BMP Signaling Regulates Mitotic Checkpoint Genes In Breast Cancer Cells

Chromosome instability (CIN) resulting from aberrant expression of mitotic checkpoint genes often contributes to tumor initiation and progression. Smad4, a pivotal tumor-suppression gene and co-mediator in TGF-βand BMP signaling pathway, is often observed to be inactivated in some human cancers. It still remains obscure, however, that what signal regulates the activity of Smad4 and implements the function of cell cycle control. In this project, we aim to explore molecular mechanism of regulation of Smad4 to cell cycle control and the effect of mitotic genes'expression to cell cycle progression.We treated MDA-MB-231 cells with dorsomorphine (DM), SB431542 (SB) to inhibit TGF-βsignaling pathway and BMP signaling pathway. After treatment with DM, we observe that the number of unsynchronized cells is dramatically increased, whereas almost of MDA-MB-231 cells are arrested in mitosis when treated with SB and DMSO. In addition, the similar phenomena occur in other breast cancer cell line, such as MCF7, T47D and colon cancer cell line HCT-116. Moreover, DM treated breast cancer cells cannot be arrested in mitosis when synchronized by Taxol, another mitosis inhibitor. Further research to test cyclin B1 by protein immuno-blotting assay, we find that cyclin B1 expression in MDA-MB-231 cells are obviously declined after releasing from interphase and treated with DM.We test the mitotic genes expression in MDA-MB-231 after treatment with DM and SB via western blotting. It is found that MAD2, the downstream gene in mitotic checkpoint, are greatly down-regulated after DM treating, and SB treatment group has no difference with control group. Furthermore, mitotic genes including MAD2, BUB3, TTK and HEC1, are expressed less with the treatment of DM in more concentration.To eliminate the possibility that DM might affect the mitotic checkpoint genes' expression through regulating other signaling pathway, we knock-down (KD) R-smads (Smad1,5) in BMP pathway and Co-smad (Smad4) by applying RNAi method. The results demonstrate that expression of mitotic checkpoint genes are obviously declined after Smadl,4,5 KD. In addition, in certain range of chemical concentration, the mitotic genes expression will be upregulated by BMPs stimulation in dose-dependent way. And stimulation of cytokine in TGF-βhas no obvious effect.Finally, we transfect mad211 added with FLAG tag into MDA-MB-231 cells through transient expression to confirm whether the ectopic MAD2 expression could rescue mitotic checkpoint defect. The result of western blotting indicates that overexpression of MAD2 could partially restore the function of mitotic checkpoint.This research enrich our understanding of regulation of signaling of smad1,5-smad4 pathway to mitotic checkpoint, and further investigate influence of BMP signaling input to mitotic genes'expression. We confirm that mitotic genes are regulated by BMP pathway not TGF-βpathway, as provides base for possible novel therapy strategy to treat related cancer.