Mechanism of Action of DNA Polymerase Inhibitors in Triple-Negative Breast Cancer Cells and Anti-Tumor Effects in Intraductal Breast Cancer Mouse Model

Abstract

Introduction: Breast cancer is the second leading cause of cancer-related fatalities globally and the most frequent malignancy among women. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and is associated with high metastasis and poor prognosis. Due to the absence of specific TNBC targeted therapies, this breast cancer type is usually treated using conventional chemotherapy. However, TNBC therapy is commonly associated with drug resistance, a high relapse incidence, and pronounced side effects, emphasizing the urgency of developing novel, effective, and targeted therapeutic strategies. DNA polymerase 1 (POLA1) and histone deacetylase 11 (HDAC11) play key roles in cell proliferation, DNA synthesis and replication, and regulation of epigenetic mechanisms. POLA1 and HDAC11 levels are commonly elevated in human breast tumors as opposed to normal breast tissues. Therefore, POLA1 and HDAC11 present potential targets for drugs in breast cancer therapy. We have previously shown that the adamantyl retinoid ST1926 is a POLA1 inhibitor in the sub-µM range, impeding proliferation and inducing cell death in human TNBC cells while sparing normal ones. Additionally, we have synthesized two ST1926 analogues, MIR002 and GEM144, which demonstrated dual inhibitory activities against POLA1 and HDAC11. The three compounds showed potent anti-tumor activities in TNBC cells but GEM144 has improved pharmacological properties. Therefore, we selected GEM144 to further investigate its mechanism of action in TNBC cell lines. Methods: POLA1 and HDAC11 levels were measured by immunoblotting techniques in different types of human TNBC cell lines and a normal-like breast MCF10A cell line. In silico analysis was performed to determine the transcript levels of POLA1 and HDAC11 levels in human TNBC versus normal breast tissues and their effects on patient overall and disease-free survival. The mechanism of action of GEM144 was studied on cell viability and cell death using the trypan blue assay, DNA damage by immunoblotting techniques, and apoptosis by TUNEL assay on human TNBC cell lines. The breast intraductal mouse model, in female NSG mice, was selected to be used as the most suitable orthotopic mouse model for breast cancer. Results: GEM144 reduced viability and increased cell death in TNBC cell lines at low micromolar (µM) concentrations while sparing normal-like breast cells. The basal-like HCC1806 cells and the luminal androgen receptor (apocrine) MDA-MD-453 cells had IC50 values of 0.5 and 1.0 µM, respectively. DNA damage was detected by the increase in γH2AX and apoptosis was confirmed by TUNEL positivity and PARP cleavage in GEM144-treated TNBC cells. In silico analysis did not indicate statistically significant differences in the transcript levels of POLA1 and HDAC11 in human TNBC versus normal breast tissues nor in patient overall and disease-free survival. However, basal protein levels of POLA1 and HDAC11 were elevated in several TNBC cell lines versus normal-like breast cells. Importantly, we successfully established the breast intraductal mouse model at the American University of Beirut and we were able to track and quantitate tumor growth in vivo using the IVIS system. Conclusion: These results highlight the effectiveness of GEM144 in TNBC management. Future in vivo experiments, using the breast intraductal mouse model, will assess the effectiveness of GEM144 versus cisplatin standard chemotherapy in TNBC treatment.