RESUMO
Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Agregação Celular/fisiologia , Proteínas Motores Moleculares/fisiologia , Cadeias Pesadas de Miosina/fisiologia , Invasividade Neoplásica/fisiopatologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Embrião de Galinha , Feminino , Técnicas de Silenciamento de Genes , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Proteínas Motores Moleculares/antagonistas & inibidores , Proteínas Motores Moleculares/genética , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/genética , RNA Interferente Pequeno/genética , Esferoides Celulares/patologia , Esferoides Celulares/fisiologia , Ensaio Tumoral de Célula-TroncoRESUMO
The simian virus 40 small t (SV40ST) oncoprotein interacts with protein phosphatase 2A (PP2A), an abundantly expressed family of serine-threonine phosphatases. This interaction is essential for the transformation of human cells by SV40, and several PP2A subunits have been implicated as tumor suppressor genes. However, the pathways controlled by specific PP2A complexes involved in cell transformation remain incompletely understood. Using a comprehensive loss-of-function approach, we identified 4 PP2A regulatory subunits [B56α, B56γ, PR72/PR130, and PTPA (protein phosphatase 2A activator)], which when suppressed replaced the expression of SV40ST in human cell transformation. We found that manipulation of complexes containing PP2A B56α, B56γ, and PR72/PR130 activates the pathways regulated by c-Myc, Wnt, and PI3K (phosphoinositide 3-kinase)/Akt in a manner that depends on their specific phosphatase activity. In contrast, suppression of PTPA disrupts the assembly of PP2A heterotrimeric complexes, which leads to the activation of these same oncogenic pathways. These observations delineate the PP2A family members and pathways perturbed by SV40ST during human cell transformation.
