Abstract 2137: Using Rac1 isogenic cells to model tumor metastasis

Abstract

Abstract The Rho family of GTPases regulate a wide range of normal cellular functions including cell growth and cytoskeleton dynamics, but it is becoming increasingly apparent that when deregulated, proteins such as Rac1 contribute to tumorigensis, tumour invasion and metastasis. Overexpression of Cdc42 and Rac1 have been reported in human breast cancer, and coincide with breast cancer progression, metastasis and resistance to chemotherapy. The aim of this study was to generate a model system to investigate how Rac1 activation affects the migratory propensity of human breast epithelial cells. We utilised X-MAN isogenic cells derived from MCF10A mammary epithelial cells which harbour a single Q61L Rac1 mutation introduced by AAV-mediated homologous recombination, paired with a parental cell line expressing the wildtype allele. This system offers several advantages over conventional methods; protein expression is driven from endogenous promoters, thereby eliminating the caveats associated with transfection-based approaches. Using the X-MAN isogenic cells we showed that introduction of the Rac1Q61L mutation results in a profound effect on cell morphology. The most striking phenotype was the appearance of large intracellular vacuoles, but immunofluorescence studies also revealed cytoskeletal abnormalities with Rac1Q61L cells showing evidence of microtubule disorganisation and accumulation of newly polymerised actin fibers. These observations, in conjunction with previous reports that activated Rac is associated with cell migration, prompted us to address whether these cells may also have a migratory phenotype. Using Transwell and scratch assays, we confirmed that Rac1Q61L cells are more motile than their parental counterparts, and this correlated with increased adhesive properties. Pathway analysis revealed Rac1Q61L cells express higher basal levels of activated MEK and ERK, both of which have been implicated in the migration of numerous cell types. In conclusion, we have generated a pair of isogenic cell lines to demonstrate how constitutive Rac1 activation results in cytoskeletal changes and increased motility. Our data highlights the advantages of being able to study clinically-relevant pathways in a phenotypically-relevant assay, and how this platform could be exploited to profile molecularly targeted agents aimed at reducing tumour invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2137. doi:1538-7445.AM2012-2137