Thesis title: Regulation and functions of nuclear Aurora-A and their dependence on TPX2
Aurora-A is a well-characterized serine-threonine kinase essential for cell division and frequently overexpressed in cancer (Nikonova et al., 2012; Lin et al., 2020; Du et al., 2021). While the mitotic functions of Aurora-A are well established, recent studies have uncovered non-mitotic, oncogenic roles during interphase, particularly in breast cancer, where a nuclear pool of Aurora-A contributes to transcriptional activation of oncogenes such as c-Myc and FOXM1, through kinase-independent mechanisms (Naso et al., 2021). Gaining insight into the mechanisms that regulate the nuclear pool of Aurora-A may therefore open new avenues for selectively targeting its oncogenic functions, which may be not tackled by canonical kinase inhibitors. However, the processes regulating Aurora-A nuclear accumulation remain largely unclear.
The best-characterized activator of Aurora-A is TPX2, a microtubule-associated protein that recruits Aurora-A to spindle microtubules, activates and protects it from degradation (Kufer et al., 2002; Bayliss et al., 2003; Gruss & Vernos, 2004; Giubettini et al., 2011; Levinson, 2018). Co-overexpression of Aurora-A and TPX2 has been reported in several cancer types, particularly breast cancer, suggesting that the Aurora-A/TPX2 complex may act as an oncogenic holoenzyme (Asteriti et al., 2010). Notably, TPX2 is localized in the nucleus during interphase.
The aim of my PhD project is to explore the regulation of nuclear oncogenic functions of Aurora-A, with a particular focus on (i) their dependence on TPX2 and (ii) the mechanisms underlying Aurora-A nucleo-cytoplasmic trafficking.
To this end, I used non-transformed hTERT-RPE-1 cell lines generated in the laboratory for inducible overexpression of Aurora-A alone or in combination with TPX2. I demonstrated that TPX2 co-overexpression significantly increases the proportion of interphase cells with nuclear Aurora-A, by protecting it from proteasome-mediated degradation. Furthermore, inhibition of the Aurora-A/TPX2 interaction using a protein–protein interaction (PPI) inhibitor generated in our laboratory leads to a marked reduction in nuclear Aurora-A in breast cancer cells, supporting the pivotal role of TPX2 in regulating Aurora-A nuclear accumulation.
To further investigate mechanisms regulating Aurora-A nuclear import, given that the kinase does no harbour a canonical NLS, I explored which protein domains are involved, by transfecting Aurora-A mutated/truncated versions in U2OS cells. Deletion of the C-terminus results in nuclear exclusion of the protein, suggesting this domain contributes to nuclear import. In parallel, I performed an interphase Aurora-A interactomic analysis in hTERT-RPE-1 cells overexpressing Aurora-A or Aurora-A/TPX2, which revealed interesting pathways to be further investigated. Importantly, data indicated interactions between Aurora-A and the nuclear import vectors importin-α3 and -α7, as well as with RANBP3, a Ran-binding protein that may mediate Aurora-A binding to importins, validated byin situ proximity ligation assays.
To assess the role of TPX2 in the reported kinase-independent oncogenic transcriptional functions of the nuclear pool of Aurora-A, I used MCF10A cells overexpressing Aurora-A alone or in combination with TPX2. TPX2 co-overexpression promotes Aurora-A-mediated formation and growth of MCF10A-derived mammospheres. Notably, disrupting the Aurora-A/TPX2 complex using PPI inhibitors impairs breast cancer mammosphere growth and viability. The obtained transcriptomic profiles of Aurora-A and Aurora-A/TPX2 overexpressing mammospheres reveal upregulation of autophagy and mitophagy pathways, supporting their involvement in mammosphere formation, and a downregulation of the p53 pathway, in line with recent findings from our lab (Naso et al., 2024).
Together, these findings highlight a central role for TPX2 in promoting the oncogenic functions of nuclear Aurora-A and identify specific importins and protein regions involved in Aurora-A nuclear import. These insights pave the way for the development of strategies specifically targeting the nuclear pool of Aurora-A in cancer.