1. Wnt/β-catenin Signaling Pathway Prospective Target for Cancer Treatment Emelia E Conte 1, Jun Yin 2, Mei Zhang 2 emeliac23@hotmail.com Western Blot Introduction 11 Conclusions Various degree of Wnt activation among different cell lines. Luminal A line MCF7 has the lowest β- catenin, p-akt, and p-p38 expression; Most basal breast cancer cell lines have p-p38 that may help regulate the Wnt pathway by stopping destruction of β-catenin The study gives us basic information for future breast cancer studies. Transduction of the Wnt reporter lentivrus into the various cell lines. Do all cells within a cell type express Wnt or only a population of cells express Wnt? How do Wnt responsive and non-responsive cell lines interact with each other? Study the effects of p38 production on formation of bone metastases in mice with active Wnt signaling pathways Continue study of basal-type breast cancers to identify more target pathways for treatment and further develop personalized medicine Future Directions 12 ma 4 My Goal Characterize various protein levels related to Wnt activity in different breast cancer cell lines Methods & Materials Plasmid miniprep BCA Protein Concentration Assay Western Blotting (WB) 1 Abstract 10 Results 3 2 5000 Plasmid Miniprep Transformation Culture on agar plate O/N Pick up colonies Culture in 5 ml LB medium O/N Miniprep Digest by BamHI/EcoRI 9 Sample # Nucleic Acid Conc. Unit260/280260/230 Sample Type 11186.2ng/µl1.92.25DNA 23060.7ng/µl1.762.07DNA 31519.2ng/µl1.872.2DNA 41111.7ng/µl1.892.21DNA 51040.2ng/µl1.922.24DNA 61117ng/µl1.92.2DNA Wnt/β-catenin signaling, p38 MAPK, and Akt are related to human breast cancer invasion and metastasis. In this study, we investigated these proteins’ levels in luminal (MCF7, BT474), basal A (HCC70, MDAMB468) and basal B (HS578, MDAMB231) breast cancer cell lines. We quantitated the proteins lysated from these cell lines by BCA protein assay. According the concentration, we ran these proteins on SDS-Polyacrylamide gels and used Western Blots to determine the protein levels. We found the different β-catenin, p38 (phosphor-) and Akt levels in these 6 different breast cancer cell lines. These results provide basic information for future study. Meanwhile, we prepared plasmid for TOP-eGFP lentivirus reporter and confirmed the DNA quality by the measurement of Nanodrop and digestion of restriction enzymes (EcoRI/BamHI). Interactions between  -catenin, p38 and Akt Edlund S et al. Mol. Cell. Biol. 2005;25:1475-1488 ma Hypothesis Wnt responsive cells may play an critical role in the tumorigenesis of breast cancer 5 6 7 Wnt/ β-catenin Signaling Pathway Most basal cell exhibit high levels of β-catenin, p- p38, and p-Akt MDMBA231 negative for both β-catenin and pAkt, but still presents a p-p38 expression MCF7-a luminal A cell line shows low levels of β - catenin BT474 shows high levels of β-catenin All basal cell lines exhibit high p-p38 expression Both Luminal A and Luminal B cell lines show low p-p38 to expression Basal cell lines vary in amount of p-Akt Luminal A cell line shows no p-Akt expression Luminal B shows high p-Akt expression HS578, HCC70, and MDMBA 231, MDMBA 468 basal breast cell lines (circled in red on WB) MCF-7 luminal A cell line BT474 Luminal B cell line Wnt activation of p38 MAPKs regulates GSK-3β inactivation (Thornton et al Science 2008) p38 regulates breast cancer invasion and metastasis (Moore, 2013) Akt oncogene promotes cell survival by inhibiting apoptosis (Franke TF, et al. Cell, 1997) Wnt can activate Akt signaling-cross talking (Perry et al. Genes & Development, 2011) 1. UPCI Summer Academy, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA, 2. Women’s Cancer Research Center,University of Pittsburgh, Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, Pittsburgh, PA BCA Protein Assay Highest Concentrations 1. HCC70 4. HS578 2. MCF7 5. MD468 3. MD231 6. Bt474 5000 Plasmid Miniprep 7-2 8 EcoRI+BamHI double cut : 5598bp and 1393bp 5000 1500 The canonical Wnt/β- catenin signaling pathway is well known for its roles in the regulation of stem cell activities, and its acti- vation has been involved in various cancers (Reya and Clevers, 2005). In the absence of Wnt ligands, cytoplasmic β-catenin complexes with Axin, APC, GSK3, and CK1 to cause phosphorylation and pro- teosomal degradation of β-catenin. In the presence of Wnt ligands, receptors such as LP5/6 and Frizzled activate the key regulator Dishevelled to inhibit the activity of Axin, APC, GSK3 and CK1. This leads to cytoplasmic accumulation of stabilized β-catenin and its translocation to the nucleus. In the nucleus, β-catenin binds with TCF/LEF family members and acts as a transcrip- tional activator to regulate target genes.

2. University of Pittsburgh Cancer Institute