1. Breast cancer affects over 200,000 new patients in the United States every year. One particularly challenging subtype is triple- negative breast cancer (TNBC), occurring in 15-25% of breast cancer and garnering its name from a lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). TNBC is clinically characterized to be more aggressive and largely unresponsive to clinically available targeted therapies resulting in poor prognosis making understanding the pathways and characteristics of TNBC to be vital importance for urgent need of developing future therapies. Increasing evidence suggests that multiple signaling pathways are abnormally regulated and involved in pathogenesis of TNBC—two of which are Wnt/  -catenin pathway and Notch pathway. Abnormal activation of Wnt/  -catenin signaling has been associated with TNBC: several important oncogenes (FZD7, LRP6, TCF) in Wnt/  - catenin were up-regulated in TNBC cells. Normally,  -catenin plays a critical role in intercellular adherent junction. However, after losing its association with cellular junction,  -catenin released from membrane will gain transcriptional function potentially promoting tumor cell invasion. Also strongly related to TNBC cells, the  B-crystallin gene is linked to neoplastic-like changes and loss of cell growth control in basal-like breast cancer/TNBC. In light of the poor outcome of TNBC case, there is an urgent clinical need of its therapeutic approaches; therefore we designed experiments to attempt to identify potential therapeutic targets in those complex pathways. Identification of Therapeutic Targets in Triple Negative Breast Cancer Cells: β-Catenin, a Critical Chemoresistant Mediator Thomas Luo 1, 2 with Bhawna Sharma 2 PhD and Dr. Vincent Cryns 2 MD Madison West High School, Madison, Wisconsin 1 ; Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 2 Acknowledgements Extended thanks to Rachel Egan, Carmen Lombard, Lisa Watchel and the Madison Metropolitan School District for providing this incredible opportunity I would like to thank Vincent Cryns and Bhawna Sharma for their patience, insight, and assistance. A thanks to Chris Schoenholz and Vincent Cryns for writing recommendations to the SRI program Introduction Conclusions Results A. B. C. A. B. C. A. B. C. E. Effects of β-catenin Inhibitor Effects of Notch Pathway Inhibitor Effects of Temozolomide Reduction of Cell Density by ICG001 Inhibition of Cell Migration/Proliferation by ICG001 Hypothesis Abnormal activity of Wnt/  -catenin pathway and/or Notch pathway leads to pathogenic alteration in TNBC cells rendering them more aggressive and chemoresistant. Objective To identify potential target molecules for TNBC, then testing the therapeutic value using a cell culture model and pathway inhibitors with cytotoxic chemotherapy drugs. Methods Cell Culture Procedures: MDA-MB-231 TNBC cell line and its modified line that were transferred with vectors cloned with and without  B-crystallin were cultured in MDEM Medium supplemented with 10 % FBS in an incubator at 37 o C with 5% CO 2. Cell Viability Assay (MTS assay): Cell viability after drug treatments was determined using MTS assay. For the assay, cells were treated with individual drugs or drug combinations for 48 hours. Pathway inhibitors and cytotoxic drugs used were Paclitaxel (200 nM), Doxorubicin (1 µM), and Temozolomide (50 µM), β-catenin inhibitor ICG001 (5 µM), and Notch pathway inhibitor DAPT (50 µM). Cell Number-Crystal Violet Assay: The effects of drug treatments on cell number were further measured with Crystal Violet assay. In this assay, cells were treated with drug and their combinations for 48 hours. The doses of the inhibitors and cytotoxic drugs were the same as those used in MTS assay experiments stated above. Wound Healing/Scratch Assay: In assessing cell migration, growth, and proliferation after drug treatment, a wound healing/scratch assay was used. Confluent cells were scratched and then treated with and without ICG (5 or 10 µM). Cell migration was monitored at 0 and 24 hours post-scratch. Figure 1. Wnt/β-catenin signaling pathway and intercellular adheren junction Figure 2. Inhibitory effects of ICG001(ICG), Doxorunicin (DOX), and Paclitaxel (PAC) on cell viability of MDA-MB-231 cells. A. Cells transfected with control vector (132-V) or vector expressing αB-crystallin (αB) (231-αB). Cells were treated with drugs and their combinations for 48 hours. B. Cells were treated for 72 hours. C. Cells without transfection were used. Cells were treated for 72 hours. DMSO was used as control treatment. Cell viability was detected with MTS assay. Results showed that ICG + DOX markedly reduced cell viability in the two types of transfected cells compared with treatment with ICG or DOX alone. Furthermore, ICG + DOX also reduced cell viability in non- transfected cells. Figure 3. Effects of DAPT, DOX, and PAC on cell viability of MDA- MB-231 cells. A. Cells transfected with (231-αB) or without αB-crystallin (αB) (231- αB). Cells were treated with drugs and their combinations for 48 hours. B. Cells were treated for 72 hours. C. Cells without transfection were used. Cells were treated for 72 hours. DMSO was used as control treatment. Cell viability was detected with MTS assay. Results showed that DAPT + DOX did not reduce cell viability of the three types of cells. Figure 4. Effects of temozolomide (TEM), ICG, and DAPT on cell viability of MDA- MB-231 cells. A. Cells transfected with (231-αB) or without αB-crystallin (αB) (231- αB). Cells were treated with drugs and their combinations for 72 hours. B. Cells without transfection were used. Cells were treated for 72 hours. DMSO was used as control treatment. Cell viability was detected with MTS assay. Results showed that TEM + ICG or DAPT did not reduce cell viability of the three types of cells. Figure 3. Reduction of cell densities by ICG, DOX, and PAC in MDA-MB-231 cells. DMSO was used as control treatment. Cell number was detected with crystal violet assay. A. Cells were not transfected with αB- crystallin (αB). B. Cells were transfected with αB. Cells were treated with drugs and their combinations for 48 hours. C. Cells densities of each treatment groups were measured with a NIH image J software, and the cell density was expressed as mean intensity detected. Results showed that ICG + DOX or PAC markedly reduced cell confluence in the two types of transfected cells compared with treatment with ICG, DOX or PAC alone. Figure 3. Inhibition of cell cell migration /proliferation by ICG in MDA-MB-231 cells. A. the wound at 0 hour after scratch. B. wound at 24 hours after scratch, and cells were cultured in control medium. C. Cells were treated 5 μM ICG for 24 hours after scratch. D. Cells were treated 10 μM ICG for 24 hours after scratch. E. the wound of each treatment groups were measured with a NIH image J software, and the effects of treatment was expressed as percentage of wound closure (%). Results showed that ICG + DOX or PAC reduced cell migration/proliferation of cells compared with treatment with control.  We demonstrated that combination treatments with ICG and DOX or PAC (high dose) a possible reduced the cell viability and cell number of MDA-MB-231 TNBC cells and inhibited migration/proliferation of those cells.  These finding showed that inhibition of β-catenin activities might sensitize TNBC to the treatment with conventional chemotherapy drugs.  The facts that TEM and DAPT did not affect those cells further showed that the effects of ICG, DOX, and PAC were specific actions.  Our data indicate that β-catenin is an important signaling molecule that mediates drug-resistance and tumorigenic effects in TNBC. Thus, Targeting of β-catenin and Wnt signaling pathway could be a promising strategy for finding novel therapy approches for treating TNBC. Discussion: Proposed Mechanism aB-Crystallin has long been associated with response to DNA damage and cell stress; however, recent data shows that aB-Crystallin actually works in complex with  -catenin. The down-expression of  -catenin in this study may actively disrupt this complex in TNBC.