β-catenin blockers enhance the effect of CDK4/6 inhibitors on stemness and proliferation suppression in endocrine-resistant breast cancer cells

Wnt/beta-catenin signaling is involved in endocrine resistance and stem cell-like properties of hormone receptor-positive breast cancer cells. Palbociclib is a well-known inhibitor of cyclin-dependent kinase 4 and 6 (CDK4/6 inhibitor) that downregulates the activation of retinoblastoma protein, thereby inhibiting the cell cycle in breast cancer cells. The inhibitory effects of a combination of palbociclib and ICG-001, a beta-catenin small-molecule inhibitor, were investigated in tamoxifen-resistant breast cancer cell lines. Tamoxifen-resistant MCF-7 (TamR) cells were established by continuously exposing MCF-7 cells to tamoxifen. The characteristics associated with the stem cell-like property of cancer were assessed using western blotting, cell cycle analysis, and the mammosphere assay. The effects of the combination of palbociclib and ICG-001 were evaluated in control MCF-7 and TamR cell lines. Compared with control cells, TamR cells exhibited elevated levels of Nanog, Sox2, ALDH1, and p-STAT3, indicating stem cell-like characteristics, and elevated beta-catenin activity. TamR cells also showed significantly higher mammosphere-forming efficiency. Several markers of stem cell-like nature of TamR cells showed reduced levels upon treatment of cells with the drug combination; there was a greater reduction in the levels of these markers when the cells were treated with the combination than in the case where cells were treated with one of the drugs individually (combination index value for 25 mu M palbociclib and 50 mu M ICG-001 was 1.1 +/- 0.02). TamR cells treated with the palbociclib and ICG-001 combination demonstrated significantly reduced cell proliferation and mammosphere-forming efficiency compared with the cells treated with one of these drugs. The combination of the drugs could additively inhibit proliferation and suppress stem cell-like characteristics. These results suggest that beta-catenin plays a role in endocrine-resistant breast cancer; the inhibition of beta-catenin and CDK4/6 together can overcome endocrine resistance in breast cancer cells.