Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) blocker, Ivabradine, a novel potential targeted treatment for triple-negative breast cancer.

Abstract

Background: Triple-negative breast cancer patients who cannot benefit from targeted therapies, receive chemotherapeutic agents with attendant side effects. Ivabradine is an FDA approved Hyperpolarization-activated cyclic nucleotide-gated channels (HCN 1–4) targeting therapeutic agent, used clinically to treat chronic angina. We found HCN2 and HCN3 overexpressed in breast cancer compared with low expression in normal breast, both from TCGA data, cell line samples and clinical tissue samples. Treatment with Ivabradine significantly suppressed proliferation of breast cancer cells. We hypothesized that Ivabradine may be used to preferentially target breast cancer. Materials and Methods: In-vitro functional assays using breast cancer cell-lines were used to characterize the mechanism of action of HCN channel inhibition on breast cancer cells. In-vivo mouse models using both breast cancer xenograft and patient derived tumor xenograft (PDTX) were also used to demonstrate the efficacy of Ivabradine treatment. Results: Subcellular fractionation experiments confirmed HCN2 and HCN3 were largely localized to the cell membrane. MCF-10A, MDA-MB-231, MDA-MB-453 and BT-474 cell-lines were treated with 200 μM Ivabradine, 40 nM Paclitaxel or 625 nM Doxorubicin. All 3 drugs caused reduced cell viability and proliferation, and induced apoptosis of breast cancer cells. In contrast, Ivabradine did not significantly alter cell viability, proliferation nor apoptosis of the normal breast epithelial cell line MCF-10A. Mechanistically, we found that Ivabradine treatment could deplete intracellular calcium ion concentration, induce ER stress, caspase-mediated apoptosis, autophagy and cell senescence. Breast cancer xenografts MDA-MB-231 and MDA-MB-453 treated with Ivabradine showed decreased tumor growth compared with control group. Knockdown of HCN2 and HCN3 showed similar results as that with Ivabradine treatment in vitro as well as for in vivo xenografts. Ivabradine treatment also effectively reduced tumor growth in 6 out of 8 PDTX models established so far of triple negative breast cancer, with no discernable side effects on the treated mice. Conclusions: Targeting HCN channels using Ivabradine can be an effective novel targeted therapy for triple negative breast cancer. Being a clinically approved drug, it can more readily pave the way for clinical trials. There is also potential to test the application of antibodies against HCN2 and HCN3 on PDTX and develop humanized antibodies for use as anti-cancer drugs.