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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

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ICI182780 treatment, nor G1, a GPR30 specific agonist, released S1P from MCF7 cells. Conclusions: E2 stimulates the rapid activation of SphK1 through non-genomic ER-alpha, but not GPR30, which leads to export of S1P out of breast cancer cells. 56.6. Estradiol-Induced Sphingosine-1-Phosphate Is Released via ABCC1 and ABCG2, and Activates ERK1/2. M. Nagahashi, R. H. Kim, J. C. Allegood, S. Ramachandran, S. Spiegel, K. Takabe; Virginia Commonwealth University, Richmond, VA Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that regulate a wide variety of important processes of breast cancer biology, including cell proliferation, survival, migration, and angiogenesis. Two specific sphingosine kinase isoenzmes, SphK1 and SphK2, which have different subcellular localizations and distinct functions, produce S1P in response to multiple stimuli including estradiol (E2) and EGF. S1P is then released out of the cell and acts in an autocrine or paracrine manner by binding to S1P specific cell surface G protein-coupled receptors. SphK1 is overexpressed in many types of cancer and promotes E2-dependent xenograft tumorigenesis of MCF7 breast cancer cells. We have found that overexpression of SphK1, but not SphK2, increases S1P release from MCF7 cells, although both increased intracellular S1P. Conversely, downregulation of SphK1, but not SphK2, with specific siRNAs, decreased release of S1P. Although both E2 and EGF activated SphK1, we found that only E2 stimulated rapid release of S1P from MCF7 cells, as determined by labeling with [3H]sphingosine and differential extraction of [3H]S1P. Similar results were demonstrated by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) that E2 dose dependently allowed immediate S1P release, when EGF did not. Since ATP-binding cassette (ABC) transporters (also known as multi-drug resistant gene family) have been reported to be involved in secretion of chemotherapeutic drugs as well as lipids, we have investigated its role in S1P release from breast cancer cells. Release of S1P was inhibited by MK571, an inhibitor of ABCC1, and Fumitrimorgin C, an inhibitor of ABCG2. This result was consistent with downregulation of ABCC1 or ABCG2 with specific siRNA, which decreased S1P release determined by LC-ESI-MS/MS. Furthermore, inhibiting these transporters also blocked E2-induced activation of ERK1/2, indicating that E2 activates ERK via downstream signaling of S1P. This is the first demonstration that the multi-drug resistant transporter ABCC1 and ABCG2 are involved in E2-induced immediate release of S1P produced by SphK1. Taken together, our findings suggest that E2-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to non-genomic signaling of E2 in breast cancer pathophysiology. 56.7. Microrna-222 Modulates Tamoxifen Resistance in Papillary Thyroid Cancer. H. Suh, S. Sur, S. Orzell, J. E. Rosen; Boston University, Boston, MA Introduction: microRNAs (miRNAs) are a class of small noncoding RNAs whose role in cancer development and progression is a rapidly expanding field of study. In papillary thyroid carcinoma (PTC), several miRNAs have been demonstrated to be significantly upregulated, including miR222. mir222 has been shown in breast cancer in vitro studies to regulate estrogen receptor (ER) expression and resistance to the anti-tumor drug tamoxifen. As PTC is more prevalent in women, we were interested in identifying gender-specific factors which may play a role in its progression. Here we hypothesized that PTC is also sensitive to estrogen blockade, and that this sensitivity can be modulated by altering the expression of miR222. Materials and Methods: BCPAP (human PTC cell line) and MCF7 (human breast cancer cell line) were grown in appropriate media. A chemical transfection of pre-miR-222, anti-miR-222 and random sequence antimiR negative control oligonucleotides was performed. After 24 hours,

each cell line was treated with serial concentrations of tamoxifen from 0-10 uM and cell proliferation was measured by MTT assay after 72 hours. All experiments were performed in triplicate. Results: Both BCPAP and MCF7 treated with our scramble negative control showed an appropriate dose-response decrease in proliferation to increasing doses of tamoxifen. Following transfection with premiR222, both BCPAP and MCF7 cells showed an increase in proliferation at each dose of tamoxifen compared to negative control. In comparison, BCPAP and MCF7 cells transfected with anti-miR222 showed a decrease in proliferation at each dose of tamoxifen in comparison to negative control, resulting in a 5-fold difference in proliferation between ectopic and knock-down expression of miR222. Discussion: Here we demonstrate for the first time that modulation of microRNA 222 can alter tamoxifen sensitivity in our in vitro model of papillary thyroid carcinoma; knock-down of miR222 lead to an increase in tamoxifen sensitivity while enforced expression of miR222 lead to a decrease in tamoxifen sensitivity in comparison to our scramble negative control. While the negative regulation of targeted protein-coding genes by miRNAs is well documented, the specific actions of key miRNAs found to be significantly associated with cancer are less well understood. Further investigation is underway to assess the changes in estrogen-receptor expression and function in thyroid