Our previous data illustrated that activation of the canonical Wnt signaling

Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. -catenin, is associated with reduced overall survival in all breast cancer patients [16]. We sought to determine the impact of Wnt/-catenin pathway activation on the malignant and metastatic potential of TNBC cells In the present study, our group analyzed the role of -catenin in the tumorigenic properties of TNBC cell lines to address its potential utility as a therapeutic target in this tumor type. Materials and Methods Ethics Statement All procedures were performed with prior approval (protocol #71828) from the University of Chicago Institutional Animal Care and Use Committee, which has full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care. Cell lines and -catenin knockdown MDA-MB-231 and HCC38 cell lines were from ATCC and cultured in RPMI 1640 media (Invitrogen, Grand Island, NY) supplemented with 10% FBS and 1% penicillin/streptomycin. The stable knock down lines of MDA-MB-231 and HCC38 were established by infection of MISSION shRNA Lentiviral Transduction Particles against CTNNB1 (Sigma-Aldrich, TRCN0000003845 and TRCN0000003846) and selected with puromycin (1ug/ml). Western blotting Total cell lysates were extracted with 50 mM Tris pH7.5, 0.1% IGEPAL, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA and protease inhibitors and sonicated. 25 g of lysate from each cell line was separated on a 10% SDS-PAGE gel and transferred to Immobilon P membrane (Millipore). Blots were probed with 1:1000 BMPR2 anti–catenin monoclonal antibody (BD Biosciences), 1:1000 anti-active (unphosphorylated) -catenin (EMD Millipore) and 1:1000 anti-actin monoclonal antibody (Sigma) in blocking buffer (5% nonfat dried milk in TBST). TOPFlash reporter assays Cells were plated in a 24-well plate at a density of 1×104 cells/well and transfected using Optifect (Invitrogen) with 2.6 g of or (obtained from H. Clevers at the Hubrecht Insitute) along with (Promega) for determination of transfection efficiency. Lysates were harvested after 48 hrs and analyzed using the Dual Luciferase Assay System kit (Promega). Luciferase activity was normalized for transfection efficiency and graphed as ratio of TOPflash/FOPflash activity. Mutant -cateninS37A was from Stephen Byers at Georgetown University. The breast cancer lines were transfected with stabilized mutant -cateninS37A as described previously [18]. Expression was verified by GFP fluorescence and Western blotting with a HA antibody (Santa Cruz). Cell growth, migration and soft agar growth assays Cell growth was analyzed with MTS assay using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, WI) following the protocol. Cells were seeded in 96-well culture plates at a density of 5103 cells/well. Cell viability was assessed by adding 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to the culture medium and incubated for 4 h at 37 C. The optical density was measured at 490 nm using Synergy 2 plate reader (Biotek, Mocetinostat VT). Cell migration was measured by wound healing assays as described [18]. Briefly, cells were plated at a density of 1 x 105 cells per well in a 6-well plate were grown to confluence, at which time they were scratched with a micropipette tip. Phase-contrast images were captured with a Zeiss Axiovert 35 inverted microscope using AxioVision REL 4.6 imaging software (Zeiss) every 12 hr. The Mocetinostat area of three representative wounds for each cell line was quantified at each time point using Image J software (NIH). Anchorage-independent growth soft agar assays were performed by seeding 4 x 103 cells in 0.3% agarose as described [19]. The colonies were quantified at day 14 from five fields in triplicate wells. Stem cell assays ALDEFLOUR assays (Stemcell) were used. 5×105 cells were stained with ALDEFLOUR reagent or ALDEFLOUR reagent plus ALDEFLOUR inhibitor. Stained cells were sorted by the University of Chicago Flow Cytometry Core Facility. Quantitative real-time PCR Gene expression changes were analyzed by real-time RT-PCR as described [20] except ribosomal RNA 18S was used as a normalization control. The primer sequences for models of TNBC that recapitulate some of the features of human tumors with respect to Wnt signaling, several breast cancer cell lines were analyzed for total and activated, or non-phosphorylated, -catenin by Western blotting. Total and activated -catenin were expressed at high levels in HCC1937, UACC3199 and HCC38 TNBC cell lines compared to the Mocetinostat ER-positive MCF-7 and another TNBC line MDA-MB-231 (Fig. 1A). Interestingly, despite the high levels of activated -catenin, none of the cell lines demonstrated significant basal TCF reporter activity (Fig. 1B). However, reporter activity was induced in most of the cell lines upon transient overexpression of a stabilized S37A mutant of -catenin (Fig. 1C). There was significant variability among the cell lines in the extent of pathway activation in response to stabilized -catenin overexpression, suggesting that there.

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