Increased Antiproliferative Activity of Antiestrogens and Neratinib Treatment by Calcitriol in HER2-Positive Breast Cancer Cells

HER2-positive breast cancer is an aggressive subtype, often associated with shorter progression-free and overall survival. Estrogen receptor (ER) expression within this subtype leads to distinct growth patterns and treatment responses. Calcitriol, the active form of vitamin D, induces ERα expression in ER-negative breast cancer cells, thereby sensitizing them to the antiproliferative effects of antiestrogens. When combined with neratinib, calcitriol enhanced cell growth inhibition. Therefore, we investigated whether adding calcitriol to the combined treatment with antiestrogens and neratinib could further inhibit the proliferation of HER2-positive breast cancer cells, regardless of their ER status. The BT-474 (ER-positive/HER2-positive) and SK-BR-3 (ER-negative/HER2-positive) breast cancer cell lines were pretreated with calcitriol to modulate ER expression, followed by treatment with calcitriol in combination with neratinib, with or without antiestrogens. Proliferation assays, cell cycle analysis, and Western blotting were then performed to assess treatment effects. The results demonstrated that calcitriol and neratinib, per se, significantly inhibited cell proliferation in a concentration-dependent manner in the HER2-positive cell lines. Notably, calcitriol enhanced the antiproliferative response of combined neratinib and antiestrogen treatment. Calcitriol, alone or in combination, modulated vitamin D receptor and ERα expression, reduced AKT and ERK phosphorylation, and promoted G1 phase arrest. These findings support the potential of this combinatorial approach as a therapeutic strategy for HER2-positive breast cancer.