Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis

Abstract

Colorectal cancer (CRC) is one of the most dangerous types of malignant tumors, and cancer metastasis is a major factor in the failure of CRC therapy. Recently, LOXL2 (lysyl oxidase-like 2) has been shown to represent a regulator of epithelial-mesenchymal transition (EMT) in different cancer types. However, LOXL2 has not been reported to be involved in CRC metastasis. In this study, we demonstrated that LOXL2 expression is strongly correlated with the rate of CRC metastasis, it participates in the regulation of EMT-related molecule expression in CRC cells in vitro, and it is involved in migratory potential alterations. Additionally, tissue microarray analysis of CRC patients showed an increase in the probability of developing CRC distant metastasis and a decrease in the survival rate of patients with high LOXL2 expression. The results obtained in this study indicate that LOXL2 is involved in the development and progression of CRC metastasis, and therefore, its expression levels may represent a useful prognostic marker.

Keywords: LOXL2; colorectal cancer; epithelial-mesenchymal transition; metastasis; tissue microarray analysis.

Figure 1. Correlation between LOXL2 expression levels and migratory potential of different CRC cells in vitro

(A) LOXL2 expression in five CRC cell lines. Distilled water was used as a negative control (N.C.). (B) LOXL2 protein levels in CRC cells. MDA-MB-231 was used as a positive control (P.C.). (C) Motility of different CRC cells in vitro, determined in wound healing assay. Representative figures obtained in three independent experiment are shown.

Figure 2. Migratory potential alterations induced by the changes in LOXL2 expression

(A) Transwell migration of LOXL2-knockdown SW480 cells. Images were obtained 36 h after seeding. (B) Transwell migration of LOXL2-overexpressing SW620 cells. Images were obtained 72 h after seeding. Results were obtained from three independent experiments, and bar graphs in (A) and (B) represent cell number per image field (mean ± standard deviation).

Figure 3. Effects of LOXL2 silencing/ectopic expression on the migration of CRC cells through the endothelial barrier

(A) SW480 cells transfected with siControl/siLOXL2 oligonucleotide were seeded on HUVECs, and images were obtained 36 h after seeding. (B) SW620 cells transfected with mock/LOXL2-expressing plasmids were seeded on HUVECs, and images were obtained 72 h after seeding. Results were obtained from three independent experiments and bar graphs in (A) and (B) represent cell number per image field (mean ± standard deviation).

Figure 4. Induction of EMT by LOXL2 through the activation of FAK/Src pathway and upregulation of Snail

(A) Expression of EMT-related molecules 24 and 48 h after the transfection of cells with LOXL2 siRNA/overexpressing plasmid. (B) CDH1 (E-cadherin), VIM (vimentin), and SNAI1 (Snail) expression in SW480 and SW620 cells 24 h after the transfection with siLOXL2 and LOXL2-overexpressing plasmid, respectively. GAPDH was used as an internal control. (C) Expression of EMT-related molecules in cells transfected with siLOXL2 and LOXL2-overexpressing plasmid.

Figure 5. LOXL2 IHC analysis in CRC tissues and the correlation between this parameter and the survival of CRC patients

(A) IHC analysis of LOXL2 expression in CRC tissues. Representative images of normal colon tissue, CRC tissue with low LOXL2 expression, and CRC tissue with high LOXL2 expression are presented (magnification, 200 ×). (B) Correlation between the overall survival rates of CRC patients and LOXL2 expression. High LOXL2 expression was correlated with poor patient survival (log-rank Mantel-Cox test, p = 0.024).