Snail promotes an invasive phenotype in lung carcinoma

Abstract

Background: Snail is a transcriptional factor which is known to influence the epitheliomesenchymal transition (EMT) by regulating adhesion proteins such as E-cadherin and claudins as well as matrix metalloproteases (MMP).

Methods: To evaluate the functional importance of snail, a transciptional factor involved in EMT in lung tumors, we investigated its expression in a large set of lung carcinomas by immunohistochemistry. Expression of snail and effects of snail knockdown was studied in cell lines.

Results: Nuclear snail expression was seen in 21% of cases this being strongest in small cell lung carcinomas (SCLC). There was significantly greater snail expression in SCLC compared to squamous cell or adenocarcinoma. Positive snail expression was associated with poor survival in the whole material and separately in squamous cell and adenocarcinomas. In Cox regression analysis, snail expression showed an independent prognostic value in all of these groups. In several cell lines knockdown of snail reduced invasion in both matrigel assay and in the myoma tissue model for invasion. The influence of snail knockdown on claudin expression was cell type specific. Snail knockdown in these cell lines modified the expression of MMP2 and MMP9 but did not influence the activation of these MMPs to any significant degree.

Conclusions: The results show that snail plays an important role in the invasive characteristics of lung carcinoma influencing the survival of the patients. Snail knockdown might thus be one option for targeted molecular therapy in lung cancer. Snail knockdown influenced the expression of claudins individually in a cell-line dependent manner but did not influence MMP expressions or activations to any significant degree.

Figure 1

Snail and claudin immunoreactivity in different carcinomas.A. in a small cell carcinoma of the lung, intense nuclear snail positivity, B. in a large cell carcinoma of the lung, nuclear positivity for snail, C. in a squamous cell carcinoma, membrane bound positivity for claudin 1 can be detected. The stromal tissue is negative and D. in an adenocarcinoma of the lung, membrane bound positivity for claudin 3 can be seen. Scale bar is 200 μm in the figures. Positive examples are shown in figure by arrows (→).

Figure 2

Kaplan-Meier estimates of survival.A. survival association of snail expression in whole material (p = 0.000), B. in adenocarcinomas (p = 0.004) and C. in squamous cell carcinomas (p = 0.005).

Figure 3

Claudin mRNA expression 1, 2, 3, 4 and 7 in normal cell lines and snail knockdown cell lines.A. The difference between normal and snail knockdown BEAS-2B cell lines was significant for claudin 7 (p = 0.001) and near to significant for claudin 4 (p = 0.052), B. between the SK-LU-1 cell line and snail knockdown cell line, there were statistically significant differences in claudin 2 (p = 0.026) and C. The difference between normal and slug knockdown SK-MES-1 cell lines was significant for the expressions of claudin 1 (p = 0.005), claudin 4 (p = 0.011) and for claudin 7 (p = 0.005). Nearly statistically significant difference was seen for claudin 2 (p = 0.059).

Figure 4

Snail immunoreactivity in carcinoma cell lines.A. SK-LU-1 carcinoma cell line, B. SK-LU-1 snail knockdown cell line, C. SK-MES-1 cell line and D. SK-MES-1 snail knockdown cell line. Scale bars are 200 μm. Snail positivity is showed with arrows (→) in figures A and C. lack of snail expression is pointed with arrows (→) in figures B and D.

Figure 5

Zymograms of the cell lines. MMPs were studied from the medium from A. BEAS-2B cells, B. BEAS-2B snail knockdown cells, C. EDTA treated gelatin gels of BEAS-2B and knockdown cell lines, D. SK-LU-1 cells, E. SK-LU-1 snail knockdown cells, F. EDTA treated gelatin gels of SK-LU-1 and knockdown cell lines G. SK-MES-1 cells, H. SK-MES-1 snail knockdown cells and I. EDTA treated gelatin gels of SK-MES-1 and knockdown cell lines. MMP standards are in right side of the zymogramms. 92 kDa is proMMP9, 82 kDa is active MMP9, 72 kDA is proMMP2 and 62 kDa is active MMP2.

Figure 6

Invasion assay results.A. Gel matrix invasion assays results for BEAS-2B (p = 0.039), SK-LU-1 (p = 0.004) and SK-MES-1 cells and B. Myoma organotypic invasion model results. Invasion depth in normal and snail knockdown cell lines BEAS-2B (p = 0.003), SK-LU-1 and SK-MES-1.