Expression of Syk in non-small cell lung cancer and its relationship with clinicopathological parameters

Abstract

This study aims to research the expression of spleen tyrosine kinase (Syk) in non-small cell lung cancer (NSCLC) and the relationship between Syk and clinicopathologic factors and p53. Immunohistochemistry was applied to detect the expression of Syk and p53 protein in 39 cases of NSCLC (23 cases of lung squamous cell cancer, 16 cases of lung adenocarcinoma) and tumor-surrounding normal lung tissues. The positive rate of Syk was 46.15% (18/39) and 100% (39/39) in NSCLC and tumor-surrounding normal lung tissues, respectively. The expression level of Syk in NSCLC was significantly lower than that in tumor-surrounding normal lung tissues (P = 0.000). The Syk expression was positively correlated withthe p53 expression in NSCLC specimens (P = 0.025). There was no significant association between Syk expression and lymph node metastasis, differentiation degree, tumor size and tumor node metastasis (TNM). The present study demonstrated that Syk was aberrantly expressed in the NSCLC and might have a significant impact on tumor growth and progression.

Keywords

Syk kinase / carcinoma, non-small-cell lung / tumor suppressor protein p53

Introduction

The development and progression of lung cancer are thought to occur through a complex, multistep process, including oncogene activation and mutation or loss of tumor suppressor genes. The genetic alterations in lung cancer tumorigenesis and metastasis have been the focus of intensive research for several decades. Tyrosine kinases play an important role in the signaling transduction pathway in lung cancer. Spleen tyrosine kinase (Syk) is a non-receptor type of protein-tyrosine kinase. Recent research shows the loss of Syk is correlated with the pathogenesis of neoplasm and Syk may suppress the growth and metastasis of neoplasms. Studies show the loss of Syk is correlated with the development and metastasis of breast cancer [1,2], gastric carcinoma [3-5] and pancreatic cancer [6], but there is no research about Syk expression in lung cancer. In the present study, we examined Syk, p53 protein expression in human non-small cell lung cancer (NSCLC) and the adjacent non-cancerous tissues, and the relationship between Syk protein expression in the NSCLC tissue and clinicopathologic factors.

Materials and methods

Materials

General information

Thirty-nine patients with NSCLC who underwent surgery with curative intent at Tongji Hospital were enrolled from March to August in 2007. Specimens of lung cancer tissue and tumor-surrounding normal lung tissue obtained at surgery were immediately snap frozen in liquid nitrogen and stored until use. The patients consisted of 26 males and 13 females, and their ages ranged from 43 to 74 years (mean, 55.85 ± 8.37 years). The pathological types were 23 cases of lung squamous cell cancer, 16 cases of lung adenocarcinoma (well-moderate differentiated in 26, poor-undifferentiated in 13). The pathological stages were classified as stage I in 18 patients, II in 3, III in 14 and IV in 4 according to the P-tumor node metastasis (TNM) classification of International Union Against Cancer (1997). Nineteen patients had no lymph node metastasis, whereas the remaindering 20 patients had regional or mediastinal lymph node metastasis. None of the patients received radiotherapy or chemotherapy before surgery.

Main reagents

Mouse anti-human monoclonal Syk and p53 antibody were respectively purchased from the NeoMarkers Company (USA) and Beijing Zhongshan Company (China). The SP kit and 3,3'-diaminobenzidine (DAB) coloring reagent kit were both purchased from the Wuhan Feiyi Company (China).

Methods

Immunohistochemistry

After surgical resection, the tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks. Four-μm-thick sections were prepared for immunohistochemistry. Immunohistochemical staining was performed using the S-P method. Sections were routinely deparaffinized and hydrated. Endogenous peroxidase activity was removed by incubation in methanol containing 0.3% H₂O₂ for 30 min. All sections were autoclaved in 10 mmol/L sodium citrate for 20 min and allowed to cool at room temperature for 20 min. Nonspecific immunoglobulin binding was blocked by incubating the sections in PBS containing 5% normal goat serum at room temperature for 60 min. Primary antibody was applied (anti-Syk at 1∶300 dilution, anti-p53 at 1∶50 dilution) and incubated overnight at 4°C. Following extensive washing, sections were incubated with secondary antibody and third antibody in sequence. Coloration was done using the DAB method followed by hematoxylin after stain, xylene clearing and neutral gummi mounting. PBS substituting for the primary antibody was used as the negative control.

Result evaluation

Positively Syk stained cells had shown brown granules in the cytoplasm and positively p53 stained cells had shown brown granules in the nuclear. If blank and positive controls were both satisfactory, more than 10% of the tumor cells stained were defined as (+); 30%-50% as (++), more than 50% as (+++). Tumor cells stained less than 10% were defined as (-).

Statistical analysis

SPSS statistical package and Fisher’s exact test were used to analyze the data. There was a statistically significant difference if the P value was less than 0.05.

Results

Expression of Syk

The Syk was positively expressed in all tumor-surrounding normal lung tissues with the positive rate being 100%. Among the 39 NSCLC specimens, 18 specimens (46.15%) showed cytoplasmic Syk expression (Fig. 1). The positive rate of Syk expression in the NSCLC specimens was decreased as compared with that in tumor-surrounding normal lung tissues (P = 0.000).

Syk expression and clinicopathologic findings

There was no significant association between Syk and lymph node metastasis, differential degree, tumor size and TNM (Table 1).

P53 expression

There was no p53 protein expression in all tumor-surrounding normal lung tissues. Among the 39 NSCLC specimens, 16 specimens (41.03%) showed nuclear p53 expression (Fig. 2). The positive rate of p53 expression in the NSCLC specimens was increased as compared with that in tumor-surrounding normal lung tissues (P = 0.000).

Syk expression and p53 expression

The Syk expression was positively correlated with p53 expression in NSCLC specimens (P = 0.025) (Table 2).

Discussion

Syk, a non-receptor type of protein-tyrosine kinase that is widely expressed in hematopoietic cells, is one of the two members of the Syk family (Syk and ZAP-70). The Syk protein contains a C-terminal kinase domain and two tandem N-terminal Src homology 2 (SH2) domains that bind phosphorylated immunoreceptor tyrosine-based activating motif (ITAM). Syk is involved in coupling activated immunoreceptors downstream signaling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis [7]. Syk plays an essential role in the development of lymphocytes and activation of immune cells. Recent findings reveal that expression of Syk appears to be involved in a wide variety of cellular functions and the pathogenesis of malignant cancer.

In this paper, we first explored the expression of Syk protein in NSCLC, adjacent non-cancerous tissues by immunohistochemistry. Our results show that the positive rate of Syk protein expression in NSCLC was significantly lower than that of adjacent non-cancerous tissues, which indicates that loss of Syk protein expression may be correlated to lung tumorigenesis. A study by Sada [7] et al suggests that Syk is a potent tumor suppressor and the absence of the gene expression would induce a serious disorder of the development and maturity of immune cells, which may cause severe combined immunodeficiency (SCID). Because of the disorder of the maturity of immune cells, the level of immunodetection is decreased and immunity to aim directly at mutational and paraplastic cells misses, which finally results in tumorigenesis.

Our results also show that there was no significant association between Syk and lymph node metastasis, differential degree, tumor size and TNM. Some researchers reported the loss of Syk was correlated with lymph node metastasis of breast cancer [1, 2], gastric carcinoma [3-5] and pancreatic cancer [6]. In the present study, lymph node metastasis was found in 14 out of 21 cases of Syk negative expression NSCLC specimens. Otherwise, only 6 out of 18 cases of Syk positive expression NSCLC specimen had lymph node metastasis. The rate of lymph node metastasis in Syk negative expression NSCLC specimens was higher than that of Syk positive expression NSCLC specimen, but there was no significant difference (P = 0.056).

The p53 protein expression was also detected in the NSCLC specimens and tumor-surrounding normal lung tissues. Among the 39 NSCLC specimens, 31 specimens (69.23%) show nuclear p53 expression. The expression of p53 in the NSCLC specimens was increased as compared with that in tumor-surrounding normal lung tissues. Our results were in agreement with those reported by Fujino et al [8].

Syk positive expression was detected in 11 out of 16 cases of p53 positive expression NSCLC specimens, otherwise only 7 out of 23 cases of p53 negative expression NSCLC specimens had Syk expression (P = 0.025). The Syk expression was positively correlated to the p53 expression in NSCLC specimens. Okamura [9] et al reported that Syk gene expression was repressed in a p53-dependent manner in human colon carcinoma cells, suggesting that the loss of p53 function during tumorigenesis can lead to deregulated Syk activity. Meanwhile, the findings by Ding [10] et al supported a correlation between the expression of Syk mRNA and p53 protein expression in breast cancer. Our findings were in agreement with the research above, which suggests that the expression of the Syk gene may play an important role in suppressing growth and metastasis of tumor.

Conclusion

In this study, the positive rate of Syk expression in the NSCLC specimens was decreased statistically as compared with that in tumor-surrounding normal lung tissues, which means that the loss of Syk protein expression may be associated with the development and progression of NSCLC. In conclusion, further studies of the Syk gene perhaps may provide a novel molecular target to prevent and treat NSCLC.

References

[1] DingY B, WuZ Y, WangS, FanP, ZhaX M, ZhengW, LiuX A. Expression of tyrosine kinase Syk in breast cancer and their clinical significance. Nanjing Yike Daxue Xuebao, 2004,18(3): 128-132 (in Chinese)

[2] ZhangK L, WangS F, WenM, ZhengB M, DiW, LiY Y. The expression and significance of Syk and C-erB-2, PCNA in patients with breast cancer. Zhongguo Aizheng Zazhi, 2006, 15(2): 131-133 (in Chinese)

[3] NakashimaH, NatusugoeS, IshigamiS, OkumuraH, MatsumotoM, HokitaS, AikouT. Clinical significance of nuclear expression of spleen tyrosine kinase (Syk) in gastric cancer. Cancer Lett, 2006, 236(1): 89-94

[4] QinB S, LiG C. Significance of expression of spleen tyrosine kinase (Syk) in gastric carcinoma. Zhongguo Wuzhenxue Zazhi, 2006, 6: 411-412 (in Chinese)

[5] XiaJ G, DingY B, ChenG Y. Expression of tyrosine kinase Syk and its clinical significance in gastric carcinoma. Shijie Huaren Xiaohua Zazhi, 2004, 12(4): 767-769 (in Chinese)

[6] ChenM X, DingY B, JiangK R, MaoY. Expression of tyrosine kinase Syk and its clinical significance in pancreatic cancer. Nanjing Daxue Xuebao, 2003, 23(5): 492-493 (in Chinese)

[7] SadaK, TakanoT, YanajiS, YamamuraH. Structure and function of Syk protein-tyrosine kinase. J Biochem, 2001, 130(2): 177-186

[8] FujinoM, Dosaka-AkitaH, HaradaM, HiroumiH, KinoshitaI, AkieK, KawakamiY. Prognostic significance of p53 and ras p21 expression in non-small cell lung cancer. Cancer, 1995,76(12): 2457-2463

[9] OkamuraS, NgC C, KoyamaK, TakeiY, ArakawaH, MondenM, NakamuraY. Identification of seven genes regulated by wile-type p53 in a colon cancer cell line carrying a well-controlled wile-type p53 expression system. Oncol Res, 1999,11(6): 281-285

[10] DingY B, WuZ Y, WangS, FanP, ZhaX M, ZhengW, LiuX A. Expression of tyrosine kinase Syk in breast cancer and their clinical significance. Zhonghua Wai Ke Za Zhi, 2004, 42(3): 137-139 (in Chinese)

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg