detection using ECL kit Migration of Estrogen Receptor -1 (ER-α) positive breast cancer cells to bone in response to bone cells Heena Singh, Dr. An-Dao Yang, Dr. Cay Egan & Dr. Tony Magliocco University of Calgary, and TBCC Translational Lab, 2500 University Drive NW, Calgary, AB Seed, Harvest & Lyse cells Load Sample and Run on 10% Acrylamide Gel Membrane Transfer Introduction Metastasis is gravely concerning in breast cancer patients as early diagnosis (without metastases) is associated with a 90% 5-year survival rate while the long term survival of patients with metastases drops significantly to 10% (1). Current treatment options are palliative and include surgical excision, chemotherapy and irradiation of the tumor bed (1). Patients with ER+ breast cancer (BC) develop bone metastasis 3X more often than patients negative for ER (2). Selective ER modulators (Tamoxifen) (2) have shown to be effective in decreasing risks related to BC (3) however the migration of BC cells to bone may be a result of factors present in the bone microenvironment(4). ERα is one such nuclear protein that will be investigated as it functions by promoting tumor invasiveness and cell proliferation and is commonly expressed in BC patients(3). ERα is a ligand-dependent transcription factor which upregulates gene expression by binding to DNA (3). Methods Specific Aim 1: Results Conclusion & Future Directions Slight presence of ERα protein in HTB126 has been detected Since very little expression is seen, selective cloning of stably transfected cells will be conducted. ERα expression in HTB126 cells can be checked after Src knockdown (Recent research illustrates high Src activity leads to protelysis of ERα in ERα+ BC cell lines) 1 2 3 4 KDa 170 130 95 72 67 55 43 ER1 Actin MCF-7 HTB126 1˚ Ab ER-1 Anti-rabbit monoclonal HRP Actin Anti-mouse monoclonal HRP Washes (TBS-T) 2˚ Ab Goat-anti rabbit polyclonal HRP Chemiluminescent detection using ECL kit HTB126 pEV HTB126 pER1 Figure 1: Cells were harvested from stably transfected HTB126 cell lines selected with Gentamycin. A protein band ~67 kDa is expected for ERα. Since MCF-7 cells are naturally ER positve, a band at 67kDa was expected and seen in the above results. As expected, ERα was not present in the HTB126 negative control parental cell lines (lane 2). However, a lack of ERα expression in lane 4 indicates ERα protein may not be present at the translational level in the transfected cells. The housekeeping gene Actin was present in all lanes at ~43 kDa indicating equal loading of samples. . Acknowledgements The O’ Brien Centre has been key in providing summer research funding for this project. Furthermore, this project could not have been possible without the help of everyone at the Tom Baker Translation Lab as well as my supervisors, Dr. Cay Egan, Dr. An-Dao Yang and Dr. Tony Magliocco. Specific Aim 2: Aims Specific Aim 1: Determine ERα protein expression in parental HTB126 cell lines and HTB126 cell lines transfected with ERα Specific Aim 2: Conduct a Transwell migration assay to asses breast to bone migration of the indicated breast cancer cell lines in the presence of bone cells. References 1. Suva, L. J., et al. (2011). Bone metastasis: Mechanisms and therapeutic opportunities. Nature Reviews.Endocrinology, 7(4):208-18 2. Gruvberger-Saal, et al. (2007). Estrogen receptor beta expression is associated with tamoxifen response in ERalpha-negative breast carcinoma. Clinical Cancer Research:, 13(7), 1987-1994. 3.Merrell, et al. (2010). Differential recruitment of nuclear receptor coregulators in ligand-dependent transcriptional repression by estrogen receptor-alpha. Oncogene, 30(13):1608-1614. 4. Rabbani, S. A., & Mazar, A. P. (2007). Evaluating distant metastases in breast cancer: From biology to outcomes. Cancer Met. Reviews, 26(3-4), 663-674. 5.Koro, et al.(2010). Interactions between breast cancer cells and bone marrow derived cells in vitro define a role for osteopontin in affecting breast cancer cell migration. Breast Cancer Research and Treatment, 126(1):73-83   KDa 130 95 72 Figure 1: Transwell® Migration Assay plate set up10. The migration of MCF-7, HTB126 parental and ERα transfected cells will be observed in response to Hs68 cells or BMDCs. The assay will contain a 8 μm pore insert seeded with the breast cells. The cells that were capable of successfully migrating (after a period of 24 hours) will be found on the inferior side of the membrane. These cells will be stained with haemotoxylin and counted using a microscope on a microscope slide. Image taken from: Koro, K., Parkin, S., Pohorelic, B., Yang, A. D., Narendran, A., Egan, C., Magliocco, A. (2010). Interactions between breast cancer cells and bone marrow derived cells in vitro define a role for osteopontin in affecting breast cancer cell migration. Breast Cancer Research and Treatment, 126(1):73-83. TT 24 hrs TT 72 hrs HTB126 TT 48 hrs MCF-7 HTB126 pER1 G800 HTB126 pER1 G700 HTB126 pER1 G600 Figure 2:. Slight expression of ER1 protein was seen at the translational level in G700 and G800 clones. Parental HTB126 served as negative controls while MCF-7 and Transiently transfected HTB126 cell lines taken at 24, 48 and 72 hr served as positive controls for the experiment. Faint bands at ~67 KDa indicate presence of ERα protein in the different transfected clones. . General Hypothesis Cancer cells expressing ERα will respond preferentially to bone cells with respect to migration in the presence of bone marrow derived cells (BMDCs).