1. INSERM U1240 IMoST, University of Clermont Auvergne, Clermont-Ferrand, F-63000, France.
2. Département d'Oncogénétique, Centre Jean Perrin, F-63011 Clermont-Ferrand, France.
3. Centre d'Investigation Clinique UMR 501, F-63001 Clermont-Ferrand, France.
4. Department of clinical research, Délégation Recherche Clinique et Innovation, Centre Jean Perrin, F-63011 Clermont-Ferrand, France.
5. Centre de Ressources Biologiques BB-0033-00075, Centre Jean Perrin, F-63011 Clermont-Ferrand, France.
Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate.
qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins.
We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity.
These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.
Keywords: RhoA, RhoB, basal-like breast cancer, BRCA1, triple negative breast cancer