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.

cancer cells and TAM resistance has not been clarified. The Hippo signalling core kinases MST1/2 and downstream LATS1/2 phosphorylate YAP/TAZ, and promote their cytoplasmic sequestration and degradation. 13,14 Inhibition of MST1/2 and LATS1/2 by DLG5 silencing promotes the nuclear localization of YAP. 10 Furthermore, TAZ, a YAP paralogue, is crucial for BCSCs. 15,16 Accordingly, we have been suggested that DLG5 may regulate TAM sensitivity by changing TAZ expression and nuclear translocation to modulate the stemness of breast cancer cells.
In this study, we examined DLG5 expression in paired of TAMsensitive and resistant breast cancer tissues and employed TAM-sensitive and resistant ER + breast cancer cells to determine the effect of altered DLG5 expression on TAM sensitivity, apoptosis and stemness as well as TAZ expression. Furthermore, we tested the impact of TAZ silencing on the TAM sensitivity and stemness of TAM-resistant breast cancer cells. Our data indicated that down-regulated DLG5 expression increased the breast cancer stem cell-like characteristics by enhancing TAZ expression, contributing to TAM resistance in ER + breast cancer.

| Patients and tissue specimens
The study was conducted, according to the Code of Ethics of the World Medical Association. The experimental protocol was approved by the Ethics Review Committee of the First Affiliated Hospital of Xi'an Jiaotong University. A total of 9 metastatic breast cancer tissues from patients, who were resistant to TAM, and their matched primary tumours were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. 8

| Immunohistochemistry
The paraffin-embedded breast cancer tissue sections (4 μm) were deparaffinized, rehydrated and subjected to antigen retrieval in sodium citrate buffer. The sections were treated with 3% hydrogen peroxide to inactivate endogenous peroxidase and treated with 10% goat serum in TBST at 37°C for 30 minutes. After being washed, the sections were incubated with antibodies against DLG5 (1:200, Abcam) or TAZ (1:200, Cell Signaling Technology) at 4°C overnight. Subsequently, the bound antibodies were reacted with biotinylated goat anti-rabbit IgG (ZSGB-Bio) and horseradish peroxidase (HRP)-conjugated streptavidin. After being washed, the stained signals were visualized with diaminobenzidine and counterstained with hematoxylin.
The signals were photographed using a microscope slide scanner (Leica MP, SCN400). The intensity of antibody staining in individual fields was semi-quantitatively analysed, as described previously. 8

| Western blot
The different groups of cells were lysed by RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors (Roche).

| Cell viability assay
The different groups of cells were cultured in 96-well plates and treated in triplicate with 4-OHT (0, 2, 5 mol L −1 ) for 24, 48 and 72 hours. During the last 4 hours culture, individual wells of cells exposed to 5 mg/mL of MTT reagent. The generated formazan in individual wells was dissolved in 200 μL DMSO and the absorbance was measured at 570 nm in a microreader.

| Immunofluorescence
The different groups of cells were fixed with a 4% paraformaldehyde for 15 minutes at room temperature (RT) and permeabilized in 0.2% Triton X-100 in PBS for 10 minutes. After being blocked with 5% bovine serum albumin (BSA) and 10% horse sera in PBS for 1 hour at RT, the cells were incubated with antibodies against β-catenin and TAZ (Cell Signal Technology) at 4°C overnight. The bound antibodies were detected with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen) and co-stained with DAPI. Fluorescent signals were captured under a confocal laser scanning microscope (Leica SP5II).

| Flow cytometry analysis
The different groups of cells were harvested and stained with APC-anti-CD44 and PE-anti-CD24 (Biolegend). The ALDH + cells were characterized using the ALDEFLUOR kit (Stem Cell Technologies), according to the manufacturer's instruction. The cells were analysed by flow cytometry (BD FACS Canto II) and analysed by FlowJo software, as described previously. 17 In addition, the differ-   in ultralow attachment 6-well plates (Corning). The cells were exposed to half volume of fresh medium (500 μL) every 3 days. On day 10 after incubation, the formed mammospheres in individual wells were captured under a light microscope.

| Soft-agar assay
The clonogenicity of individual groups of cells was determined by soft-agar colony formation assay, as described previously. 17 Briefly,

| Statistical analysis
Data are expressed as the mean ± standard deviation (SD). The difference between groups was analysed by Student's t test using GraphPad Prism Version 7.00. A P-value of <0.05 was considered statistically significant.   Figure 4E). Collectively, such data demonstrated that DLG5 reduced the frequency of CD44 + /CD24 − BCSCs.

| DLG5 suppresses the breast cancer stem celllike characteristics to restore TAM sensitivity by inhibiting TAZ expression and nuclear translocation
Given that the Hippo transducer TAZ is required for sustained selfrenewal of BCSCs, 16

| DISCUSSION
TAM has been widely used for treatment of ER + breast cancers in the clinic. 20 However, TAM resistance is a huge challenge for clinical practice, and promotes breast cancer metastasis and death. 21,22 Our previous studies and those of others have shown that DLG5 acts as a tumour suppressor in breast cancer, and its expression is up-regulated in Luminal type, but not basal-like, breast cancer. 10,11 DLG5 is a primary target of progesterone receptor 23  (f) endocrine adaptation (just in a minority of patients) 26,27 In additional, increased stemness and cancer stem cell-like characteristics can contribute to drug resistance. 5