Abstract 3879: YAP (Yes-associated protein), a proposed tumor suppressor in breast cancer, is regulated by cyclin D1

Abstract

Abstract In breast cancer, cyclin D1 over-expression and amplification has been linked to a worse prognosis. Conversely, lower levels of cyclin D1 have also been correlated to an unfavorable outcome. We have in our previous studies observed that lower levels of cyclin D1 correlate to a more migratory phenotype of breast cancer cells. In order to specifically delineate the possible effectors downstream of cyclin D1, we performed a whole-genome microarray experiment where cyclin D1 was downregulated in MDA-MB-231 cells. To avoid selecting for pure cell cycle-dependent changes, the cell cycle-related binding partners of cyclin D1; cyclin-dependent kinases 4 and 6 (Cdk4/6), were also downregulated and included in the microarray experiment. One of the top downregulated genes upon cyclin D1 silencing was YAP, a recently proposed tumor suppressor gene implicated in regulation of breast cancer cell migration. We set out to clarify the link between cyclin D1 and YAP, both in an in vitro setting and by examining the expression of the two proteins in a clinical material of 179 primary invasive breast cancers. To validate the microarray results, cyclin D1 and Cdk4/6 protein levels were transiently silenced up to 96 h in MDA-MB-231 and MCF-7 breast cancer cells using siRNA, and YAP protein levels were observed by western blot. The clinical material was stained using YAP and cyclin D1 antibodies, and intensity of the signal was scored. Also, chromogenic in situ hybridization was performed allowing for assessment of amplification of the CCND1 gene. Transient downregulation of cyclin D1 in MDA-MB-231 and MCF-7 cells resulted in decreased YAP protein levels, as predicted by the microarray results. Downregulation of Cdk4/6 did not result in decreased YAP levels. In the clinical material, cyclin D1 intensity was inversely correlated to YAP intensity (Spearman's rho −0.220, P=0.031). Amplification of CCND1 was significantly correlated to none or low intensity of YAP expression. We conclude that on protein level, the silencing of cyclin D1 decreases levels of YAP independently of cyclin D1's cell cycle-dependent association with Cdk4/6. On the chromosomal level, CCND1 gene amplification is correlated to loss of YAP expression which might explain the inverse correlation seen between cyclin D1 and YAP in the clinical material. Studies to further characterize the link between cyclin D1, YAP and migration are ongoing. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3879.