Abstract
Triple Negative Breast Cancer (TNBC) represents 10-15% of all breast cancers, is characterised by its lack of hormone and HER2 receptor expression and exist as clinically high grade and aggressive tumours with poor prognosis. A major challenge of these tumours is the lack of effective targeted therapies and the resistance to conventional therapies. Homeobox (HOX) genes are a family of homeodomain-containing transcription factors, which are primarily known for establishing and maintaining the identity and fate of cells and tissues during normal embryogenesis and organ development. Recent studies have highlighted HOX genes to be dysregulated in most cancers including breast, ovarian, prostate, colorectal and melanoma, and possess an established role in driving key hallmarks in carcinogenesis, including the regulation of the cell cycle, apoptosis, angiogenesis, and metastasis. In this study, the HOX expression profiles between TNBC and all other breast cancer subtypes was evaluated relative to normal HOX expression and showed TNBC tumours possess unique and specific HOX expression profiles and were indicative of prognosis. Given their cancer-specific expression, targeting HOX proteins for cancer therapy would potentially provide a novel therapeutic option, as yet unexploited. Rather than targeting individual HOX genes, which is ineffective due to the functional redundancy of HOX genes, targeting multiple HOX proteins post-translationally becomes feasible by inhibiting the interaction between HOX and their PBX co-factors by disrupting protein-protein interactions.
In this study, we evaluated the anti-tumour efficacy of a novel inhibitor of HOX protein function, HTL-001, which antagonizes the interactions between HOX and PBX proteins, thus preventing the binding of this complex to DNA to promote tumour growth. HTL-001 is a synthetic peptide comprised of a hexapeptide sequence, which resembles the interaction site between HOX proteins of paralogs 1-9 and PBX proteins. Targeting HOX/PBX dimers with HTL-001, induced apoptotic cell death in all breast cancer cell lines including MCF-7, ZR-75-1, MDA-MB-231, BT-20 and SK-BR-3, with the highest sensitivity seen in brain seeking MDA-MB-231-BR cells. HTL-001 also showed high synergistic anti-cancer effects in TNBC cell lines when combined with conventional chemotherapies and significantly translated these effects in vivo with significant inhibition of tumour growth by HTL-001 alone and in combination with Paclitaxel. The mechanism of action of HTL-001 was explored and the downstream effects on apoptosis was examined in detail. Disruption of HOX-PBX dimers by HTL-001 resulted in the upregulated expression of key apoptotic and anti-survival proteins that form an interconnecting network of signalling pathways initiated by ROS mediated ER stress which causes calcium influx and calpain activation to regulate caspase-independent activation of AIF, and release of immunogenic DAMPs. Mechanisms of synergy were evaluated to additionally show the main mechanism of synergistic apoptosis results from activating HTL-001 mediated signalling pathways and enhancing these by a chemotherapeutic agent. Resistance to HTL-001 was found to be mediated by autophagy and can be overcome by autophagy inhibitors. HTL-001 is currently the only therapy which targets HOX gene dysregulation and in doing so, may help address the current urgent unmet therapeutic need in TNBC through its unique mechanism of action and high levels of synergy with conventional anti-cancer agents.