 αvβ6 and transcriptional factor Ets-1. 5-year follow-up was conducted among all the 158 patients, and the follow-up rate was 100%. Among these, 71 (44.9%) were confirmed cancer specific death within 5 years of prognosis and 87 (55.1%) were censored as their case follow up was discontinued or patients were alive beyond 60 months or died of reasons other than colon cancer.115 patients (72.8%) relapsed from cancer during 5-year follow-up, and 43 (27.2%) were censored. Follow-up based on patient’s history, physical examination, complete blood count, liver function tests, tumor markers monitoring, ultrasound scan of the abdomen every 3 months, abdominal pelvic CT scans and colonoscopy every 6 months. Patients with high-risk factors were encouraged to take adjuvant chemotherapy. Patients with liver metastasis tumor accepted the radiofrequency ablation (RFA) or super γ knife radiotherapy. 87 patients received fluorouracil-based adjuvant chemotherapy, and 28 patients received the RFA or radiotherapy.
In the 158 tissue specimens, 57 (36.07%) were positive for αvβ6 expression, and 91 (57.59%) were positive for Ets-1 expression. Clinicopathological features and αvβ6 & Ets-1 expression of all specimens were expounded in details in Table 1. And the expression levels of αvβ6 and Ets-1 in 10 samples of normal colon mucosa were both negative. A following analysis showed that there was no evidence of a statistical difference between αvβ6 or Ets-1 positive and negative tumors regarding age, gender, location, tumor size, Duke’s phase, T stage, and histopathological type, but significant statistical differences existed regarding differentiation, N stage, M stage and TNM stage. αvβ6 and Ets-1staining patterns in the tissue specimens are shown in Figure 1. High αvβ6 expression was detected at the invading edge of the tumor, and staining was observed predominantly in the cell membrane. Ets-1 mainly exhibited a nuclear and cytoplasmic immunostaining in tumor cells.
Images of αvβ6 and Ets-1 immunohistochemistry staining in human colorectal cancer samples. αvβ6 staining was observed predominantly in the cell membrane. A-C the immunohistochemistry photomicrographs of integrin αvβ6. A: integrin αvβ6 weak staining, B: integrin αvβ6 moderate stainning, C: integrin αvβ6 high staining; Ets-1 mainly exhibited a nuclear and cytoplasmic immunostaining in tumor cells. D-F the immunohistochemistry photomicrographs of transcriptional factor Ets-1. D: Ets-1 weak staining, E: Ets-1 moderate staining, F: Ets-1 high staining.
In the 158 tissue specimens, 101 (63.92%) were negative for αvβ6 expression, 30 (18.99%) were low positive for αvβ6 expression, 27 (17.09%) were high positive for αvβ6 expression; 67 (42.41%) were negative for Ets-1 expression, 30 (37.34%) were low positive for Ets-1 expression, 32 (20.25%) were high positive for Ets-1 expression. The expression of αvβ6 and Ets-1 were positively correlated (r = 0.711, P = 0.000), Specific data was shown in Table 2.
According to the expression of αvβ6 and Ets-1, all patients were divided into four categories: both αvβ6 and Ets-1 positive group (Group 1), αvβ6 positive with Ets-1 negative group (Group 2), αvβ6 negative with Ets-1 positive group (Group 3), and both αvβ6 and Ets-1 negative group (Group 4), respectively. The Kaplan-Meier survival analysis showed that patients who were both αvβ6 and Ets-1 positive relapse earlier (32.59 ± 2.37) than those who were in Group 2 (43.82 ± 2.57), Group 3 (46.68 ± 1.83), and Group 4 (53.30 ± 1.10) (P = 0.000). And patients who were in either αvβ6 or Ets-1 positive (Group 2 and 3) also relapsed earlier than those who were both αvβ6 and Ets-1 negative (P < 0.0001) (Figure 2). Likewise, the Kaplan-Meier survival analysis showed that patients who were both αvβ6 and Ets-1 negative live longer (56.82 ± 0.95) than those who were both αvβ6 and Ets-1 positive (46.23 ± 1.71), and those who were in Group 2 (52.31 ± 2.41) or Group 3 (53.62 ± 1.71) (P < 0.0001). And patients who were in Group 2 or Group 3 live shorter than those who were both αvβ6 and Ets-1 negative (P < 0.0001) (Figure 3).
Disease relapse time is also shown by Kaplane-Meier survival analysis using a Log-rank test. The samples were grouped according to αvβ6 and Ets-1 expression. (P = 0.000).
The Cox proportional hazards regression model were used to conduct Univariate and multivariate data analyses to determine the prognostic value of αvβ6 and Ets-1 expression. Positive expression of αvβ6 and Ets-1, as well as senior grade of N stage, M stage, and TNM stage were the factors to predict a poor prognosis in univariate analysis (Shown in Table 3). Then the variables with P < 0.05 were chosen to conduct multivariate analysis, we revealed that positive expression of αvβ6 and Ets-1 were unfavorable independent prognostic factors (RR = 2.175, P = 0.012 and RR = 3.903, P < 0.001, respectively) (Shown in Table 3).
#: TNM stage IV is linearly dependent on M stage M1.
Colorectal cancer has a higher morbidity and mortality in the world. Despite improvements made in screening and treatments during the past years, the clinical outcome of colon cancer remains unsatisfactory. Understanding the molecular mechanism of tumorigenesis would contribute to treatment of patients with this disease.
The data in the present study indicated that the total expression percentage of integrin αvβ6 and Ets-1 in colorectal cancer was 36.07% and 57.59% respectively. The expression level of αvβ6 and Ets-1 was associated with the differentiation, N stage, M stage and TNM stage of the tumors. Correlation analysis showed that the expression of αvβ6 and Ets-1 were positively correlated, and their strong stains were always detected at the invading edge of the tumor, weak or no stain always in the center of the tumor. These findings about integrin αvβ6 expression were similar with our previous study in gastric carcinoma, and Ets-1 expression pattern and its role look like Ito. The phenomenon above could be explained as following: High cell density in tumor induces integrin αvβ6 and Ets-1 expression, which promote MMP-9 secretion for colon cancer cells,increasing the degradation of extracellular matrix (ECM),which constitutes the molecular biological basis for a self-perpetuating system of tumor invasive growth in colon cancer progression.
In the present study, survival analysis showed that patients who were both αvβ6 and Ets-1 positive relapsed earlier than others, and their survival time was also obviously shorter. Based on these data, we confirmed that the concomitant expression of αvβ6 and Ets-1 in colorectal cancer cells could be used as an independent predictor to determine early relapse, metastases and prognosis in CRC patients. The important conclusion of this study is similar with our previous study and Zsuzsanna.
We have previously shown a direct linkage between ERK2 and the cytoplasmic domain of β6, and have delineated the binding domain for ERK2 within the cytoplasmic tail of the β6-integrin subunit, 746EAERSKAKWQTGTNPLYR G764 (the ERK2 binding sequence is italic). Through this physical interaction, integrin αvβ6 transmitted a growth-enhancing signal to cancer cells. In vivo studies of colon cancer xenografts expressing a β6-mutant lacking the ERK2 binding domain indicated that deletion of the β6-ERK2 binding motif greatly compromised tumor growth. Taken all the factors into consideration, these results suggested that tumor growth was, at least in part, dependent on direct αvβ6-ERK binding. Meanwhile, we also have known that in colorectal cancer cells αvβ6-ERK2 direct linkage could increase the phosphorylation level of ERK2, and ERK2 conformation would be changed as soon as binding with αvβ6. To some extent, this process helped ERK2 to be phosphorylated easily, and protected phosphorylated ERK2 not to be dephosphorylated. The effective targets of phosphorylated-ERK2 were localized in the nucleus. Unfortunately, the nuclear factors which could be regulated by phosphorylated-ERK2 still remained unclear.
Park showed that the expression of Ets-1 has intimate correlation with tumor cell invasion and metastasis. Ets-1 and MMPs co-expressed in colon cancer and other malignant tumor, and its expression degree would be increased with tumor invasion and metastasis extent accordingly, but its expression was very low in benign and non-invasive tumor. Hashiya confirmed that Ets-1 played an important role in promoting the tumor angiogenesis through regulating angiogenesis factor, such as VEGF, Ang