“Centrosome Amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation”

Centrosome amplification (CA), a prominent feature of human cancers linked to genomic instability and tumourigenesis in vivo, is observed as early as pre-malignant metaplasia, increasing with progression from dysplasia to neoplasia. However, the mechanistic contributions of CA to tumourigenesis are not fully understood. Using non-tumourigenic breast cells (MCF10A), we demonstrate that induction of CA (by CDK1 inhibition or PLK4 overexpression) increased both the migration and invasion of non-tumourigenic cells. Mechanistically, we found small GTPase Rap-1 was activated upon CA induction. Rap-1 inhibition (using GGTI-298) blocked CA-induced migration, invasion and ECM attachment, demonstrating the role of Rap-1 in CA induced tumourigenesis. Induction of CA in a long-term cell culture system disrupted epithelial cell-cell junction integrity, via dysregulation of expression and subcellular localisation of cell junction proteins (ZO-1, Occludin, JAM-A & β-catenin). Physically, CA inhibited apical junctional complex formation, visualised by transmission electron microscopy. Furthermore, CA induction in non-tumourigenic cells elevated β-integrin 3 expression, potentially mediating increased cell attachment to the extracellular matrix (ECM) induced by CA. Simultaneously, CA induced elevated expression of matrix metalloprotease MMP1 and MMP13 facilitating ECM degradation and cell invasion. In vivo validation in a Chicken Embryo xenograft model, showed CA+ MCF10A cells invaded into the chicken mesodermal layer, characterised by inflammatory cell infiltration and a marked focal reaction between chorioallantoic membrane and cell graft. This reaction was inhibited by pre-treatment of CA+ MCF10A cells with Rap-1 inhibitor GGTI-298. Inhibition of CA in metastatic breast cancer cells with high levels of endogenous CA (triple negative cell line MDA-MB-231), using PLK4 inhibitor Centrinone B, abrogated their metastatic capacity in vitro. Here, we demonstrated CA induction in normal cells confers early tumourigenic changes which promote tumour progression, mediated by ECM disruption, altered cell-cell contacts, and Rap-1-dependent signaling. These insights fundamentally demonstrate the mechanism of how CA induces tumourigenesis in normal cells, alone and without requiring additional pre-tumourigenic alterations.