Abstract P4-06-05: Silencing of Cyclin D1 Increases Breast Cancer Cell Migration through Increased Id1 Expression and Induction of Epithelial-Mesenchymal Transition

Abstract

Abstract Backround: The oncogene cyclin D1 is overexpressed in many breast cancers and despite proliferation activating properties, has been linked to a less malignant phenotype. We have previously shown that down-regulation of cyclin D1 in actively cycling breast cancer cells significantly increased their migratory capacity whilst also decreasing proliferation. Gene expression analysis and animal modeling have highlighted an increase in the inhibitor of differentiation-1 (Id1) gene following cyclin D1 gene (CCND1) silencing. Notably, increased Id1 is associated not only with enhanced migratory and invasive features of breast cancer cells but also with prevention of cell differentiation and induction of an epithelial-mesenchymal-like (EMT) phenotype. Here we sought to determine if the increase in cell motility following cyclin D1 silencing was through increased Id1 expression and induction of an EMT phenotype, and confirm our findings in a large patient cohort. Materials and Methods: Expression of cyclin D1, Id1 and SNAI2 was determined in MDA-MB-231 and ZR75 cells by qPCR and western blot, whilst cell migration was monitored by transwell assay. Gene expression of CCND1, ID1 and EMT markers was analysed from a pooled cohort of batch-mean centered publically available data sets, with data analysis performed in SPSS. Results: Silencing of cyclin D1 increased cell migration 1.38 and 1.72 fold in MDA-MB-231 (P<0.001), and ZR75 (P<0.001) cells respectively, an effect abolished by silencing of Id1. qPCR analysis confirmed an increase in Id1 (1.73) and SNAI2 (1.50) expression following siRNA treatment of cyclin D1, an effect reversed following siRNA treatment of Id1. CCND1 gene exression was negatively, while Id1 was positively, associated with the EMT markers Vimentin (P<0.001), SNAI2 (P<0.001) and TWIST1 (P<0.001) in 1,100 breast tumors. In ER+ patients CCND1 was negatively correlated to Id1 expression (0.050) and in CCND1 low patients increasing Id1 expression was predictive of worse cumulative survival (0.027). Conclusion: Decreased CCND1 expression enhances MDA-MB-231 and ZR75 cell migration through increased Id1 expression and induction of EMT. Moreover, both genes are correlated to EMT markers in breast cancer patients and in a subset of the cohort, confer a worse survival. These results may explain the conflicting data regarding cyclin D1 expression in breast carcinomas and solidifies the rationale of the protein as a clinical target. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-05.