Overexpression of SCC-S2 correlates with lymph node metastasis and poor prognosis in patients with non-small-cell lung cancer

Abstract

The objective of the current study was to investigate the expression pattern and clinicopathological significance of SCC-S2 in patients with non-small-cell lung cancer (NSCLC). The expression profile of SCC-S2 in NSCLC tissues and adjacent noncancerous lung tissues was detected by real-time RT-PCR, western blot analysis, and immunohistochemistry. In 25 lung cancer tissues examined, 18 (72%) of them exhibited stronger levels of SCC-S2 mRNA compared with their corresponding normal tissues. SCC-S2 protein level was up-regulated in cancerous lung tissues compared to adjacent normal tissue. Moreover, the expression level of SCC-S2 in 93 archived NSCLC tissues was measured by immunohistochemical staining. SCC-S2 was found to be overexpressed in 71 of 93 (76.3%) human lung cancer samples and correlated with lymph node metastasis (P = 0.0181), p-TNM stage (P = 0.0042), Ki-67 expression (P = 0.0028), and poor survival (P = 0.012). In addition, depleting SCC-S2 expression by small-interfering RNA inhibited growth and invasion in lung cell lines. These results indicate that SCC-S2 plays an important role in NSCLC and might be a useful therapeutic target of NSCLC.

Figure 1

The expression of SCC‐S2 in primary non‐small‐cell lung cancer tissues (T) and in adjacent noncancerous lung tissues (N) from the same patient. (a) Real‐time PCR analyses of SCC‐S2 mRNA in primary lung cancers and their adjacent normal tissues. Squamous cell carcinoma included cases 2, 3, 5, 7, 8, 9, 11, 13, 14, 15, 19, 22. Adenocarcinoma included cases 1, 4, 6, 10, 12, 16, 17, 18, 20, 21, 23, 24, 25. (b) Expression of SCC‐S2 protein in each of the primary lung tumors (T) and normal lung tissues (N) paired from the same patient by western blotting. (c) SCC‐S2 expression levels were up‐regulated in primary lung cancers (T) in comparison to the paired normal lung tissue (N) from the same patient as examined by immunohistochemistry. SCC‐S2 immunostaining was negative or was detected weakly in adjacent normal bronchial epithelia.

Figure 2

Immunohistochemical staining of SCC‐S2 in lung cancer tissue sections. (a) Negative staining in lung squamous cell carcinoma (−). (b) Positive immunostaining in lung adenocarcinoma (+), SCC‐S2 was localized mainly in the cytoplasm of cancer cells. (c) Strong SCC‐S2 staining in adenocarcinoma (++). (d) Weak SCC‐S2 immunostaining in lung squamous cell carcinoma. (e) SCC‐S2 was negative or was detected weakly in adjacent normal bronchial epithelia. (f) Negative controls were prepared by non‐immune rabbit IgG at the same dilution as for the primary antibody in tumor sample.

Figure 3

SCC‐S2 expression in non‐small‐cell lung cancer (NSCLC) tissues was associated positively with the expression of Ki‐67. Immunohistochemical staining for SCC‐S2 and Ki‐67 in two representative NSCLCs. One showed both strong expressions of SCC‐S2 (a) and Ki‐67 (b). The other showed both weak expressions of SCC‐S2 (c) and Ki‐67 (d).

Figure 4

Overall survival of non‐small‐cell lung cancer (NSCLC) patients in relation to SCC‐S2 status. The overall survival of patients who had NSCLC with low SCC‐S2 expression versus high SCC‐S2 expression.

Figure 5

SCC‐S2 depletion in A549 and H1299 cell lines. (a) Western blot analyses of SCC‐S2 expression in lung cancer cells transfected with the siRNA. (b) Real‐time PCR analyses of SCC‐S2 depletion efficiency in cancer cells. Two cases of adjacent normal lung tissue were included. The level of SCC‐S2 mRNA in siRNA treated cells was comparable with normal lung tissues. Columns, mean; bars, SD.

Figure 6

SCC‐S2 depletion impaired cancer cell proliferation. (a) MTT assay was performed after SCC‐S2 siRNA treatment. A reduction of absorbance was observed (P < 0.05). (b) Assessment of clonogenic potentials of the SCC‐S2‐depleted cancer cells. Numbers of colonies were counted. The number of colonies formed by cells treated with SCC‐S2 siRNA was far less than that of control cells (P < 0.05). Columns, mean; bars, SD.

Figure 7

Invasion assays of A549 and H1299 cells transfected with control and SCC‐S2 ‐specific siRNA. SCC‐S2 siRNA treatment had a measurable blocking effect on cell invasion in both cell lines. Numbers of cells invading onto the lower surface of the filter were counted, a significant difference was observed (P < 0.05). Columns, mean; bars, SD.

Figure 8

Expression of vascular endothelial growth factor receptor 2 (VEGFR‐2), matrix metalloproteinase 1 (MMP‐1), and MMP‐9 in SCC‐S2 ‐depleted H1299 and A549 cells. (a) Real‐time PCR and (b) western blot analysis showed the expression changes after silencing SCC‐S2 in H1299 and A549 cells. The expression of VEGFR‐2, MMP‐1, and MMP‐9 was explored. Columns, mean; bars, SD.