ATX‑LPA axis facilitates estrogen‑induced endometrial cancer cell proliferation via MAPK/ERK signaling pathway

Abstract

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). ATX is a crucial factor that facilitates cancer progression; however, the effect of ATX on endometrial cancer has not been explored. The aim of the present study was to investigate the role of ATX in the progression of endometrial cancer. The immunohistochemical results revealed higher protein expression levels of ATX and LPA receptors (LPA 1, 2 and 3) in human endometrial cancer tissue than in non‑carcinoma tissue. In addition, reverse transcription‑quantitative polymerase chain reaction and western blotting analysis demonstrated that ATX and LPA receptor mRNA and protein expression was greater in Ishikawa cells, which are positive for estrogen receptor (ER), than in Hec‑1A cells that exhibit low ER expression. Short interfering RNA knockdown of ATX in Ishikawa cells led to decreased cell proliferation and cell colony number, as determined by Cell Counting kit‑8 and colony formation assays. Estrogen stimulated ATX mRNA expression. Inhibition of ATX decreased estrogen and LPA‑induced cell proliferation. High LPA levels markedly elevated the phosphorylation levels of extracellular signal‑regulated kinase (ERK). ATX downregulation moderately decreased estrogen‑ and LPA‑induced phosphorylation of ERK. In addition, the ERK inhibitor, PD98059, reduced cell proliferation with estrogen, ATX and LPA treatment. The present study suggested that the ATX‑LPA axis may facilitate estrogen‑induced cell proliferation in endometrial cancer via the mitogen‑activated protein kinase/ERK signaling pathway. The present study may provide ideas and an experimental basis for clinicians to identify new molecular targeted drugs for the treatment of endometrial cancer.

Keywords: endometrial cancer; autotaxin; lysophosphatidic acid; proliferation; estrogen.

Figures

Figure 1.

Greater expression of ATX and LPA1, 2 and 3 in 10 endometrial carcinoma tissues than in adjacent non-cancerous tissues. Immunohistochemistry staining of (A) ATX and (B) LPA 1, 2 and 3 protein expression in endometrial carcinoma tissues and adjacent non-cancerous tissue by inverted microscopy. All images of the stained sections were captured at ×400 magnification. Arrows (→) show the localization of the staining in cancer cells and stromal cells. (C) Quantification of immunohistochemical staining of 10 endometrial carcinoma tissues and adjacent non-cancerous tissues. *P

Figure 2.

Expression levels of ATX and LPA1, 2 and 3 in two different endometrial cancer cell lines. (A) Reverse transcription-quantitative polymerase chain reaction analysis of ATX mRNA expression levels in Ishikawa and Hec-1A cells. (B) ATX localization by immunocytochemistry and (C and D) protein expression levels by western blot analysis in Hec-1A and Ishikawa cells. *P

Figure 3.

Knockdown of ATX with siRNA inhibited the proliferation of Ishikawa cells. (A) Ishikawa cells were transfected with siRNA-NC or siRNA1 and siRNA2 specific to the ATX gene for 48 h. The mRNA and protein expression of ATX were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The mRNA were detected by reverse transcription-quantitative polymerase chain reaction. (B) The protein expression of ATX was analyzed by western blot. Quantification of siRNA-NC, siRNA1 and siRNA2 groups. (C) Cell Counting kit-8 assay of cell proliferation of transfected cells at 1, 2, 3 and 4 days following siRNA knockdown of ATX. (D) Colony formation assay of proliferation of Ishikawa cells with siRNA knockdown of ATX. Data are shown as the mean ± standard deviation (n=3). *P

Figure 4.

Reduced ATX levels protect against proliferation of Ishikawa cells induced by estrogen and LPA. (A) Reverse transcription-quantitative polymerase chain reaction of the mRNA expression of ATX in Ishikawa cells stimulated with 17β-estradiol (10 nM) for 24 h. ***P

Figure 5.

ATX-LPA axis is involved in estrogen-induced proliferation via ERK signaling. (A) Reverse transcription-quantitative polymerase chain reaction of the mRNA expression levels of LPA1, 2 and 3 in Ishikawa cells following siRNA knockdown of ATX for 24 h. **P