Androgen receptor increases CD133 expression and progenitor-like population that associate with cisplatin resistance in endometrial cancer cell line

Abstract

Endometrial cancer (EMC) is a sex steroid hormone-related female malignancy. Androgen and androgen receptor (androgen/AR) signals have been implicated in EMC progression. Cancer stem/progenitor cells (CSPCs) are suspected to link to chemoresistance in patients with EMC. In this study, we examined the androgen/AR roles in cisplatin resistance and CSPC population. We found AR expression increased naive EMC side population, CSPC population, cell migration, and epithelial-mesenchymal transition. Meanwhile, it decreased cisplatin cytotoxic effect on EMC cells. Collaterally, endogenous AR expressions in EMC cells were upregulated in the cisplatin-resisting state. Moreover, AR expression could further enhance CD133 expression, CSPC-related markers, and drug-resistance gene messenger RNA expression in EMC cells. Finally, the AR-associated gene expression might go through indirect regulation. This is the first report revealing AR function on EMC cells' CSPC and cisplatin resistance.

Keywords: AR; cancer stem/progenitor cells; cisplatin resistance; endometrial cancer; side population cell.

Figure 1.

The AR expressing Ishikawa cells and AR increases the S/P cells. A, Stable transfectant of AR complementary DNA (cDNA) in Ishikawa cells was confirmed by Western blot assay. par: Ishi-par (vector transfectant of Ishikawa cells); AR: Ishi-AR (AR expressing transfectant of Ishikawa cells). B, AR transactivation function measured by androgen response element (ARE)-driven luciferase reporter activity. The Ishi-par and Ishi-AR cells were treated with 10 nmol/L 5α-dihydrotestosterone (DHT) for 18 hours after cotransfecting with ARE-luciferase/pRL-TK plasmids. The readings of Ishi-AR were compared to Ishi-par cells and presented as fold changes. C, Hochest incorporation assay by Flow cytometry. The verapamil cotreatment were subjected and compared with the readings of Hochest treatments only to illustrate S/P. Representative images were presented, and higher S/P was noticed in Ishi-AR than in Ishi-par. D, Quantitative results of (C). All the experiments were calculated from 3 independent experiments. AR indicates androgen receptor; S/P, side population.

Figure 2.

Androgen/AR enhances basal levels of CD133 expression and population. A, Cell surface protein CD133 expression was measured in Ishi-par and Ishi-AR cells using flow cytometry assay. The par-CD133 and AR-CD133 represented CD133-PE-conjugated antibody staining of cells. The dashed lines (green dash line: par-CD133 and orange dash line: AR-CD133) indicated the mean CD133 fluorescence intensities. B, CD133+ populations in Ishi-par and Ishi-AR cells. The overlapping of isotype IgG-PE histogram graphs in (A) was excluded and the residual areas were counted as CD133+ populations. The data were the mean values from 3 independent experiments. AR indicates androgen receptor; IgG-PE, immunoglobulin G-phycoerythrin.

Figure 3.

The AR has little effect on cell growth, yet promotes cell migration and EMT. A, The WST-1 assay for Ishi-par and Ishi-AR cells. The OD 590 nm absorbance readings on 0, 2, 4, and 6 days were recorded and plotted on the graph. B, Cell counting assay for Ishi-par and Ishi-AR cells. The cell numbers on 0, 2, 4, and 6 days were recorded and plotted on the graph. The mean values were from 3 independent experiments. C, The AR promotes cell migration in the cisplatin-resistance EMC cells. The Ishi-par and Ishi-AR cells were treated with cisplatin for 14 days and then measured using wound-healing assay. Wound closure area (μm²) was measured as described in the Methods section. D, The AR promotes EMT in the cisplatin-resistant EMC cells. The Ishi-par and Ishi-AR cells were treated with cisplatin for 14 days and then measured using EMT markers (E-cadherin [E-Cad] and vimentin) expressed by immunoblot assay. The expression of AR was also measured to confirm overexpression of Ishi-AR, while actin expressions were served as loading controls. AR indicates androgen receptor; EMC, endometrial cancer; EMT, epithelial–mesenchymal transition; OD, optical density.

Figure 4.

The AR enhances CSPC in cisplatin-resistant Ishikawa cells. A, The AR mRNA expressions in naive and 7-day cisplatin-treated Ishi-par and Ishi-AR cells. The mRNA expressions were examined by AR-specific primers. β-Actin expressions served as the loading control. The values were calculated by comparing naive Ishi-par cells’ AR expression and presented as fold changes. B, Cell surface CD133 expressions in cisplatin-resistant Ishi-par and Ishi-AR cells were measured using flow cytometry assay. The par-iso and AR-iso represented the IgG-PE-conjugated isotype antibody stainings of the cells. The par-CD133 and AR-CD133 represented CD133-PE-conjugated antibody staining of cells. The dashed lines (orange dash line: par-CD133 and green dash line: AR-CD133) indicated the mean CD133 fluorescence intensity. C, Table shows AR reduces cisplatin cytotoxicity and CSPC characteristics. The naive state Ishi-par and Ishi-AR cells’ IC₅₀ against cisplatin were tested. The cisplatin-resistant state (14 days treatment), Ishi-par and Ishi-AR cells IC₅₀, and CD133 population were also measured. AR indicates androgen receptor; CSPC, cancer stem/progenitor cell; mRNA, messenger RNA.

Figure 5.

The mRNA expression of CSPC marker genes. A, CSPC-related transcription factors SCF, Bmi-1, Nanog, Oct-4, and Notch mRNAs were measured. The expression levels were calculated by comparing Ishi-AR to Ishi-par cisplatin-resistant cells and presented in fold changes. B, CSPC-related markers CD24, CD133, CD44, ABCG2, CD117, and Nestin mRNAs were measured. C, The AR enhances cisplatin-resistant genes MDR1, ABCB5, and ABCG2 mRNA expressions in endometrial cancer (EMC) cells. The expression levels were calculated by comparing Ishi-AR to Ishi-par cisplatin-resistant cells and presented in fold changes. The mean values were from 3 independent experiments. AR indicates androgen receptor; CSPC, cancer stem/progenitor cell; mRNA, messenger RNA.