Abstract
Calreticulin (CRT) and vascular endothelial growth factor-A (VEGF-A) are crucial for angiogenesis, and mediate multiple malignant behaviors in gastric cancer. In this study, we report that CRT is positively correlated with VEGF-A in gastric cancer patients. Moreover, high expressions of both CRT and VEGF-A are markedly associated with the pathological stage, progression, and poor prognosis in the patients. Therefore, we sought to elucidate the mechanism by which CRT affects VEGF-A in gastric cancer. Firstly, we demonstrate the novel finding that knockdown of CRT reduced VEGF-A mRNA stability in two gastric cancer cell lines, AGS and MKN45. The AU-Rich element (ARE) is believed to play a crucial role in the maintenance of VEGF-A mRNA stability. Luciferase reporter assay shows that knockdown of CRT significantly decreased the activity of renilla luciferase with VEGF-A ARE sequence. Additionally, competition results from RNA-binding/electrophoretic mobility shift assay indicate that CRT forms an RNA-protein complex with the VEGF-A mRNA by binding to the ARE. In addition, the proliferation rate of human umbilical vein endothelial cells (HUVEC) was significantly reduced when treated with conditioned medium from CRT knockdown cells; this was rescued by exogenous VEGF-A recombinant protein. Our results demonstrate that CRT is involved in VEGF-A ARE binding protein complexes to stabilize VEGF-A mRNA, thereby promoting the angiogenesis, and progression of gastric cancer.
Highlights
Gastric cancer is the third leading cause of cancer-related death worldwide [1], and the seventh leading cause of cancer-related mortality in Taiwan
The clinicopathological parameters showed that the expression levels of both CRT and Vascular endothelial growth factor-A (VEGF-A) were significantly associated with TNM stage progression and nodal status (N stage) of the patients
Both CRT and VEGF-A were positively correlated with T stage, and VEGF-A was positively significantly correlated with distant metastasis (M stage)
Summary
Gastric cancer is the third leading cause of cancer-related death worldwide [1], and the seventh leading cause of cancer-related mortality in Taiwan. We previously demonstrate that calreticulin (CRT) can be a prognostic marker of gastric cancer. Overexpression of CRT enhances angiogenesis and malignant behavior in gastric cancer cells, and further associated with microvessel density, tumor invasion, lymph node metastasis, and survival in patients [2]. Correlations between CRT and metastasis have been reported in multiple cancers. The expression of CRT is especially higher in aggressive breast cancer cells and positively correlated with distant metastasis in tissue samples [4]. CRT has been shown previously to promote angiogenesis via activating the nitric oxide signaling pathway [5]. A positive correlation between CRT and Vascular endothelial growth factor-A (VEGF-A) has been addressed in neuroblastoma, bladder cancer, and gastric cancer cell lines [2, 6, 7]
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