Enrichment of ovarian cancer stem-like cells is associated with epithelial to mesenchymal transition through an miRNA-activated AKT pathway

Abstract

Objectives: Evidence has indicated that ovarian epithelial cancer-type cells under serum-free culture conditions can form spheroid cells and exhibit characteristics expected of cancer stem-like cells (CSCs). However, the mechanism by which differentiated ovarian cancer cells acquire stem-cell properties during CSC enrichment has needed to be elucidated. Recent studies have demonstrated that induction of epithelial to mesenchymal transition (EMT) can generate CSCs and be associated with tumour aggressiveness and metastasis.

Materials and methods: Ovarian epithelial cancer cell lines, SKOV3 and HO8920, were cultured for spheroid cells and adherent cells. CSC enrichment was investigated using MTT assay, flow cytometery and qRT-PCR and expression level of PI3K/AKT pathway components was analysed by western blotting.

Results: Compared to adherent cells, the spheroid cells expressed mesenchymal markers highly and exhibited significantly more motility; we also observed increases in phosphate AKT1 levels in the spheroid cells. Moreover, transfection of miR-20a or miR-200c led to corresponding reduction in endogenous PTEN protein, while AKT1 and phosphate AKT1 levels were upregulated in miRNAs-transfected cells. Finally, PI3K/AKT pathway inhibitor LY294002 reduced expressions of mesenchymal markers and stem-cell gene activity in spheroid cells, enhancing sensitivity of spheroid cells to paclitaxel treatment.

Conclusions: Our findings demonstrate that EMT contributed to enrichment of ovarian CSCs in vitro, making EMT targeting in epithelial ovarian cancer a novel therapeutic option.

Figure 1

Ovarian epithelial cancer spheroid cells have the properties of CSCs. (a): I) SKOV3 and III) HO8910 cells grown as adherent cells in McCoy's 5A media supplemented with 10% FBS. II and IV) These cell lines also formed spheroid cells, as seen suspended in the dishes coated by 1.2% poly‐Hema and containing serum‐free medium for 7 days. V and VI) ABCG2, Oct4 and Nanog were detected by qRT‐PCR using 18S rRNA as a reference gene (*P < 0.05). (b) Cell cycle distribution analysis of SKOV3 and HO8910 cells in different culture systems was performed by flow cytometry. The percentages of cells in the G0/G1 phase were 79.5% (SKOV3) and 70.2% (HO8910) of spheroid cells, and 68.3% (SKOV3) and 58.9% (HO8910) of adherent cells. (c) The spheroid cells were more drug‐resistant compared with the adherent cells after 48 and 72 h of treatment with paclitaxel (20 μm). (d) Spheroid cells form more and larger colonies than adherent cells in soft agar. I) Representative images of the colonies derived from the adherent and spheroid cells. II) Corresponding quantitative data depicting the colony number/field (**P < 0.01).

Figure 2

The expression of mesenchymal markers increased in ovarian epithelial cancer spheroid cells. (a) As shown by qRT‐PCR, SKOV3 or HO8910 spheroid cells, under stem cell‐selective conditions, exhibited higher expression of mesenchymal markers compared with SKOV3 or HO8910‐adherent cells under differentiating condition (*P < 0.05). (b) Representative immunofluorescent staining of Twist and vimentin in SKOV3 or HO8910 cells. Nuclei were stained with DAPI.

Figure 3

The ovarian spheroid cells exhibit greater cell motility. (a, b) The invasion and migratory abilities of the adherent and spheroid cells were both analysed by transwell assay. The spheroid cells showed greater invasion and migratory capabilities compared with the adherent cells. (a): I) Representative images of invading adherent and spheroid cells. Corresponding quantitative data are shown in II. (b): I) Representative images of migrating adherent and spheroid cells. Corresponding quantitative data are shown in II. Corresponding quantitative data depicting the cell number/field (**P < 0.01).

Figure 4

The AKT pathway was activated by miRNA‐mediated PTEN gene downregulation. (a) PTEN, AKT1 and phospho‐AKT1 expression in adherent and spheroid cells of SKOV3 and HO8910 lines. Phospho‐AKT1 was activated in spheroid cells and PTEN was downregulated. (b) Sequence matching between PTEN 3′‐UTR and miRNA was bioinformatically predicted. Pre‐miR‐20a and pre‐miR‐200c were amplified and inserted into the expression vector. pSilencer 4.1 cmv puro by BamHI and HindΙΙΙ. (c) SKOV3 and HO8910 cells were transfected with a miR‐20a or miR‐200c expression vector. PTEN mRNA expression was downregulated by miR‐20a and miR‐200c. (d) PTEN, AKT1 and phospho‐AKT1 expression were analysed in SKOV3 cells that were transfected with a miR‐20a or miR‐200c expression vector. Both AKT1 and phospho‐AKT1 were upregulated in miR‐20a‐ and miR‐200c‐transfected cells, while PTEN was downregulated (*P < 0.05, **P < 0.01).

Figure 5

PI3K inhibitor LY294002 blocked EMT during ovarian epithelial cancer stem‐like cells enrichment through the AKT pathway. (a) AKT1 and phospho‐AKT1 expression in SKOV3 and HO8910 spheroid cells were blocked due to treatment with PI3K inhibitor LY294002. (b) Results from qRT‐PCR showed that the expression of mesenchymal markers was significantly lower (*P < 0.05) and the expression of epithelial markers CK19 or E‐cadherin changed little in spheroid cells treated with PI3K inhibitor LY294002. (c) LY294002 treatment decreased the mRNA expression of stem‐cell markers, including Oct4, Nanog and ABCG2 in spheroid cells. (d) Cell cycle distribution analysis of SKOV3 and HO8910 spheroid cells treated with or without LY294002 was performed by flow cytometry. The percentage spheroid cells in the G0/G1 phase declined after treatment with LY294002 (from 76.8% to 63.9% of SKOV3 cells or from 71.3% to 64.9% of HO8910 cells). (e) PI3K inhibitor LY294002 enhanced the sensitivity of spheroid cells to paclitaxel. The SKOV3 or HO8910 spheroid cells were treated with LY294002 and paclitaxel as indicated. The cell survival rate was determined by MTT assay. (f) Spheroid cells without LY294002 treatment form more and larger colonies compared with LY294002 treated in soft agar. I) Representative images of the colonies derived from the spheroid cells with or without LY294002 treatment. II) Corresponding quantitative data depicting the colony number/field (*P < 0.05).