In this study, we found that increased BRF2 protein expression was prevalent in NSCLC. Overexpression of BRF2 correlated with abnormal expression of E-cadherin, N-cadherin, and snail. Additionally, expression of BRF2 was found to be an independent prognostic factor in NSCLC patients. Furthermore, we showed that targeted knockdown of BRF2 expression could inhibit the migratory and invasive abilities of NSCLC cells and induced loss of the epithelial-mesenchymal transition of NSCLC cells. These results suggested that BRF2 overexpression in tumor tissues is significantly associated with the poor prognosis of NSCLC patients through promoting epithelial-mesenchymal transition (EMT) program.
Lung cancer has remained the leading cause of cancer-related death worldwide for several years. Every year, there are 1.35 million new lung cancer cases in the world [
Epithelial-mesenchymal transition (EMT) initially occurs during normal embryonic development [
RNA polymerase (pol) III is responsible for the transcription of small, less than 300 nucleotides, untranslated RNAs including microRNAs (miRNAs) [
Here, we evaluated the prognostic value of BRF2 expression in patients with resectable NSCLC and found that high expression of BRF2 in NSCLC predicted decreased overall 5-year survival and a higher risk of recurrence. Furthermore, disruption of BRF2 transcripts through small interfering RNA in NSCLC cells results in a reduced capacity of migration and invasion in vitro, inhibiting EMT related invasion in association with increased E-cadherin expression. We suggest that BRF2 may play an important role in the migration and invasion of NSCLC.
A total of 77 consecutive patients who were diagnosed with NSCLC and treated with pulmonary lobectomy plus regional lymph node dissection from January 2005 through December 2006 at the Department of Thoracic Surgery, Qilu Hospital, were studied retrospectively. And they all had the clear pathological diagnosis without preoperative radiotherapy and chemotherapy. 37 cases of adjacent tissue samples were taken from about 0.5 cm away from the outer edge of the lung tumor tissues, and the other 43 cases were taken more than 5 cm from the tumor margin of normal lung tissues as a negative control. For RT-PCR and western blotting analysis, 14 matched pairs of tumors tissue and adjacent noncancerous tissue samples were obtained from pulmonary lobectomy specimens of patients diagnosed with NSCLC immediately after surgery between October 2010 and September 2011 in our department and stored at −80°C.
The clinical characteristics of the patients are summarized in Table
The correlation of clinicopathologic variables of NSCLC with BRF2 expression.
BRF2 (overexpression) | ||||
---|---|---|---|---|
Variable | No. of patients | No | Yes |
|
Age | 0.424 | |||
|
38 | 17 | 21 | |
|
39 | 21 | 18 | |
Gender | ||||
Male | 46 | 26 | 20 | 0.125 |
Female | 31 | 12 | 19 | |
Smoking | ||||
No or little | 60 | 30 | 30 | 0.830 |
more | 17 | 8 | 9 | |
Histology | 0.427 | |||
Adeno | 37 | 20 | 17 | |
Squamous | 40 | 18 | 22 | |
Differentiation | 0.210 | |||
Well | 35 | 14 | 11 | |
Moderate | 18 | 11 | 7 | |
Poor | 34 | 13 | 21 | |
T classification | 0.437 | |||
T1 | 17 | 9 | 8 | |
T2 | 26 | 12 | 14 | |
T3 | 24 | 14 | 10 | |
T4 | 10 | 3 | 7 | |
N classification | 0.087 | |||
N0 | 33 | 20 | 13 | |
N1 | 44 | 18 | 26 | |
Clinical stage | 0.839 | |||
I | 20 | 11 | 9 | |
II | 32 | 15 | 17 | |
III | 25 | 12 | 13 |
The human lung cancer cell lines A549 and SK-MES-1 were routinely obtained from the Shanghai Institute of Biochemistry and Cell Biology of China. The adenocarcinoma cell line A549 was cultured in Roswell Park Memorial Institute (RPMI) 1640 (Sigma, St Louis, USA) containing 10% FBS and the SK-MES-1 squamous cell carcinoma cell line was normally maintained in MEM supplemented with 20% fetal bovine serum. The cell lines were all cultured in 1% penicillin-streptomycin at 37°C in a 5% CO2 humidified cell culture incubator.
All specimens were collected during the surgery, fixed by 10% formalin, and embedded in paraffin. The tissues were cut as 4
All sections were reviewed independently by three independent observers blinded to all clinical and pathologic information. Discordant cases were resolved by choosing the value that is consistent between two observers or the average of the scores. A reproducible semiquantitative method that considered both staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the percentage of positively stained cells (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, >76%) was adopted [
The cutoff value for high and low expression was determined based on a heterogeneity value measured through log-rank statistical analysis with respect to overall survival [
Cells were transfected with BRF2 siRNA (Qiagen, Valencia, CA) or control siRNA by HiPerFect transfection reagent (Qiagen). The siRNA transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The following siRNA sequences were used in this study: BRF2 siRNA1: sense 5-GCACUUACAUGCAGAUAGUTT-3; antisense 5-ACUAUCUGCAUGUAAGUGCTT-3; BRF2 siRNA2: sense 5-GGUGGGAAAUAAUUCCUUATT-3; antisense 5-UAAGGAAUUAUUUCCCACCTT-3; BRF2 siRNA3: sense 5-GCCACCAACAUUUGAGGAUTT-3; antisense 5-AUCCUCAAAUGUUGGUGGCTT-3; negative control: sense 5-UUCUCCGAACGUGUCACGUTT-3; antisense 5-ACGUGACACGUUCGGAGAATT-3.
RNA and protein were obtained 48 h after transfection for qRT-PCR or western blot analysis. The BRF2 siRNA that could most effectively deplete BRF2 was used in the following experiments.
Surgical specimens were processed immediately after operation. Total RNAs were extracted from tissues by using Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol and treated with RQ1 RNase-free DNase (Promega); cDNA was synthesized. The expressions of BRF2 and HIF-1
The fresh tissues were washed three times with ice-cold phosphate-buffered saline (PBS) and lysed on ice in RIPA (radio immunoprecipitation assay) buffer (Cell Signaling Technology, Danvers, MA, USA) containing complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Protein from tissues or cells was separated via SDS-PAGE and transferred to a PVDF membrane (GE healthcare, USA). Membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1.5 h at room temperature; the membranes were then incubated overnight at 4°C with anti-BRF2 (1 : 1000, Abcam, Cambridge, MA, USA), anti-E-Cadherin (1 : 1000, Cell Signaling, Danvers, MA, USA), anti-N-Cadherin (1 : 1000, Cell Signaling, Danvers, MA, USA), anti-snail (1 : 1000, Cell Signaling, Danvers, MA, USA), or anti-GADPH (1 : 1000, Abcam, Cambridge, MA, USA) antibodies. Followed by anti-rabbit horseradish peroxidase conjugated IgG, an ECL kit (GE healthcare, USA) was used for detection.
The wound healing assay was performed as described previously [
The invasion assay was performed using transwell chambers (Corning, New York, USA) with 50
Chi-square test was used to test the associations between BRF2 expression and clinicopathological factors. The correlation between BRF2 protein immunoreactivity and E-Cadherin, N-Cadherin, or snail was analyzed by nonparametric test (Mann-Whitney
We detected the expression of BRF2 protein in the normal lung tissues, adjacent nontumor tissues, and tumor tissues by immunohistochemistry. As shown in Figure
The expression of BRF2 in lung cancer, adjacent lung cancer tissues and normal tissues.
Variable |
|
Positive ( |
Positive rate (%) |
|
|
---|---|---|---|---|---|
Cancer tissue | 77 | 39 | 50.65 | 6.155 |
|
Adjacent tissue | 37 | 12 | 32.43 | ||
Normal tissue | 43 | 13 | 30.23 |
The expression pattern of BRF2 in NSCLC tissues. (a) Immunohistochemical analysis of BRF2, E-cadherin, N-cadherin, and snail in nonsmall cell lung carcinoma (×400). (b) Quantitative real-time PCR analyses of BRF2 mRNA in eight pairs of matched NSCLC and noncancerous tissues with GAPDH as a loading control in both panels. (c) Protein levels of BRF2 expression were evaluated by western blotting from paired noncancerous tissue and NSCLC (T: tumor tissue; N: normal tissue).
To investigate the status of BRF2 gene expression in NSCLC, we used real-time PCR to measure the mRNA expression in 14 pairs of primary cancer tumors and adjacent noncancerous specimens. Compared with their adjacent noncancerous specimens, 9 of 14 NSCLC had upregulated expression (Figure
Of the 77 patients, 49 (63.63%) cases died within 5 years after operation, and tumor relapse developed during follow-up in 57 (74.03%) patients. Kaplan-Meier analyses compared by the log-rank test were used to calculate the effect of the clinicopathologic factors with lung cancer on overall survival and disease-free survival. Univariate analysis demonstrated that BRF2 protein overexpression (
Univariate and multivariate analyses of prognostic variables.
Variable |
|
95.0% confidence interval | Exp ( |
|
---|---|---|---|---|
PFS Univariate analysis | PFS Multivariate analysis | |||
Gende | 0.923 | 0.855–3.389 | 1.702 | 0.130 |
Age | 0.353 | 0.423–1.748 | 0.860 | 0.677 |
Smoking | 0.243 | 0.465–2.472 | 1.072 | 0.871 |
Histology | 0.792 | 0.346–1.229 | 0.652 | 0.186 |
T status |
|
0.283–1.089 | 0.555 | 0.087 |
Clinical stage |
|
2.177–19.997 | 6.597 |
|
Differentiation | 0.139 | 0.521–1.129 | 0.767 | 0.178 |
BRF2 protein |
|
0.121–0.561 | 0.260 |
|
E-cadherin |
|
1.192–5.534 | 2.568 |
|
N-cadherin | 0.919 | 0.905–3.327 | 1.735 | 0.097 |
Snail | 0.295 | 0.548–2.124 | 1.079 | 0.826 |
|
||||
OS Univariate analysis | OS Multivariate analysis | |||
Gender | 0.780 | 0.691–3.018 | 1.444 | 0.328 |
Age | 0.296 | 0.507–2.334 | 1.088 | 0.829 |
Smoking | 0.370 | 0.369–2.146 | 0.890 | 0.795 |
Histology | 0.878 | 0.500–1.902 | 0.975 | 0.941 |
T status | 0.082 | 0.327–1.492 | 0.698 | 0.354 |
Clinical stage |
|
1.267–14.989 | 4.357 |
|
Differentiation | 0.057 | 0.410–0.992 | 0.638 |
|
BRF2 protein |
|
0.168–0.888 | 0.387 |
|
E-cadherin |
|
1.215–7.613 | 3.042 |
|
N-cadherin | 0.381 | 0.618–2.525 | 1.250 | 0.535 |
Snail | 0.221 | 0.575–2.579 | 0.698 | 0.354 |
Kaplan-Meier curves of disease-free and overall survival according to the status of BRF2 protein expression (a, c). Mann-Whitney
We further analyzed the prognostic significance of BRF2 protein in selective patient subgroups stratified according to the clinicopathologic factors of ESCC. Univariate analysis demonstrated that the overall 5-year survival rate of patients with BRF2 protein high expression was significantly lower than that of the remaining patients among N1, T1-2, and adenocarcinoma (
Kaplan-Meier survival curves of patients stratified according to lymph node metastasis (a), tumor size (b), and pathological type (c).
Typical immunohistochemical staining patterns observed for the EMT marker proteins in NSCLC are shown in Figure
Epithelial-mesenchymal transition factors score grade in relation to BRF2 protein immunoreactivity. Mann-Whitney
To find out whether BRF2 mediates prognosis in lung cancer through promoting metastasis, we applied small interfering RNA technology to knock down the BRF2 expression on the migratory and invasive ability of A549 and SK-MES-1 cells (Figure
Targeted knockdown of BRF2 expression inhibited the metastatic potentials of NSCLC A549 cells. (a): (A): the levels of BRF2 protein in the A549 cells separately treated with different siRNAs; (B): the detection of the knockdown of BRF2 in A549 cells by WB and RT-PCR, respectively (
Enhanced cell migration and invasion properties are important consequences of EMT [
Targeted knockdown of BRF2 expression resulted in a gain of E-cadherin and a loss of N-cadherin and snail in A549 cells and SK-MES-1 cells. The expression of E-cadherin, N-cadherin, and snail was detected by western blot analysis. GADPH was used as loading control.
The ability to proliferate uncontrollably is the dominant characteristic of many types of cancer cells. And it has been demonstrated that overexpression of
In this study, our results showed that there was no significant correlation between BRF2 expression and the clinicopathological features of NSCLC in the statistical analysis. Notably, our survival analysis demonstrated that BRF2 protein overexpression significantly predicted decreased overall 5-year survival and higher recurrence rate. Moreover, further analysis using the Cox regression model confirmed that BRF2 expression was an independent factor in predicting progression-free survival for NSCLC patients, suggesting that BRF2 protein may be potential prognostic factors for the relapse of NSCLC patients. So we conclude that BRF2 possesses a certain effect in invasion and metastasis of lung cancer and is an independent prognostic factor of recurrence and metastasis in lung cancer. Considering the oncogenic role of BRF2 in the tumor cell, Lockwood et al. identified BRF2 as a novel lineage-specific oncogene in lung squamous cell carcinoma. In our study, however, we found that BRF2 plays critical role in prognosis in 37 patients with AC; however, the log-rank test showed a trend towards better overall survival in the high BRF2 group in 40 patients with SCC but this did not reach statistical significance (
EMT is a key process in tumor metastasis [
Snail is one of the transcriptional regulators of this E-cadherin to N-cadherin switch [
In conclusion, this study demonstrates that the overexpression of BRF2 in NSCLC is a strong indicator of more aggressive tumors and poor clinical outcome. Moreover, overexpression of BRF2 could increase the migratory and invasive abilities of NSCLC cells. These results suggest that the BRF2 expression is critical for the invasiveness of malignant NSCLC cells, possibly through EMT involving upregulation of snail and consequent aberrant expression of E-cadherin and N-cadherin. Thus, it may be a candidate biomarker for NSCLC prognosis and a target for new therapies.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Project supported by the National Natural Science Foundation of China (no. 30571844), the Science and Technology Development Foundation of Shandong Province (no. 2009GG10002007), the National Natural Science Foundation of Shandong Province (no. ZR2013HM089), and the Wu Jie Ping Foundation (no. 320.6750.12393). Sponsors had no involvement in the study design, collection, analysis, and interpretation of data, writing of the paper, and decision to submit the paper for publication.