AIM: To study the differential expression of Annexin A1 (ANXA1) protein in human gastric adenocarcinoma. This study was also designed to analyze the relationship between ANXA1 expression and the clinicopathological parameters of gastric carcinoma.

METHODS: Purified gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC) were obtained from 15 patients with gastric cancer by laser capture microdissection. All of the peptide specimens were labeled as ¹⁸O/¹⁶O after trypsin digestion. Differential protein expressions were quantitatively identified between GAC and NGEC by nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry (nano-RPLC-MS/MS). The expressions of ANXA1 in GAC and NGEC were verified by western blot analysis. The tissue microarray containing the expressed ANXA1 in 75 pairs of gastric carcinoma and paracarcinoma specimens was detected by immunohistochemistry (IHC). The relationship between ANXA1 expression and clinicopathological parametes of gastric carcinoma was analyzed.

RESULTS: A total of 78 differential proteins were identified. Western blotting revealed that ANXA1 expression was significantly upregulated in GAC (2.17/1, P < 0.01). IHC results showed the correlations between ANXA1 protein expression and the clinicopathological parameters, including invasive depth (T stage), lymph node metastasis (N stage), distant metastasis (M stage) and tumour-lymph node metastasis stage (P < 0.01). However, the correlations between ANXA1 protein expression and the remaining clinicopathological parameters, including sex, age, histological differentiation and the size of tumour were not found (P > 0.05).

CONCLUSION: The upregulated ANXA1 expression may be associated with carcinogenesis, progression, invasion and metastasis of GAC. This protein could be considered as a biomarker of clinical prognostic prediction and targeted therapy of GAC.

Gastric cancer (GC) is a common digestive tract cancer because of the lack of early diagnosis strategies; a previous study revealed that GC cases are usually diagnosed when the disease is at an advanced stage[1]. On the basis of metastasis, recurrence and other causes, the treatment and prognosis of GC remain poor. Therefore, effective biomarkers of GC should be determined; the mechanism of incidence and development should also be investigated to promote early diagnosis, effective treatment and prognosis improvement of GC.

Annexin A1 (ANXA1) is a key member of the A subfamily and belongs to the multi-gene family of Annexins. ANXA1 exhibits calcium-mediated phospholipid binding properties and participates in many physiological and pathological processes. Further studies have shown that ANXA1 is abnormally expressed in various tumours. This abnormal expression is closely related to tumourigenesis, development, invasion and metastasis of human tumours. The expression of ANXA1 in tumours is tissue specific; for instance, a low ANXA1 expression is observed in oesophageal squamous cell carcinoma[2], whereas a high expression is found in colorectal cancer[3]. The relationship between ANXA1 expression in GC and GC invasion as well as metastasis remains unclear. ANXA1 also exhibits a low expression in GC and negatively correlates with invasion and metastasis[4]. However, other studies have revealed opposite results[5,6].

We performed laser capture microdissection (LCM) to investigate the relation of ANXA1 expression in GC to the clinical parameters and to obtain purified gastric adenocarcinoma cells (GAC) and normal gastric epithelial cells (NGEC). ¹⁸O/¹⁶O was used to label the digested peptides in the mixture of GAC and NGEC. Nanoliter-reverse-phase liquid chromatography-mass/mass spectrometry (nano-RPLC-MS/MS) was performed to identify and quantify the differentially expressed proteins. Nano-RPLC-MS/MS was also conducted to validate the results of proteomics. To verify the differential protein expression of ANXA1, we performed western blot. Immunohistochemistry (IHC) was performed to detect the expression of ANXA1 in 75 pairs of tissue microarray of GC tissues and paracarcinoma tissues. This study aimed to analyze the correlations of ANXA1 with clinical pathological parameters, including age, gender, differentiation degree, metastasis, invasion depth, tumour-lymph node metastasis (TNM) staging and tumour size (maximum diameter). This study also investigated the relations and possible mechanisms of the expression differences of ANXA1 protein in carcinogenesis, progression and prognosis of GC.

The mortality rate of GC ranks second among the mortality rates of malignant tumours and approximately 75 million people die of stomach cancer worldwide yearly[9]. The five-year survival rate of GC is only 20%-30%, and the five-year survival rate of radical resection of early GC can reach 90%-95%[10]. Molecular markers are considered as one of the most sensitive and effective indicators of tumour diagnosis, recurrence, metastasis and prognosis prediction. Therefore, the molecular markers closely associated with the development of GC and the relative pathogenesis should be determined in the early diagnosis of GC to improve treatment outcomes. Proteomics technology has an important function in the identification of tumour-related protein species in cancer development as well as in the discovery of tumour molecular markers and therapeutic targets. The present study employed simple LCM to solve problems about tumour proteomics heterogeneity, in which the purified cells exhibiting > 90% homogeneity were rapidly obtained. Using the advanced ¹⁸O stable isotope labelling/MS quantitative proteomics technique, we set the normal gastric mucosa and GC tissue of humans as the targets in this study to screen and identify the differentially expressed proteins. A total of 78 differentially expressed proteins were found and 42 of such proteins were highly expressed in GC cell compared with NGEC. By contrast, 36 differentially expressed proteins were found at lower concentrations. These results provided information about differentially expressed proteins and the mechanism of GC carcinogenesis, development and screening of molecular markers.

During the screening of the identified differential proteins, ANXA1 was significantly upregulated in GC. ANXA1 is involved in various physiological and pathological processes, such as cell signal transduction, cell proliferation, differentiation and apoptosis as well as inflammation and immune response. Previous studies found that ANXA1 is upregulated in breast cancer[11], lung cancer[12], pancreatic cancer[13], colorectal cancer[3,14] and bladder cancer[15]. By contrast, ANXA1 is downregulated in oral squamous cell carcinoma as well as nasopharyngeal, laryngeal and other head and neck cancer[16-18], oesophageal squamous cell carcinoma[2] and prostate cancer[19]. The dysfunction of ANXA1 is closely related to breast cancer, lung cancer, and pancreatic cancer as well as in other tumour invasion and metastasis. Therefore, ANXA1 is considered as a risk factor affecting the survival of patients. Wang et al[20] used a immunohistochemical method and found that ANXA1 expression is 39% higher in the stomach/gastroesophageal junction adenocarcinoma and closely correlated with the pathological staging and distant metastasis of tumour, i.e., higher clinical stages, particularly in distant lymph node metastasis, correspond to higher ANXA1 expression levels; higher tumour recurrence rates correspond to lower survival rate. This result suggested that the upregulation of ANXA1 could be used as a prognosis indicator in the stomach-oesophageal junction adenocarcinoma. Currently, the expression and function of ANXA1 in GC remain controversial.

In this study, quantitative proteomics was performed to screen ANXA1, revealing that ANXA1 was expressed 2.17 times higher in GC than in normal gastric mucosa. Western blotting and IHC of the tissue microarray also revealed the same results as proteomics analysis. In particular, the results showed that ANXA1 was significantly expressed at a higher extent in GC tissues than in paratissues; further analysis about the relations of ANXA1 and clinical parameters of human GAC revealed that ANXA1 expression was upregulated in tumour-penetrating serosa and thus invaded the paratissues. This result is different from human GAC normally confined to the mucosa, submucosa and muscularis; human GAC also possibly invaded the tumour in the lower serosa. ANXA1 was also highly expressed in human GAC with lymph node metastasis and distant metastasis compared with human GAC without lymph node metastasis and distant metastasis. As TNM staging increased, the positive expression rate of ANXA1 increased. However, no relationship was observed among ANXA1, patient’s age, gender, histological grade and tumour size. This result suggested that ANXA1 was closely related to the biological behaviour of human GAC and involved in the development, invasion and metastasis of human GAC. Currently, the mechanism by which ANXA1 functions in the biological behaviour of GC remains unclear. Cheng et al[21] reported that ANXA1 can regulate GC invasion by mediating formyl peptide receptor (FPR)/extracellular signal-regulated kinase/integrin protein β-1 bind protein pathway; all of the three FPRs are involved in the regulation process. Kang et al[11] further found that ANXA1 gene can decrease the activity and protein expression of matrix metalloproteinase-9 (MMP-9) transcriptional promoter by inhibiting the activity of nuclear factor κ-B. MMP-9 also has an important function in GC invasion and metastasis. Vascular endothelial growth factor (VEGF) is another important factor regulating angiogenesis, and angiogenesis is important in tumour growth and metastasis. Pin et al[22] found that VEGF-induced cell migration and angiogenesis of miR-196a can change the expression level of ANXA1, which is controlled by p38-ANXA1 signal conduction pathway. The function of Helicobacter pylori (H. pylori) infection in GC has also been demonstrated, showing that H. pylori can change the cellular ANXA1 localization[23]. This result suggests that ANXA1 may be involved in H. pylori infection-induced GC. Furthermore, these results can explain the possible mechanism by which ANXA1 participates in GAC biological behaviour to some extent.

A few reports about ANXA1 in GC have been published. For instance, Yapar et al[4] conducted an immunohistochemical study and found that ANXA1 expressed in GC is 56.3% higher than that in paratissues; this expression is also positively correlated with tumour invasion and lymph node metastasis; a high ANXA1 expression suggests poor prognosis of GC. Jorge et al[6] performed RT-PCR and immunohistochemistry, revealing that ANXA1 mRNA and protein expressions are increased in GC compared with normal gastric mucosa. The results of the present study are consistent with those in the aforementioned previous studies, although other studies have revealed contrasting results. In some studies, ANXA1 is downregulated in GC[4,24] and negatively correlated with tumour staging and lymph node metastasis[4]. The reasons may be described as follows: (1) different test conditions, antibodies, and test methods; and (2) different samples or different types and pathological staging parameters of GC samples. The intracellular ANXA1, which is mainly in the cytoplasm, is possibly redistributed at different stimulations. For example, ANXA1 likely enters the nucleus as induced by an epidermal growth factor (EGF) under oxidation conditions or heat shock; by contrast, ANXA1 is transferred to the membrane and then secreted out of the cells as stimulated by GC or phorbol-12-myristate-13-acetate[25,26]. Therefore, the upregulation and downregulation of ANXA1 in GC may be associated with different stages of GC pathology. Different levels of EGF and GC in vivo may possibly induce cellular ANXA1 relocalization. ANXA1 may also be involved in GC occurrence and development via different pathways and mechanisms.

In summary, the present study provided valuable information to clarify GC pathogenesis. This study also presented the basic foundation to screen cancer biomarkers. After the differential proteins of GC were initially screened, the results suggested that identification and function in vivo and in vitro of some important differential proteins require further studies. Given that ANXA1 was also expressed in gastric stromal cells, ANXA1 expression in GC may be difficult to assess by IHC. Nevertheless, the study on immunohistochemical tissue microarray revealed the relationship between ANXA1 and GC clinicopathological parameters. We found that the upregulation of ANXA1 in human GAC was closely related to the depth of tumour invasion, lymph node metastasis, distant metastasis and TNM stage. The results also suggested that ANXA1 was possibly involved in tumour invasion and metastasis. The high expression of ANXA1 suggested poor prognosis of GC. The mechanism by which ANXA1 participated in the GC biological behaviour should be further studied. With the continuous development of this research, ANXA1 may be used in early cancer detection, diagnosis and treatment. ANXA1 may also become an indicator of cancer prognosis and a new target of cancer therapy, thereby providing new ideas of GC diagnosis and treatment.