The expression of metastasis-associated in colon cancer-1 and KAI1 in gastric adenocarcinoma and their clinical significance

Background The most common reason for malignant tumor treatment failure is recurrence and metastasis. Metastasis-associated in colon cancer-1 (MACC1) was originally identified as a metastatic and prognostic biomarker for colon cancer and later other solid tumors. Kangai 1 (KAI1), a marker of suppressor of metastasis, is also associated with metastasis and poor prognosis in many tumors. However, the prognostic value of either MACC1 or KAI1 in gastric adenocarcinoma (GAC) is unclear. In this study, we explored the relationship between MACC1 and KAI1 expression, as well as their respective correlation with clinicopathological features, to determine if either could be helpful for improvement of survival prognosis in GAC patients. Methods The expression levels of both MACC1 and KAI1 in 325 whole-tissue sections of GAC were examined by immunohistochemistry. Clinical data was also collected. Results MACC1 was significantly overexpressed in GAC tissues when compared to levels in normal gastric tissues; KAI1 was significantly down-expressed in GAC tissues when compared to levels in normal gastric tissues. Investigation of association between MACC1 and KAI1 protein levels with clinicopathological parameters of GAC indicated association between the expression of each with tumor grade, lymph node metastasis, invasive depth, and TNM stages. The overall survival time of patients with MACC1- or KAI1-positive GAC tumors was significantly shorter or longer than that of those who were negative. Importantly, multivariate analysis suggested that positive expression of either MACC1 or KAI1, as well as TNM stage, could be independent prognostic factors for overall survival in patients with GAC. Conclusions MACC1 and KAI1 may represent promising metastatic and prognostic biomarkers, as well as potential therapeutic targets, for GAC.


Background
There were approximately 950,000 new stomach cancer cases and 720,000 deaths that occurred in 2012 worldwide [1]. In general, China is one of the highest incidence countries worldwide [1]. Gastric adenocarcinoma (GAC) accounts for approximately 90 % of all diagnosed stomach cancers. It was also one of the most frequent causes of cancer-related deaths. The 5-year survival rate is less than 5 % for GAC patients with distant metastasis, as well as is less than 60 % for patients with only localized malignancies [2]. In China, the majority of patients diagnosed with GAC have advanced stages of disease and are unsuitable for curative surgery.
Tumor recurrence and metastasis are the most common cause of treatment failure. Tumor recurrence and metastasis involve in multiple steps with a high degree of complexity and require the contribution of many molecules. Metastasis-associated in colon cancer-1 (MACC1) is a gene which contributes to these processes. MACC1 was first identified in colon cancer in 2009 and was bound to the promoter of the mesenchymal-epithelial transition (MET) gene to control its transcriptional activity [3,4]. In vitro, MACC1 may drive proliferation, migration, invasion, and dissemination [5]; in vivo, it may regulate gene transcriptionally for metastasis, such as tyrosine kinase MET [5][6][7]. Furthermore, accumulating evidence has indicated that MACC1 should contribute to apoptosis and epithelial-mesenchymal transition (EMT) via hepatocyte growth factor/mesenchymal-epithelial transition (HGF/ MET) pathways [8]. MACC1 was also considered as a decisive driver for metastasis and tumorigenesis [9]. MACC1 was also an independent prognostic factor for colon cancer [3,5]. Now, more and more studies have demonstrated that MACC1 could also be a metastatic and prognostic factor for various human cancers, including pancreatic [10], liver [11], lung [12], ovary [13], breast [14], gastric [8], malignant glioma [15], and cervical carcinoma [16].
The involvement of MACC1 and KAI1 in the recurrence and metastasis of GAC suggests that they should be valuable biomarkers for measuring cancer progression and developing higher accurately therapeutic targets. To our knowledge, a correlation between MACC1 and KAI1 in GAC has not yet been reported. In this study, we detected the association between MACC1 and KAI1 expression in patient cancer tissues as well as compared their expression with clinicopathology, metastasis, and prognosis of GAC.

Biopsy specimens
GAC tissues and adjacent noncancerous gastric tissues were collected at the Department of Pathology of the First Affiliated Hospital of Bengbu Medical College, from January 2008 to December 2010. Patients who had received preoperative chemo-or radio-therapy were excluded. All tissue specimens were obtained with patient consent, and the research was approved by the ethical committee of Bengbu Medical College and conducted in accordance with the ethical guidelines of the Declaration of Helsinki. The adjacent noncancerous gastric tissues were removed from the same patient, avoiding necrotic tissue, and from surrounding gastric tissue at least 5 cm away from the carcinoma edge. The research group consisted of 325 patients, 214 males and 111 females, aged from 26 to 78 years; the average age was 57.7 ± 10.9 years. All patients who had completely clinical, pathological, and follow-up (at 8-month intervals by phone, mail, or email) data were sporadic cases. Overall survival (OS) time was collected from surgery to death or December 2015 (mean OS time 42.0 months; range 8-95 months). Tumor node metastasis stage was evaluated according to the 7th edition of the American Joint Committee on Cancer (AJCC). Grade of tumor differentiation was according to the World Health Organization (WHO) standard.
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Immunohistochemistry
All GAC and corresponding normal gastric tissues were fixed in 10 % buffered formalin and embedded in paraffin. Then continuous 4-μm-thick tissue sections were cut. Subsequently, all sections were deparaffinized and dehydrated with xylene, graded ethanol, and washed for 10 min in PBS (pH 7.2). Immunohistochemistry was performed according to the Elivision Plus detection kit instructions (Lab Vision, USA). Endogenous peroxidase activity was blocked by incubation of sections in methanol containing 3 % H 2 O 2 for 10 min at room temperature, then placed in citrate buffer (pH 6.0) and heated to 95°C for 30 min for antigen repair. After several washes in PBS, the sections were quenched with goat serum for 20 min at room temperature, then incubated with rabbit polyclonal antibody against human MACC1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal antibody against human KAI1 (Abcam, Cambridge, MA, USA) for 1 h at 37°C. All sections were counterstained with hematoxylin, dehydrated, air-dried, and mounted. Negative controls were prepared by deleting primary antibodies from the staining procedure. MACC1-positive staining was mainly confined in the cytoplasm of cancer cells, and KAI1positive staining was mainly confined in the membrane and cytoplasm of cancer cells.

Evaluation of staining
Staining results were evaluated by two experienced pathologists who were blind to the clinical data and assessed by semi-quantitative scores. Because of intratumoral heterogeneity of antibody expression, we randomly chose ten visual fields from different areas of each section of GAC. If there was a disagreement, the pathologists would reexamine the immunostaining and reach a consensus [27][28][29]. To assess MACC1 and KAI1 expression, both the extent and intensity of immunostaining were thought [27]. The staining extent score was graded as follows: none, 0; weak, 1; moderate, 2; and strong, 3. The intensity of positive staining was graded as follows: <10 %, 1; 11-50 %, 2; 51-75 %, 3; and >75 %, 4. Then the score was determined by multiplying the extent and intensity of immunostaining to reach a range of scores from 0 to 12. For tumors that were positive for both MACC1 and KAI1, an average of the final of each sample was taken. Immunostaining was thought positive when the score was ≥3.

Statistical analysis
Relationship between either MACC1-or KAI1 protein expression and clinicopathological parameters were compared using Fisher's exact test or chi-square test. The correlation between MACC1 and KAI1 expression was compared using Spearman's coefficient test. The effects of MACC1 and KAI1 expression on OS time were determined using Kaplan-Meier method for univariate analysis. Independent prognostic indicators were determined using the Cox regression model for multivariate analysis. The association between the positive expression of either MACC1 or KAI1 and clinicopathological parameters was determined using SPSS 19.0 software for Windows (Chicago, IL). A value of P < 0.05 was determined as statistically significant.

Results
All GAC patient clinicopathological characteristics could be seen in Table 1.

Expression of MACC1 and KAI1 in GAC and their association with clinicopathology
To assess the contributions of MACC1 and KAI1 to GAC, their expression levels were evaluated in both GAC and normal gastric tissue slides using immunohistochemistry. MACC1-positive staining was mainly confined in the cytoplasm of cancer cells, and KAI1-positive staining was mainly confined in the membrane and cytoplasm of cancer cells. These data were compared to clinicopathological characteristics. The positive rate of MACC1 protein expression was 60.3 % (196/325) in GAC tissues and 9.2 % (30/325) in normal gastric tissues (Fig. 1a, b), and this difference was shown to be statistically significant (P < 0.01). There were also significant differences between the expression of MACC1 and tumor grade (P = 0.009), size of tumor (P = 0.009), invasion of depth (P < 0.001), lymph node metastasis (LNM) (P < 0.001), and tumor-node-metastasis (TNM) (P < 0.001). In contrast, there were no associations detected between MACC1 expression and patient age (P = 0.295), gender (P = 0.482), location (P = 0.072), and gross type (P = 0.108).

Multivariate analysis
Multivariate analysis demonstrated that positive expression of either MACC1 or KAI1, as well as TNM stage, was an independent prognostic indicator for GAC (Table 4).

Discussion
Gastric adenocarcinoma (GAC) is a highly heterogeneous tumor. This heterogeneity may affect the reproducibility of biomarker evaluation [5,30]. So, thorough investigation of the metastatic and prognostic values of a candidate biomarker is thus required to ensure validity. In our study, we investigated MACC1 expression in GAC and matched normal tissues from 325 patients and compared it to clinicopathological characteristics. We found that MACC1 expression was significantly higher in GAC tissues than that in the control tissues. Furthermore, MACC1 expression was positively correlated with tumor size, grade, invasion of depth, LNM, and TNM stage. Our results were consistent with those of previous studies in GAC [8,[31][32][33] demonstrating that MACC1 should be useful as a clinical candidate biomarker of GAC.
KAI1, a cell membrane protein that binds to ECM or adhesion proteins [34,35], is widely considered as a suppresser gene of metastasis in many cancers [19][20][21][22][23][24][25][26]. KAI1 has been correlated with carcinogenesis [20] and showed to predict a poor metastasis and prognosis [19][20][21][22][23][24][25][26]. In this study, we also found that KAI1 expression was significantly related to tumor grade, invasion of depth, LNM, and TNM stage. In addition, Kaplan-Meier survival analysis indicated that GAC patients with positive KAI1 expression had significantly increased survival time compared to those with negative KAI1. These results indicated that KAI1 should play a key role in tumorigenesis, invasion, metastasis, and prognosis of GAC. Several other immunohistochemical studies that investigated the metastatic and prognostic significance of KAI1 in GAC patients obtained similar results [24,36,37]. Thus, our results supported the conception that KAI1 should be a credible biomarker of GAC, especially for predicting metastasis and prognosis of cancers. Metastasis and recurrence are the most common reasons of cancer-related deaths in GAC. TNM staging system is well-known as the guide for devising therapeutic strategies for patients with GAC. However, the TNM staging system cannot provide comprehensive information about the biological behavior of the cancer. Thus, it is urgent to seek novel and effective metastatic and prognostic biomarkers to predict biological behavior (metastasis and recurrence) in GAC patients. In our study, multivariate Cox model analysis showed that the positive expression of either MACC1 or KAI1, as well as TNM stage, was an independent prognostic indicator for patients with GAC.
In our study, we found that MACC1 expression was negatively correlated with KAI1 expression. Furthermore, we also found that there was a negative correlation between the high expression of MACC1 and low expression of KAI1 in the same GAC patient. Abnormal (decreased or lost) expression of KAI1 may be involved in the initiation, development, invasion, metastasis, and recurrence of GAC through lost of function of tumor suppressor gene or suppressor gene of tumor metastasis. Indeed, KAI1, as a suppressor of tumor metastasis, could inhibit β-catenin tyrosine phosphorylation and stabilize E-cadherin-β-catenin complexes to suppress tumor metastasis [38]. In addition, KAI1 could inhibit the process of β-catenin-mediated EMT to prevent tumor angiogenesis and lymphangiogenesis [39]. Meanwhile, MACC1 could be bound to the promoter of the MET gene and activate the HGF/MET signaling pathway to promote cancer cell proliferation, invasion, and metastasis [3,4]. Furthermore, it could promote angiogenesis and lymphangiogenesis to lead to cancer cell invasion and metastasis [40,41]. Moreover, KAI1 is able to be bind to c-MET to form a complex or quench the activation of HGF, thus preventing the activation of MACC1 to inhibit the migration of tumor cells [42,43]. Decreased or lost expression of KAI1 might lose inhibiting the activation of MACC1, angiogenesis and lymphangiogenesis, and stabilization of Ecadherin-β-catenin complexes to promote cancer cell invasion and metastasis. At the same time, abnormal expression of MACC1 could further promote cancer cell invasion and metastasis. However, the methodology of subjects in our study was relatively simple; further studies with more methodologies (such as assessing the effect of target molecules on biological properties in vitro and in vivo models) are needed to verify the present observation.