18]. These results suggested that RASSF4 could regulate cell proliferation through modulation of cyclin D1 status. The role of RASSF4 on cell invasion has not been reported previously. In the present study, we found that RASSF4 inhibited invading ability of lung cancer cell lines. RASSF4 restoration decreased MMP2 and MMP9 levels, which are important mediators of invasion in many types of cancers [19–22]. Thus, the role of RASSF4 on invasion inhibition may be due to its role on MMP2 and MMP9 downregulation.

In conclusion, this study demonstrated downregulation of RASSF4 protein expression in NSCLC and its correlation with TNM stage, nodal status, and poor prognosis. Our results demonstrated that RASSF4 restoration in lung cancer cells could inhibit proliferation and invasion, possibly through regulation of cyclin D1 and MMP family proteins. Given these findings, RASSF4 serves as an important tumor suppressor in nonsmall cell lung cancer.