Efficacy of DNA Polymerase Inhibitors in Triple-Negative Breast Cancer Cells

Abstract

Introduction: Breast cancer is the most common malignancy among women and the second leading cause of cancer-related mortality globally, posing a significant public health burden. Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with high associated metastasis and an overall poor prognosis. Due to the lack of long-established scarcity of specific effective therapies, TNBC is usually treated using conventional chemotherapy leading to drug resistance and relapse pressing the urge to develop novel and effective therapeutic strategies. DNA polymerase 1 and histone deacetylase 1, play a key role in cell proliferation, DNA synthesis, and replication, and regulation of epigenetic mechanisms, are significantly highly expressed in breast tumors compared to normal breast tissues. Therefore, these two enzymes present potential targets for drugs that block DNA synthesis suppressing tumor growth. We have shown that the adamantyl retinoid, ST1926, a potent cancer therapeutic compound, to be a POLA1 inhibitor. Sub-micromolar concentrations of ST1926 suppress the proliferation and induce cell death in human breast cancer cells while sparing normal ones. We have synthesized two ST1926 analogues, MIR002 and GEM144, with improved pharmacological properties and dual POLA1 and HDAC11 inhibitors activities forming a novel class of inhibitory compounds. In this study, we accessed the anti-tumor effects of ST1926, MIR002 and GEM144 and investigated cell death mechanism, and their mode of action on a panel of human TNBC cell lines and normal-like breast cells.

Methods: Basal POLA1 and HDAC11 levels were measured by immunoblotting technique in different types of human TNBC cell lines (HCC1806, MDA-MB-453, and MDA-MB-231) and normal-like MCF10A breast cells. The selected cell lines have lost or mutant p53 and represent different TNBC subtypes. The mechanism of action of the different treatments was studied on cell growth by sulforhodamine B (SRB). Cell cycle arrest was determined by using propidium iodide-based flow cytometric analysis of DNA content. DNA damage was assessed by immunoblotting techniques, and apoptotic cell death using the TUNEL assay and PARP cleavage.

Results: ST1926, MIR002, and GEM144 suppressed the growth of all tested TNBC cell lines at low micromolar concentrations while sparing normal-like breast cells as shown by SRB. The different treatments induced an early effect on HCC1806 and a more delayed one on MDA-MB-453 cells. Cell cycle analysis showed that ST1929, MIR002 and GEM144 induced cell death and a massive accumulation of treated cells in the sub-G1 phase. DNA damage and double strand break was detected by the increase in γ-H2AX in GEM144-treated TNBC cells. Apoptosis was confirmed by TUNEL positivity and PARP cleavage in HCC1806 suggesting a caspase-dependent cell death. On the other hand, apoptotic cell death identified by the TUNEL positivity in GEM144-treated MDA-MB-456 cells did not show PARP cleavage suggesting a caspase-independent mode cell death. Finally, the protein levels of POLA1 and HDAC11 were reduced in GEM144 treated TNBC cells.

Conclusion: These results demonstrate the effectiveness of ST1926, MIR002, and GEM144 in inhibiting TNBC cell growth and inducing apoptosis independently of p53 signaling pathways. This research may shed the light on a new potential therapeutic approach for TNBC management. Future in vivo studies using the intra-ductal breast cancer mouse model may be used to further validate this new therapeutic approach.