Oncogenic function of angiopoietin-2 in vitro and its modulation of tumor progression in colorectal carcinoma

Abstract

Angiopoietin-2 (Ang-2) has been investigated in cancer primarily in terms of its angiogenic function, and its role as an oncogene has yet to be elucidated. The current study hypothesized that Ang-2 may be an oncogene and have a function in tumor progression. An investigation of the function of Ang-2 in the LoVo colorectal cancer (CRC) cell line in vitro, which expresses a high level of Ang-2, was performed by knocking down endogenous expression with a targeted short hairpin RNA. The aggressive phenotypic effects of Ang-2 on experimental and control group cells were assessed using cell proliferation, migration and invasion assays. The association between Ang-2 expression levels and clinicopathological factors was evaluated in 415 CRC tissues using immunohistochemistry. Suppressing Ang-2 expression decreased cellular proliferation, invasion and migration in an in vitro study. Ang-2 overexpression was observed in 46% of patients with CRC and was significantly associated with pT (P=0.048), pN (P<0.001), venous invasion (P=0.023), lymphatic invasion (P<0.001) and tumor-node-metastasis stage (P=0.022). Furthermore, Ang-2 overexpression was an independent prognostic factor in pN stages 1 and 2. These results reveal that Ang-2 may be an oncogene in colorectal carcinogenesis and its expression may exert aggressive phenotypic effects during tumor progression. In addition, Ang-2 expression may serve as a prognostic marker and a potential drug target.

Keywords: Angiopoietin-2; Colorectal carcinoma; Oncogenic function; shRNA.

Figure 1.

Ang-2 expression was investigated by reverse-transcription polymerase chain reaction and western blot analysis in colorectal cancer cell lines, HUVEC and normal cell line (CCD-841 CoN). (A) Among 5 colorectal cancer cell lines, 3 cell lines expressed Ang-2 mRNA, as did the HUVEC cell line. (B) LoVo cells expressed a markedly higher level of Ang-2 protein compared with other colorectal cancer cell lines, as detected by western blotting. (C) The LoVo cell line transfected with Ang-2 target shRNA did not express Ang-2 mRNA. (D) The LoVo cell transfected with Ang-2 target shRNA exhibited markedly decreased expression levels of Ang-2 protein. Ang-2, Angiopoietin-2.

Figure 2.

(A) The proliferation of LoVo cells following Ang-2-targeted shRNA transfection was significantly decreased compared with control LoVo cells and LoVo cell transfected with non-targeted shRNA. (B) The invasion ability of Ang-2 target shRNA transfected LoVo cells was decreased markedly compared with the control and non-target shRNA-transfected LoVo cells. ***P<0.05. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Figure 3.

The migration of Ang-2 target shRNA transfected LoVo cells was decreased compared with the control and non-target shRNA transfected LoVo cells. ***P<0.01. Ang-2, Angiopoietin-2; shRNA, short hairpin RNA.

Figure 4.

(A) Ang-2 was not expressed in N cells. However, Ang-2 expression was markedly increased in T cells. (B) The cumulative survival was significantly decreased in patients with high levels of Ang-2 compared with patients with low levels of Ang-2 expression, as determined by Kaplan Meier analysis (log rank test, P<0.001). T, colorectal cancer cells; N, normal colonic epithelial cells; Cum, cumulative; Ang-2, Angiopoietin-2.