DLG5 suppresses breast cancer stem cell-like characteristics to restore tamoxifen sensitivity by inhibiting TAZ expression

Abstract

Tamoxifen (TAM) is a primary drug for treatment of estrogen receptor positive breast cancer. However, TAM resistance remains a serious threat to breast cancer patients and may be attributed to increased stemness of breast cancer. Here, we show that discs large homolog 5 (DLG5) expression is down-regulated in TAM-resistant breast cancer and cells. DLG5 silencing decreased the sensitivity to TAM and increased the frequency and stemness of CD44⁺ /CD24^- breast cancer stem cells (BCSCs) and TAZ, a transducer of the Hippo pathway, expression in MCF7 cells while DLG5 overexpression had opposite effects. TAZ silencing restored the sensitivity to TAM and reduced the frequency and stemness in TAM-resistant breast cancer cells. Taken together, our data indicate that down-regulated DLG5 expression increases the stemness of breast cancer cells by enhancing TAZ expression, contributing to TAM resistance in breast cancer.

Keywords: DLG5; TAZ; stemness; tamoxifen resistance.

Figure 1

Down‐regulated DLG5 expression is associated with the TAM resistance in breast cancer. (A) The levels of DLG5 expression were analysed by a gene expression profile in the GEO database (GSE26459); (B, C) Immunohistochemistry analysis of DLG5 protein expression in nine paired of adjacent non‐tumour breast, TAM‐sensitive and resistant breast cancer tissues. (D, E) Western blot and qRT‐PCR analysis of DLG5 expression in MCF7, MCF‐TamR and LCC2 cells; Data are representative images (magnification ×200 upper panels, 400 bottom panels) or expressed as the mean ± SD of each group from three separate experiments. **P < 0.01, ***P < 0.001

Figure 2

Altered DLG5 expression changes the sensitivity of breast cancer cells to TAM in vitro. MCF7 and LCC2 cells were transduced with lentivirus for DLG‐specific shRNA and DLG5 overexpression, respectively and the levels of DLG5 expression were determined by Western blot (A, C). The sensitivity of different groups of cells to 2.5 or 5.0 μmol L⁻¹ 4‐OHT was determined longitudinally by MTT (B, D). Subsequently, the cells were treated with vehicle or 5.0 μmol L⁻¹ 4‐OHT for 48 hours and the percentages of apoptotic cells were determined by flow cytometry following PE‐Annexin V and 7‐AAD staining (E‐H). Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 3

DLG5 reduces the frequency of CD44⁺/CD24⁻ BCSCs in breast cancer cells. The percentages of CD44⁺/CD24⁻ BCSCs or ALDH ⁺ cells in MCF7, MCF7‐shDLG5, LCC2 and LCC2‐oxDLG5 cells were determined by flow cytometry. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. (A, B) The frequency of CD44⁺/CD24⁻ BCSCs in MCF7 cells. (C, D) The frequency of CD44⁺/CD24⁻ BCSCs in LCC2 cells. (E, F) The frequency of ALDH ⁺ cells in MCF7 cells. (G, H) The frequency of ALDH ⁺ cells in LCC2 cells. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 4

DLG5 reduces the breast cancer cell stemness in vitro. The formation of mammospheres and clones of MCF7, MCF7‐shDLG5, LCC2 and LCC2‐DLG5 cells were determined by mammosphere formation and soft‐agarose colony formation assays, respectively. The relative levels of DLG5, Oct4 and c‐MYC to GAPDH were determined by Western blot. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. (A, B) The mammosphere formation. Scale bar, 50 μm. (C, D) The colony formation. (E) Western blotting analysis of DLG5, OCT4 and c‐MYC expression in the different groups of cells

Figure 5

Down‐regulated DLG5 expression promotes TAZ expression and nuclear localization in breast cancer cells. (A) Analysis of TAZ expression in the GEO database (GSE26459). (B) Immunohistochemistry analysis of TAZ expression in TAM‐sensitive and resistant breast cancer tissues. Scale bar, 50 μm (C) Western blot analysis of TAZ expression and phosphorylation in the indicated cells. (D) Western blot analysis of TAZ expression in the cytoplasm and nuclei of breast cancer cells. (E, F) Immunofluorescent analysis of TAZ protein distribution in breast cancer cells. Scale bar, 25 μm. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. **P < 0.01

Figure 6

TAZ silencing restores TAM sensitivity in TAM‐resistant breast cancer cells by inhibiting the breast cancer stemness. TAM‐resistant MCF7‐shDLG5 and LCC2 cells were transfected with control scramble or TAZ‐specific siRNA. The relative levels of TAZ expression were determined by Western blot (A). The sensitivity of different groups of cells to 5 mol L⁻¹ 4‐OHT was analysed by MTT (B). The frequency of CD44⁺/CD24⁻ BCSCs was determined by flow cytometry (C‐E). The formation of mammospheres was examined by mammosphere formation assays (F‐H). Scale bar, 50 μm. Data are representative images or expressed as the mean ± SD of each group from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001