Lysine demethylase 2A promotes the progression of ovarian cancer by regulating the PI3K pathway and reversing epithelial‑mesenchymal transition

Abstract

Metastasis is the most common cause of death in ovarian cancer patients but remains largely untreated. Epithelial‑mesenchymal transition (EMT) is critical for the conversion of early‑stage ovarian tumors into metastatic malignancies. Thus, investigating the signaling pathways promoting EMT may identify potential targets for the treatment of metastatic ovarian cancer. Lysine demethylase 2A (KDM2A), also known as FBXL11 and JHDM1A, is a histone H3 lysine 36 (H3K36) demethylase that regulates EMT and the metastasis of ovarian cancer. However, the function and underlying mechanisms of EMT suppression in ovarian cancer have not been thoroughly elucidated to date. In the present study, we used Gene Expression Omnibus (GEO) databases to determine that KDM2A is significantly upregulated in human ovarian cancers. KDM2A expression was assessed by immunohistochemistry of epithelial ovarian cancer (EOC) borderline ovarian tumors and normal ovary tissues. Seven fresh EOC tissues and 3 fresh normal ovary tissues were collected for western blot analysis. Kaplan‑Meier survival curves were constructed to identify genes related to EOC prognosis from the TCGA data portal. Stable KDM2A‑knockdown cell lines were established to study the biological functions and underlying mechanisms of KDM2A in EMT in vitro. GEO database analysis revealed that KDM2A was highly upregulated in EOC tissues; this analysis was accompanied by immunochemistry and western blot analysis using samples of human tissues. High expression of KDM2A was associated with poor survival in EOC patients. KDM2A knockdown promoted apoptosis and suppressed the proliferation, migration and invasion of tumor cells in vitro. EMT and the PI3K/AKT/mTOR signaling pathway were suppressed in KDM2A‑silenced cells. Inactivation of the PI3K/AKT/mTOR signaling pathway in A2780 cells induced EMT inhibition. Our data revealed that KDM2A functions as a tumor oncogene, and the downregulation of KDM2A expression regulates EMT and EOC progression, providing a valuable prognostic marker and potential target for the treatment of EOC patients.

Figure 1.

KDM2A expression in EOC tissues. (A) Differential genes obtained from the GSE14407 dataset were visualized using a (A) heatmap and (B) volcano plot. (C) Representative immunohistochemical staining of KDM2A in representative EOC tissues and normal ovarian epithelia. The inset is a zoomed image. Original magnification, ×200. Scale bars, 50 µm. (D) The overall survival curves of patients with KDM2A-low and -high indices. A significant difference was observed between groups (P=0.022). (E) The expression level of KDM2A was higher in 7 fresh EOC tissues compared with 3 fresh normal ovary tissues. EOC, epithelial ovarian cancer.

Figure 2.

Effects of KDM2A on EOC cell proliferation. (A and B) shKDM2A was introduced into SKOV3 and A2780 cells, and the silencing efficacy was confirmed by western blot analysis. Scale bar, 50 µm. (C) Cell viability was determined by CCK-8 assay after shKDM2A knockdown treatment at various time-points (24, 48 and 72 h) in SKOV3 and A2780 cells. (D) KDM2A knockdown reduced the ability of colony formation of SKOV3 and A2780 cells. Scale bar, 100 µm. (E and F) Cell cycle distribution analysis by flow cytometry. KDM2A arrested G2 progression delay. Data are presented as the mean ± SD of at least 3 independent experiments. *P<0.05, **P<0.01, ****P<0.0001, compared with the untreated controls (Control) and the negative controls (sh-Control). EOC, epithelial ovarian cancer.

Figure 3.

KDM2A silencing increases cell apoptosis. (A and B) Flow cytometric analysis of PE/7-ADD-stained A2780 and SKOV3 cells after transfection. (C) Bax protein was upregulated and Bcl-2 protein was downregulated after transfection. (D) Downregulation of KDM2A increased the apoptosis rate of the A2780 and SKOV3 cells as detected by Hoechst 33342 staining. Scale bar, 20 µm. **P<0.01, ****P<0.0001.

Figure 4.

Downregulation of KDM2A suppresses cell migration and invasion of the A2780 and SKOV3 cells. (A and B) The 3 cell groups were collected and assayed for their capabilities of invasion using a Transwell system. Cell invasion of the sh-KDM2A was significantly decreased compared to the 2 control groups. Scale bar, 50 µm. (C and D) The 3 cell groups were calculated for cell migration using the wound healing assay. Cells in the sh-KDM2A group had decelerated scratch wound healing ability. Scale bar, 50 µm. (E) Western blot analysis was used to assess the changes in MMP2 and MMP9 in the 3 groups. The levels of both proteins were decreased in the sh_KDM2A groups. *P<0.05, **P<0.01.

Figure 5.

KDM2A facilitates cancer cell EMT via the PI3K/AKT/mTOR pathway. (A) Expression of E-cadherin, N-cadherin and vimentin, EMT phenotype markers, in KDM2A-knockdown cells and the 2 control groups. (B) Activation of the PI3K/AKT/mTOR pathway in the 3 groups. (C) Expression of KDM2A, the EMT phenotype and proteins of the PI3K/AKT/mTOR pathway in A2780 cells were administered gradient concentrations of the PI3K inhibitor (LY294002, 0, 5, 10 and 20 µmol/l). (D) The migration ability of SKOV3 and A2780 cells gradually decreased with 10 µmol/l LY294002. Scale bar, 50 µm. (E) LY294002 inhibited the colony formation of SKOV3 and A2780 cells. Fewer colonies were formed in the treated group compared with the control group. EMT, epithelial-mesenchymal transition. Scale bar, 100 µm.