Mechanism Of The Small Molecule Compounds Which Inhibit The Canonical Wnt Signaling Pathway In MDA-MB-231Breast Cancer Cells

Wnt signalling is involved throughout development and may be inappropriately activated in a variety of human cancers. Wnt signaling also plays an important role in many processes of life, including regulating early embryonic development, cell differentiation, cell proliferation and growth. In the canonical pathway, secreted Wnt proteins induce the stabilization of β-catenin. In addition, identifying novel upstream modulators would be of importance to elucidate the mechanisms involved in regulating (3-catenin activity. The development of small molecule inhibitors may offer a novel therapeutic opportunity. In this study, we focus on the roles of how small chemical regulate Wnt signaling.Part one:Mechanism of migrazole regulate the canonical Wnt signaling pathway.The phenotypes induced by migrazole are consistent with disruption of Wnt signalling. In addition, mutations which upregulate components of the pathway that migrazole affects may also suppress a migrazole induced phenotype. In this manner, the target of migrazole may be elucidated. Further characterization and target identification will be pivotal in establishing migrazole as a small molecule tool. The aim of this work was to find whether migrazole inhibits canonical Wnt signalling and where is the target. Migrazole is used for stimulating β-catenin degradation by Western blot, Topflash reporter in different kinds of cell lines, including breast cancer cells MDA-MB231, colon cancer cells RKO Stable, brain cancer cell lines N1E115and N2A, colon cancer cell lines DLD1and SW480. We use LiCl which is the inhibitor of GSK3β to induce Topflash expression and confirmed that migrazole works downstream of GSK3β. Because of DLD1and SW480are APC mutations colon cancer cells, it is suggested that migrazole’s target should work between GSK3β and APC components. By Western blot in HEK293T and N2A, We confirmed that migrazole can also inhibit BMP signaling pathway but have no much effect on NF-KB or TGFp.Part two:Mechanism of21H7/M110inhibit the canonical Wnt signaling pathway in MDA-MB231cells21H7/M110is an inhibitor of Wnt signaling in colon cancer cells which are APC mutations. Compounds can through PIM to inhibit Wnt signaling pathway in colon cancer cells. MDA-MB231are breast cancer cells which are not APC mutations. The aim of this work was to find whether21H7/M110effectively inhibits Wnt signalling in breast cancer cells. The results show that21H7/M110can increase PIMs’ expression in MDA-MA231and RKO cells which are breast cancer and colon cancer cells. And knockdown PIMs have no effect on MDA-MB231cells.21H7/M110is used for stimulating P-catenin degradation by induced HIF1α expression by Western blot, Topflash reporter, hypoxia-inducible VEGF-luc reporter assays and Real-time PCR in both colon cancer cells and breast cancer cells. Instead,21H7/M110works as an iron chelator to inhibit Wnt signaling. Our results reveal a critical requirement for iron in Wnt signalling and they demonstrate that iron chelation serves as an effective mechanism to inhibit Wnt signaling.Part three:Inhibition of tankyrases induces AXIN stabilization and blocks Wnt signalling in breast cancer cellsXAV939is an Inhibitor of Wnt signaling which induces an increase in Axin protein levels and also promotes β-catenin phosphorylation by stabilizing Axin-scaffolded destruction complexes in colon cancer cells. The aim of this work was to find whether tankyrases through AXIN1/2stabilization effectively inhibits Wnt signalling in breast cancer cells. XAV939is used for stimulating β-catenin degradation by stabilizing AXIN1/2by Western blot, Topflash and Fopflash reporter assays and Real-time PCR in both colon cancer cells and breast cancer cells. XAV939does not inhibit the growth of these cells in high percentage serum even after Wnt signalling has been turned off but does inhibit the growth of these cells in low percentage serum when21H7and M110were used as positive controls by Sulforhodamine B (SRB) growth inhibition assays. XAV939can inhibit cells migrating, colon formation and proliferation in MDA-MB231cells.The results show that tankyrases effectively inhibits Wnt signalling through AXIN1/2stabilization in breast cancer cells and also XAV939inhibits MDA-MB231cells migration by adding for16h. It was also found that XAV939is effective for Wnt signaling in a different way compared to21H7and M110which work by SRB growth inhibition. Our study provides new mechanistic insights into the regulation of Axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies in breast cancer cells.Overall, the development of small molecule inhibitors may offer a novel therapeutic opportunity. We focus on the roles and clarified of how those three difference small chemical regulate Wnt signaling.