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. 2022 Feb 1;23(2):715-721.
doi: 10.31557/APJCP.2022.23.2.715.

Annexin A1 Is a Potential Prognostic Marker for, and Enhances the Metastasis of, Cholangiocarcinoma

Kwuntida U Kotepui  1   2   3 , Sumalee Obchoei  1   2   4 , Kulthida Vaeteewoottacharn  1   2 , Seiji Okada  5 , Sopit Wongkham  1   2 , Kanlayanee Sawanyawisuth  1   2
Affiliations

Annexin A1 Is a Potential Prognostic Marker for, and Enhances the Metastasis of, Cholangiocarcinoma

Kwuntida U Kotepui et al. Asian Pac J Cancer Prev. .

Abstract

Objective: Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein which contributes to proliferation, cancer progression and metastasis. Overexpression of ANXA1 is closely associated with metastasis in numerous types of cancer. Cholangiocarcinoma (CCA) is a bile-duct cancer which has high rates of metastasis. Previously, we demonstrated up-regulation of ANXA1 in a highly metastatic CCA cell line (KKU-213AL5). Here, we investigated the functions of ANXA1 in the progression of CCA cell lines and evaluated its clinical impacts in human CCA tissues. Methods: Effects of ANXA1 on metastatic potential of CCA cell lines were evaluated using cell-proliferation, clonogenic, migration and invasion assays. The expression of ANXA1 in 44 intrahepatic human CCA tissues was investigated using immunohistochemistry (IHC). The association of ANXA1 with clinicopathological features of CCA patients was analyzed.

Results: Silencing of ANXA1 expression using siRNA significantly decreased cell proliferation, colony formation, cell migration and invasion in the KKU-213AL5 cell line. IHC results showed low expression of ANXA1 in normal bile ducts in the non-tumor area. In contrast, high expression of ANXA1 in human CCA tissues was associated with advanced tumor stage, tumor size and presence of lymph-node metastasis.

Conclusion: These findings strongly imply that ANXA1 contributes to the progression of CCA. ANXA1 can serve as a potential prognostic marker for CCA. Ablation of ANXA1 action may be an alternative strategy to prevent metastasis of CCA.

Keywords: ANXA1; Annexin A1; Metastasis; cholangiocarcinoma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
ANXA1 Expression in KKU-213A and KKU-213AL5 Cell Lines and Metastases of These in Mouse Lungs. Comparison of ANXA1 expression at (A) the mRNA level; and (B) protein level in KKU-213A and highly metastatic KKU-213AL5 cell lines. The expression of ANXA1 in KKU-213A was normalized with β-actin and set = 1. (C) The expression of ANXA1 and CK19 in lung metastasized tissues from mice injected with KKU-213A and KKU-213AL5 was determined using IHC (magnification 40x). (D) The percentages of cells staining positive for ANXA1 in lung tissues from mice injected with KKU-213A or KKU-213AL5 CCA cells (n =6 each group). *P < 0.05
Figure 2
Figure 2
Suppression of ANXA1 Reduced Cell Proliferation, Colony Formation, Cell Migration and Invasion of KKU-213AL5 Cells. (A) Expression of ANXA1 protein was depleted by siRNA (si-ANXA1). (B) The rate of cell proliferation was determined daily for 3 days using the MTT assay. Relative proliferation was normalized to day 0 and compared between scramble control cells and si-ANXA1-treated cells. Data represent mean ± SD of three replicates from one of two independent experiments. (C) Silencing of ANXA1 reduced colony formation in the KKU-213AL5 cell line. Crystal violet-stained colonies of KKU-213AL5 compared between the scramble control and si-ANXA1-treated cells. Data are presented as the mean ± SD of numbers of colonies in three replicates from one of two independent experiments. *P < 0.05, ***P < 0.001. The numbers of migrating cells (D) and invading cells (E) of KKU-213AL5 compared between scramble control and si-ANXA1-treated cells. Data are presented as the mean ± SD of numbers of migrating/ invading cells in three replicates from one of three independent experiments. ***P < 0.001
Figure 3
Figure 3
Immunohistochemistry of ANXA1 in Human CCA Tissues. (A) Negative or low expression of ANXA1 is apparent in non-tumor tissues. CCA tissues with low ANXA1 expression (IHC index ≤6) and high ANXA1 expression (IHC index >6). NBD = normal bile duct, magnification 200x. (B) ANXA1 protein expression levels in each of 44 CCA patients. The IHC-index is shown of ANXA1 in normal bile duct (NBD) and CCA tissues from individual CCA patients. (C) Comparison of IHC indices of ANXA1 in NBD and CCA tissues. ***P < 0.001. (D) Kaplan-Meier survival plot between low and high ANXA1 expressing CCA patients
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