Characterising LINC complex roles in 3D epithelial migration and breast cancer metastasis

Abstract

Cell migration is essential for the development of multicellular organisms; with disruptions in this process contributing to diseases such as cancer, neurological disorders and musculoskeletal diseases. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex is an evolutionary conserved proteinaceous structure, critical for maintaining proper cellular migration. This multifunctional complex provides a physical connection between the nuclear interior and the cytoskeleton, with disruptions stimulating loss of directed cell migration, compromised nuclear structure and abnormal cellular signalling. As it is also noted that the nucleus in many cells is the stiffest cellular component, it is suggested that LINC complex disruptions may be key in increasing the migration potential of cells through both in vivo and in vitro 3D environments.

This project aimed to investigate the roles of LINC complex disruptions on keratinocyte morphological and migratory behaviours in 2D, and both non-restrictive and space-restrictive 3D culture environments, through the application of dominant negative SUN1 mutants. Through extensive analysis, it was identified that these mutants exhibited altered cell-cell and cell-substratum attachment phenotypes, alongside increased nuclear heights. It was also demonstrated that the LINC disrupted mutants displayed a migration advantage in space-restricted 3D environments, which was attributed to a decrease in nuclear stiffness. Through fibroblast incorporation to the 3D scaffolds used, it was further shown that LINC disrupted keratinocytes displayed increased levels of differentiation markers, alongside increased cellular stacking phenotypes across scaffolds surface regions, potentially attributed to alterations in Hippo pathway signalling.

The migratory phenotypes observed in DN mutants closely resemble that of high-grade cancer cells, able to migrate through space-restrictive environments during metastasis. Comprehensive protein expression and localisation analysis across a range of breast cancer cell lines and tissues suggested that several LINC complex components display altered expression levels closely linked to cancer progression, most significantly a down-regulation of lower nesprin-1/-2 isoforms was identified. As following investigations later suggested Nup88 as an upstream regulator of nesprin-2, able to bind C-terminal regions, it’s suggested that these phenotypes link closely to that observed in high-grade cancers.

Together, the data presented suggests that LINC complex disruptions increase migration potential of cells through restrictive 3D environments due to a decrease in nuclear stiffness, comparable to that observed across high-grade breast cancer cell lines.