Background
The annexin superfamily consists of 13 calcium or calcium and phospholipid binding proteins expressed in most eukaryotic cell types. Despite their high biological and structural homology (40-60%), annexins have diverse functions in cellular activities including vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation. In certain clinical conditions, the expression levels of annexins or their localization changed remarkably, which suggests that annexins may contribute to the pathological consequence and sequelae of most serious human diseases including cardiovascular disease and cancer [
1]. As the first characterized member of the annexin superfamily, Annexin-1(ANXA1) gene located on chromosome 19q24, encoding a 37 kDa protein functioning as a strong inhibitor of glucocorticoid-induced eicosanoid synthesis and PLA2. Recently, increasing evidences implicated that ANXA1 contributes to a variety of cellular biological activities, including anti-inflammatory effects, cell proliferation inhibition, the regulation of cell death and differentiation, phagocytic clearance of apoptosing cells, and most importantly the process of carcinogenesis [
2].
These diverse biological activities of ANXA1 make it a potential target for novel therapeutic intervention. However, more recently, ANXA1 protein has been recognized to be differentially expressed in various human tumors, e.g., breast cancer, prostate cancer, esophageal cancer, gastric cancer, endometrial carcinoma, pancreatic cancer, and colorectal cancer [
2‐
13]. Loss/aberrant expression pattern of ANXA1 in esophageal squamous cell carcinoma, prostate cancer, and endometrial carcinoma could be correlated with altered tumor behavior, e.g., dedifferentiation of tumor cells, increased invasiveness, and thus with tumor progression [
6,
8‐
10,
12]. On the other hand, increased expression pattern of ANXA1 in hepatocellular carcinoma, colorectal cancer, and pancreatic cancer was shown to be associated with tumor growth, lymph node metastasis, and advanced disease stages, and consequently with poor patient outcome [
11,
13,
14]. However, contradictory descriptions on ANXA1 expression were reported in certain human tumors, e.g., breast cancer [
3,
5,
15], bladder cancer [
16,
17], and gastric cancer as well [
18‐
21]. Hence, although the importance of ANXA1 in cancer is apparent and antibodies for therapeutic invention were easily prepared, researchers and clinicians are hampered by the conflicting expression pattern of ANXA1 in human solid cancers and by the lack of complete data sets describing the tissue-specific expression of this gene/protein.
We therefore sought to systematically investigate the expression pattern of ANXA1 in human gastrointestinal solid cancer and matched non-cancerous tissues. Generally, real-time RT-PCR was used for the detection of ANXA1 mRNA expression, while Western blotting and immunohistochemistry were established to visualize the tissue-specific expression pattern and quantification of ANXA1 protein in these specimens. Next, we restored ANXA1 expression in AGS gastric cell lines and the possible mechanism of ANXA1 in gastric carcinogenesis was further explored. These differentially expression patterns of ANXA1 in gastrointestinal carcinomas set a solid groundwork for further ANXA1-targeted molecular cancer therapy and as a diagnostic and prognostic marker.
Discussion
In the present study, a systematic expression profile of ANXA1 in human gastrointestinal tumors was explored by real-time RT-PCR, Western blot, immunohistochemistry, and/or immunofluoresncece. We provided comprehensive evidences that (1) ANXA1 is differentially expressed in healthy human tissues; (2) ANXA1 expression is tumor type-specific: downregulation in squamous cell carcinoma, upregulation in pancreatic carcinoma, but controversial in other gastrointestinal tumors; (3) enforced expression of ANXA1 reduced cell viability by inhibiting the production of COX-2, while COX-2 overexpression could abolish this effect induced by ANXA1. Our findings can be helpful in disclosing the functional diversity of ANXA1 on the basis of organ-specific expression profiles.
In contrast to various studies that various gastrointestinal epithelial tissues showed weak or absent expression of ANXA1, our findings revealed that ANXA1 displays very ubiquitous expression in all investigated benign tissues, suggesting a critical role of ANXA1 in the maintenance of gastrointestinal organized tissues. Although ANXA1 expression seems quite unique among healthy human tissues, each tissue shows rather restrictive expression of ANXA1: e.g., highest in the liver, 6.5-fold compared with the calibrator tissue (bile duct). One possible interpretation is that ANXA1 is predominantly localized in the cytoplasm of the epithelial cells, but weak/absent in the stroma. Therefore, the concentration of ANXA1 in certain organ is determined by the ratio of the main cellular components within healthy human tissues.
Although ANXA1 is attracting more attention in cancer research, the conflicting reports on the expression of ANXA1 limited its importance as a therapeutic and/or prognostic biomarker in cancer (Additional file
3: Table S1). Therefore, a systemic investigation with standard methods was necessary to identify the ANXA1 expression profile in six gastrointestinal tumor entities. In the present study, we provided preliminary evidence that ANXA1 expression seems to be tumor type-specific in these malignant tissues. Squamous cell carcinoma of esophagus showed a significant reduction of ANXA1 mRNA and protein expression, which is strongly expressed in the normal squamous epithelium. This finding is compatible with previous studies that identified ANXA1 as a marker of differentiation in squamous cell carcinoma of the cervix, and head and neck [
8‐
10]. Down-regulation of ANXA1 seems to be exceptional in adenocarcinomas, since many clinical cases documented overexpression in adenocarcinoma of various organs, e.g. esophageal and esophagogastric junction, stomach, liver, colon, and pancreas [
7,
11,
13,
14,
18]. Our results confirmed overexpression of ANXA1 mRNA and protein in pancreatic ductal adenocarcinoma, suggesting ANXA1 up-regulation involved in pancreatic tumorigenesis. However, the trend of ANXA1 up-regulation in tumors was not significant in colorectal and hepatocellular adenocarcinoma, which may be due to limited sample numbers. Interestingly, loss of ANXA1 expression has also been described in some tumors, for example, in breast cancer, cholangiocarcinoma, and gastric cancer [
5,
21,
23]. In present study, however, we observed a significant loss of ANXA1 expression in cholangiocarcinoma and gastric cancer. Taken together, the concept that ANXA1 is “a tumor suppressor” and therefore is usually down-regulated in squamous cell carcinoma can be identified. However, whether ANXA1 is oncogenic in adenocarcinoma requires a new look on the basis of our data.
Using tissue microarray analysis, we previously demonstrated a significant loss of ANXA1 expression in human primary gastric cancer compared with that in adjacent non-neoplastic mucosa. Recently, Cheng et al showed lack of immunoreactivity for AnxA1 in adjacent clinically normal gastric mucosa and overexpression of ANXA1 in GC [
18]. Therefore, to clarify the status of ANXA1 expression in gastric mucosa is of great value in determining whether ANXA1 is “oncogenic” or “tumor suppressor”. Firstly, in the immunohistochemistry analysis, we used whole tissue sections of GC to avoid tissue heterogeneity and selected two anti-ANXA1 antibodies after antibody specificity confirmation by Western blotting. Moreover, we applied RT-PCR and immunofluoresncece analysis to evaluate ANXA1 mRNA level and localization. Our study is the first comprehensive study which combined four methods to investigate ANXA1 expression pattern in non-neoplastic mucosa. All these methods uniformly revealed a higher expression of ANXA1 in these gastric epitheliums. Thus, we cannot rule out the possibility of ANXA1 as a tumor suppressor even if ANXA1 staining could not be observed in normal gastric mucosa in those studies. Next, we found that most of the cases investigated showed a lower expression of ANXA1 compared with adjacent non-neoplastic mucosa. In particular, liver metastases showed a lower level of ANXA1 expression than surrounding liver tissues, which serves as an internal control. In line with our previous study, loss of ANXA1 is a frequent event in gastric carcinogenesis. However, upregulation of ANXA1 mRNA was observed in 3 of 8 (37.5%) cases, which might be attributable to an abundant accumulation of cells in the tumor tissue compared with the corresponding benign tissue. Thus, the up-regulation of ANXA1 mRNA would be an epiphenomenon without any functional relevance with carcinogenesis. Taken together, the concept that ANXA1 is “oncogene” and therefore upregulated in GC requires a new looks on the basis of our data.
One of the most important finding of our study is that the level of ANXA1 expression is inversely associated with the cell viability and migration: AGS cells with low ANXA1 expression had higher ability of cell growth and invasion than N87 cells with high ANXA1 expression. By upregulation of ANXA1 with plasmid containing full-length ANXA1, we could significantly inhibit the anchorage-independent cell growth of gastric cancer cells, further supporting the importance of ANXA1 in the progression of gastric cancer.
Mechanically, ANXA1 exerts its antiproliferative activity via the inhibition of cyclin D1 and various signal transducing kinases [
2]. Cyclooxygenase-2 (COX-2) is an inducible enzyme and accumulates in activated macrophages and other cells at sites of inflammation. Increasing evidences showed that COX-2 was upregulated in various carcinomas and plays a key role in tumorigenesis. Previously, studies on ANXA1 knockout mice revealed constitutively increased expression of COX-2 [
24]. In this context, we found that overexpression of ANXA1 aborgated COX-2 expression in gastric cancer cells. Notably, knockdown ANXA1 expression with ANXA1-specific shRNA leads to an increase of COX-2 expression, suggesting ANXA1 mediating many diverse cellular functions, such as inflammation and proliferation. Moreover, we found that ANXA1 expression inversely co-localized with COX-2 expression in caner specimen, suggesting a cross-talk between ANXA1 and COX-2. This notion is supported by a previous study showing that IL-1beta increased the expression of COX-2 and concomittantly decreased the expression of lipocortin 1 (ANXA1) on the surface of A549 cells [
25]. Taken together, our data suggested that the cross-talk between ANXA1 and COX-2 might play a critical role in cell proliferation and tumor growth. However, whether COX-2 is a feedback of cell growth induced by ANXA1 or a direct downstream target of ANXA1 still needs further investigation.
Competing interest
There are no competing interests for all authors.
Authors’ contributions
YG participated in the design of the study, carried out the mRNA and protein expression of ANXA1 in human tissues and cancers and analyzed the data. YC and DX participated the immunohistochemistry analysis of ANXA1 and COX-2 in gastric cancer patients and assisted the analysis of data. YG and DX performed the cell biology study and the Western Blotting test. YC and GY participated evaluation of immunostaining and assisted the collection of clinical data. JW and GY participated in its design and coordination, and supervised the study. GY drafted the manuscript. YG, YC and DX contributed equally to this work. All authors read and approved the final manuscript.