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

Abstract

Centrosome amplification (CA) is a prominent feature of human cancers linked to genomic instability and tumourigenesis in vivo. CA is observed as early as pre-malignant metaplasia, with CA incidence increasing as the disease progresses from dysplasia to neoplasia. However, the mechanistic contributions of CA to tumourigenesis (tumour architecture and remodelling) are poorly understood. Using non-tumourigenic breast cells (MCF10A), we demonstrate that CA induction (by CDK1 inhibition or PLK4 overexpression) alone increased both cell migration, invasion and Extracellular Matrix (ECM) remodeling. Mechanistically, CA induction activated small GTPase Rap-1. We demonstrated the key role of Rap-1 mediated signalling in CA induced tumourigenesis through Rap-1 inhibition (using GGTI-298) which blocked CA-induced migration, invasion and ECM attachment. CA induction in a long-term MCF10A 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 & {beta}-catenin). At the ultrastructural level, CA significantly inhibited apical junctional complex formation, as visualized by transmission electron microscopy. CA induction in the luminal A breast cancer cell line MCF7 revealed similar trends in cell junction disruption. Furthermore, CA induction in MCF10A elevated expression of integrin {beta}-3, matrix metalloprotease MMP1 and MMP13 facilitating the observed ECM attachment, degradation and cell invasion phenotype. In vivo validation using a Chicken Embryo xenograft model, showed CA positive (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 GGTI-298. Interestingly, in metastatic breast cancer cells with high levels of endogenous CA (triple negative cell line MDA-MB-231) inhibition of this CA-signalling pathway (using PLK4 inhibitor Centrinone B) abrogated their metastatic capacity in vitro. This demonstrates dual roles for CA signalling, for initiating and maintaining the CA-induced metastatic phenotype. Here, we demonstrated that CA induction in normal non-tumourigenic cells acts through Rap-1-dependent signaling to confer early pro-tumourigenic changes promoting tumour progression, mediated by ECM disruption, and altered cell-cell contacts. These insights reveal that in normal cells, CA induction alone (without additional pro-tumorigenic alterations) is sufficient to induce tumourigenesis and CA-mediated signaling supports a metastatic phenotype. StatementCentrosome amplification alone drives early tumourigenic change in normal breast epithelial cells