Development of CP506 a novel hypoxia-selective cytotoxin for the treatment of triple negative breast cancer (TNBC)

Abstract

Breast cancer is the most prevalent form of cancer and the leading cause of cancer death in women. Triple- Negative Breast Cancer (TNBC), which accounts for approximately 15-20 percent of breast cancers, has a much more aggressive clinical phenotype when compared with hormone receptor-positive breast tumours. Clinically, TNBC represents a highly relevant breast cancer subgroup and an area of intensive research in the cancer research field, with an urgent requirement for novel approaches to improve patient survival. Another important factor in the treatment of TNBC is the high prevalence of hypoxia associated with this disease which portends poor treatment outcome. However, hypoxia may represent an ideal investigative target for the newly developed CP- 506, a novel hypoxia-selective cytotoxin designed and developed at the Auckland Cancer Society Research Centre. In vitro assays were performed in this thesis to evaluate the mechanism of action, efficacy, and metabolism of CP-506. To validate CP-506 DNA damaging activity as a nitrogen mustard in TNBC cell lines compared with other established DNA damaging agents (Cisplatin and Chlorambucil), a reliable DNA damage marker (gH2AX) was selected and used forward in a flow cytometry assay. CP-506H and CP-506M were established both capable of inducing DNA damage in a dose-dependent manner. Then a series of antiproliferative IC50 assays were undertaken to investigate the antiproliferative efficacy of CP-506 metabolites (CP-506H and CP-506M) relative to cisplatin and chlorambucil. Results showed distinct cytotoxicity profile across the panel of TNBC cell lines, with a significant correlation between the IC50 values for CP-506H and cisplatin (R2 = 0.723, p = 0.029), suggesting a common mechanism of cytotoxicity and shared DNA damage repair pathway. IC50 assays conducted under oxic and anoxic conditions, showed on-mechanism activation of prodrug CP-506. Further, the TNBC lines were able to metabolise the HAP candidate CP-506 under anoxic but not aerobic conditions, indicating resistance to the aerobic reductases. Overall, CP-506 displayed hypoxic selectivity across all selected TNBC cell lines, within particular MDA-MB-468 (BRCA1 WT) and MDA-MB-436 (BRCA1 MUT) revealing high sensitivity to CP-506H/M metabolites and therefore showing that BRCA1 status per se does not necessarily determine the sensitivity of a TNBC cell line to DNA damaging agents.