Role of LaNt α31 in breast cancer

Abstract

Invasion and metastasis of tumour cells are complex processes involving the interaction of cancer cells with laminins and other extracellular matrix components. Laminin N-terminus α31 is a laminin-derived, netrin-like protein produced by alternative splicing from the LAMA3 gene. To date, LaNt α31 has been established to be expressed in most tissues and is upregulated in breast cancer, with further upregulation in distal metastases. In addition, cell culture studies have established that LaNt α31 overexpression alters the mode of MDAMB231 breast cancer cell invasion from multicellular streaming to individual cell invasion when invading a laminin-containing matrix. However, no further experiments have been reported beyond these observations, leaving a substantial gap in knowledge. This thesis describes investigations designed to increase understanding of the mechanisms driving LaNt α31-induced changes and to determine the impact on cancer cell behaviour, including invasion and migration. Firstly, LaNt α31 was overexpressed using adenoviral-mediated transduction in invasive and non-invasive breast cancer cell lines. The effects of LaNt α31 on the activity of crucial cell signalling cascades known to modulate matrix remodelling and invasive behaviour and be regulated by laminin-mediated signalling, including FAK, SRC, and PI3K/AKT, were assessed by western blot and antibody arrays targeting phosphorylated protein kinases. These analyses revealed that the upregulation of LaNt α31 led to a sustained increase in Akt phosphorylation, with less pronounced changes observed in the other pathways. RNA sequencing was used to explore changes in the expression of genes that influence matrix remodelling, integrity, and adhesion as a consequence of induced LaNt α31 expression. Elevated mRNA expression of laminin α3, β3, and γ2 subunits was observed in both cell lines . Collagens that anchor laminins to the matrix, including basement membrane collagen IV and anchoring fibril collagen VII, were elevated in invasive breast cancer cell lines. In addition, the genes for proteins involved in hemidesmosomes, focal adhesion, desmosomes, and tight junctions were increased in MDAMB231-LaNt α31 . The expression of the major receptors and co-receptors that bind to either laminins or collagens, such as integrins α2, α3, α5, α6, and β1, was also increased. To perform longer-term experiments, including three-dimensional (3D) culture and impedance-based cell analysis , lentiviral expression was used to drive stable expression. 3D cultures were conducted to study the invasive behaviour of LaNt α31-expressing cells in a tumour microenvironment model. In this assay, laminin-enriched matrix (Matrigel) and collagen I were used as substrates, with assays performed in the presence or absence of pathway inhibitors. The results demonstrated that MDA-MB-231-LaNt α31 cells assembled compact, aggregated spheroids when embedded in a collagen I matrix. In contrast, in laminin-containing matrices, LaNt α31 induced ductal-like tube formation resembling a process known as vasculogenic mimicry, with increased central core sizes. Targeting the PI3Ks and MMPs impaired the observed effects of LaNt α31 expression in spheroids, but not those of ROCK inhibitors consistent with the requirement of PI3K/AKT pathway activation for these outcomes. Impedance-based phenotypic assays were performed using various substrate coatings, including media secreted from untreated cells, media from LaNt α31-expressing cells, or from purified extracellular matrix proteins LM111, LM511, LM332, rat tail collagen I, or netrin-4. These studies identified that MDAMB231-LaNt α31 cells increased barrier function and covered the electrodes more quickly than MDAMB231-WT. Interestingly, the coverage and barrier integrity of MDAMB231-untreated and MDAMB231-LaNt α31 cells on plates coated with either netrin-4 or conditioned media from LaNt α31-expressing cells were very similar. Together, these results reveal that LaNt α31 induces clustering, cell-cell interaction, and proliferation in invasive breast cancer cell lines in 2D and 3D environments by enhancing LM332 levels alongside α6β1 and α3β1 integrins, co-receptors, and adhesion protein-mediated signalling pathways. Furthermore, increased expression of LaNt α31 is linked to altered cellular behaviour in tumour microenvironment models potentially driven by and dependent on modulation of the PI3K/Akt signalling pathway leading to a hypoxic-like state and vascular mimicry in invasive breast cancer cells. These findings identify a complex interplay between LaNt α31, molecular signals, and extracellular matrix proteins that contribute to breast cancer proliferation and invasive behaviour. This research has implications for understanding LaNt α31 and laminin-mediated regulation of tumour progression.