Abstract P5-07-08: Restoration of APC decreases tumorigenic potential in breast cancer cells

Abstract

Abstract Mutations of the Adenomatous Polyposis Coli (APC) tumor suppressor gene have been reported in 5-70% of human breast cancers. APC is most well known for its role as a negative regulator of the Wnt/b-catenin pathway, where APC sequesters β-catenin and targets it for degradation via the proteasome. The breast cancer cell line, DU4475, has a single point mutation in the mutation cluster region of APC, coding for an early stop and subsequent truncated protein, lacking the β-catenin binding domain. Preliminary data using a TOPFlash reporter assay suggested that Wnt/β-catenin pathway was active, which was validated with immunofluorescence (IF) showing nuclear localization of β-catenin. Our overall hypothesis is that restoration of APC will decrease tumorigenic potential in DU4475 breast cancer cells. To test our hypothesis, we have generated DU4475 cells stably expressing GFP (control) (DU4475-GFP), a GFP-tagged internal fragment of APC that includes the β-catenin binding domain (DU4475-APCmid), and a GFP-tagged C-terminal fragment of APC that includes the microtubule binding domain of APC (DU4475-APCC-ter). After selection using G418, the GFP positive cells were sorted using a cell sorter. Results from TOPFlash reporter assay and IF demonstrated a modest decrease in Wnt/β-catenin activity in DU4475-APCmid cells. In addition, we observed decreased proliferation as measured in a growth assay in DU4475-APCmid cells compared to the control cells. We also observed a 17% increase in cleaved caspase-3 and only a minimal decrease in cell proliferation as measured by phospho-Histone H3 using IF in 3D cultures upon APCmid re-introduction. We also observed decreases in Cyclin D1, Cyclin B1, Early Growth Response 1, and HIF1α in DU4475-APCmid using real time PCR. These changes suggest that the re-introduction of the central domain of the APC tumor suppressor results in alterations in cell growth and gene expression changes indicative of an increased tumorigenic phenotype in vitro. Ongoing studies in the laboratory are dissecting the signaling pathways downstream of APC restoration to identify therapeutic targets that may be used to restore the functional alterations of mutant APC. These studies have generated a system to be utilized for in vivo tumor and metastasis formation under the control of APC mutation in a human cell culture model. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-08.