Figure 7
Attenuation of tamoxifen-induced breast cancer proliferation and metastasis by targeting ALDH1A1 or ERα36. (A) Reduction of mammosphere formation by MCF7/ER36 cells in the presence of 4-OHT (1 μM) by ALDH1 inhibitors, diethylaminobenzaldehyde (DEAB, 10 nM) or disulfiram (DSF, 0.1 μM). DMSO was used as a vehicle control. Data were presented as mean ± SEM. Statistical significance was determined by two-tailed Student's t test. *P < 0.01. (B) Attenuation of the invasive ability of MDA-MB 436 cells after 4-OHT treatment (1 μM) in transwell assays by ALDH1 inhibitors DEAB and DSF. n = 3. (C) Reduction of tumor-initiating frequency of MCF-7/ERα36 cells treated with tamoxifen by DSF. Tumor-initiating frequency was analyzed with limiting dilution of tumor cell transplantation (seven mice each group). (D) Reduced number of lung metastases originated from orthotopical xenograft tumors formed by FACS-sorted 4T1-ERα36+ cells after DSF treatment (5 mg/kg body weight). Tamoxifen (1 mg/kg body weight), DSF or DMSO was intragastrically administered (ig) every 3 or 4 days for 18 days (arrows). Quantitation is presented as mean ± SEM. Statistical significance was determined by two-tailed Student's t test. (E) Growth inhibition of xenografted tumors in the presence of tamoxifen by a monoclonal anti-ERα36 antibody. Decreased tumor volume was observed after ERα36 antibody treatment. NOD/SCID mice (7/group) were orthotopically injected with human Bcap-37 cells (1 × 106). When tumor size reached 200 mm3, an anti-ERα36 monoclonal antibody (20 mg/kg body weight) was administered through the tail vein every 3 or 4 days. An irrelevant IgG was injected as a control. *P < 0.05. (F) Reduction of lung metastases formed by FACS-sorted 4T1-ERα36+ cells in the presence of tamoxifen by monoclonal anti-ERα36 antibody. Tamoxifen (1 mg/kg body weight) was intragastrically administrated after orthotopical injection of 4T1-ERα36+ cells. Anti-ERα36 monoclonal antibody (20 mg/kg body weight) or control IgG (20 mg/kg body weight) was iv administered through the tail vein every 3 or 4 days for 18 days. *P < 0.05. (G) IHC staining of decreased levels of ALDH1A1 in the lung metastatic lesion of breast cancer in nude mice treated with tamoxifen together with monoclonal ERα36 antibody. Hematoxylin was used for counterstaining. Scale bars, 50 μm. (H) Failure of anti-ERα36 monoclonal antibody to inhibit the growth of xenograft tumors formed by MCF-7/ERα36-Δ cells (with mutant of aa285-310 in ERα36). The sequence of ERα66 from aa 456 to 481 was used in the mutant ERα36. MCF-7/ERα36 cell-formed xenografts treated with anti-ERα36 antibody or IgG were used as controls. *P < 0.05.
