Investigating The Role of a Signature of Active DUBs in Modulating the Metastatic and Tumourigenic Behaviour of Triple-Negative Breast Cancer

Abstract

Triple-Negative breast cancer (TNBC) is still ranked as the top most difficult breast cancer type due its highest tendency to spread, severely impacting patients. The metastatic steps of TNBC are still not fully understood, requiring extensive effort to delineate the biological pathways regulating its tumourigenesis and metastasis. Our lab proposes the role of the ubiquitin system, particularly the deubiquitinating enzymes (DUBs) as potential TNBC targets. DUBs are versatile enzymes known for their catalytic activity in reversing ubiquitination events, regulating almost all molecular pathways. Their deregulation, however, is increasingly linked to cancer. In this study, I have identified active candidate DUBs using a data mining approach followed by experimental validation using HA-Ahx-Ahx-Ub-VME activity-based probe assays in TNBC cell lines. My analysis identified ATXN3, UCHL1 and UCHL3 as active DUBs in TNBC cell lines. Here, I studied candidate DUBs in TNBC using a combination of in vitro assays and in vivo using a humanised animal model of TNBC, zebrafish xenograft model (ZTX). To this end, ATXN3-/- MDA-MB-231 cell line or PR-619 (a pan DUB inhibitor and a strong modulator of ATXN3) were tested on TNBC tumourigenicity using clonogenic and cell viability assays. A significant reduction was observed in colony count and cell viability. Furthermore, Both ATXN3-/- or PR-619 did not show any effect on tumour cell intensity or tumour size at the Yolk sac but showed a significant reduction in cell dissemination at 3hr, 24hrs and 48hrs post-injection. PR-619 showed a reduction of cell dissemination at only 3hrs post-injection. As there are no selective inhibitors designed to target ATXN3, I have identified few significant hits against ATXN3. In this study I have also examined the role of UCHL1 and UCHL3 on cancer cell clonogenicity and viability, and I have observed no changes. Overall, ATXN3 might be a promising target in TNBC. More validations are needed to confirm my data in mice xenograft models.