Involvement of Scribble protein in breast cancer invasion and metastasis

A common feature of cancer cells is the loss of cell polarity. Scribble is a cell polarity protein with an unknown mechanism for its role in tumour suppression. Changes in the phosphorylation pattern of four serine sites at the C-terminal of scribble, implicate scribble in breast cancer invasion and metastasis. This was due to CD74 overexpression in lymph node metastatic triple negative breast cancers. Investigating how changes in serine phosphorylation status at these sites affect the regulation of invasion and metastasis in breast cancer was the focus of this study. These sites were mutated by site-directed mutagenesis to generate mutant scribble genes grouped into A-mutants and D-mutants, based on the amino acid change to the serine sites to mimic unphosphorylated and phosphorylated scribble, respectively. Widefield and confocal bioimaging of the expression and localization of these mutants in cell line HEK293T revealed good expression but varied localization to cytoskeletal elements, intercellular contact site, microtubules, centrosome and vesicles. Wound healing, MTT and flow cytometry assays were conducted on HEK293T transfected with these mutant genes to study how their expression affected cell migration, proliferation and the cell cycle, respectively. Notable differences emerged: 1.) A-mutants migrated more than D-mutants. 2.) D-mutants proliferated more than A-mutants and 3.) There were significantly more D-mutant expressing cells in the G2 phase of the cell cycle. Protein interaction study was also conducted. The binding partners of S[1306+1309]A and S[1306+1309]D mutants of scribble were captured and analysed by Co-IP and mass spectrometry. Bioinformatics analysis identified the pathways these binding proteins were significantly involved in: S[1306+1309]A binding partners were involved in cell migration via regulation of actin cytoskeleton pathway but contribute to intercellular adhesion via the tight junction pathway. S[1306+1309]D binding partners were involved in the TSH signalling and cell cycle pathways. The results show a significant possibility that unphosphorylated and phosphorylated hScrib support cell invasion and cell proliferation, respectively, and that these processes are antagonistic.