Simple SummaryBreast cancer is a global burden with the most severe subtype being triple negative breast cancer (TNBC). Despite advances in conventional therapies, treatment for TNBC is currently lacking. Interestingly, cholesterol has gained interest as a potential therapeutic target due to cancer cells’ increased reliance on this macromolecule. In this study we aimed to assess the effects of cholesterol depletion as a therapeutic target in TNBC. We show that treatment with the cholesterol-depletory agent 2-hydroxypropyl-β-cyclodextrin (HPβCD) impeded the growth of cancer cells and further led to cancer cell death, which could be attributed to an altered cellular cholesterol profile following treatment. Furthermore, mice xenograft studies indicated complete eradication of early-stage tumours with no relapse, followed by a remarkable reduction in intermediate- and late-stage tumours, respectively. We have also identified SFRP1 as a possible molecular target facilitating the therapeutic action of HPβCD. These findings consequently potentiate cholesterol depletion as a novel anticancer strategy to be pursued.AbstractCholesterol accumulation is documented in various malignancies including breast cancer. Consequently, depleting cholesterol in cancer cells can serve as a viable treatment strategy. We identified the potency of 2-hydroxypropyl-β-cyclodextrin (HPβCD), a cholesterol-depletor in vitro against two breast cancer cell lines: MCF-7 (Oestrogen-receptor positive, ER+) and MDA-MB-231 (Triple negative breast cancer (TNBC)). The results were then compared against two non-cancerous cell lines using cytotoxic-, apoptosis-, and cholesterol-based assays. Treatment with HPβCD showed preferential and significant cytotoxic potential in cancer cells, inducing apoptosis in both cancer cell lines (p< 0.001). This was mediated due to significant depletion of cholesterol (p< 0.001). We further tested HPβCD in a MF-1 mice (n= 14) xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late-, intermediate-, and early-stage TNBC, respectively. We also detected molecular-level perturbations in the expression patterns of several genes linked to breast cancer and cholesterol signalling pathways using RT2-PCR arrays and have identified SFRP1 as a direct binding partner to HPβCD through SPR drug interaction analysis. This work unravels mechanistic insights into HPβCD-induced cholesterol depletion, which leads to intrinsic apoptosis induction. Results from this study potentiate employing cholesterol depletion as a promising unconventional anticancer therapeutic strategy, which warrants future clinical investigations.