Regulation of Estrogen related receptor alpha localization and signaling by the kinesin KIF17 Nilsa M. Méndez 1 and Geri E. Kreitzer 2 1 Industrial Biotechnology Department, University of Puerto Rico and 2 Developmental and Cell Biology, Weill Cornell Medical College Abstract Kinesins are motor proteins that transport a variety of cargos such as organelles, vesicles, RNA, protein complexes and even viruses, to specific destinations in the cell in a microtubule and ATP dependent manner. KIF17 is part of the kinesin family and it was found - by the methods of yeast two- hybrid assay and immunoprecipitation - that one of the cargos that interacts with is Estrogen related receptor alpha (ESRRA). ESRRA is an orphan nuclear receptor structurally and functionally similar to the classic estrogen receptors (ER) but its activity is independent of any known ligands, like estrogen or estradiol. However, phosphorylation of ESRRA by EGF signaling pathway can make ESRRA to change its conformation and enhance DNA- binding. ESRRA activity is regulated in part by direct competition with ERs and it must go to the nucleus in order to activate transcription of its target genes. It has been previously reported that KIF17 is involved in regulating CREM mediated transcription by interacting with and controlling the intracellular localization of the transcriptional activator ACT in murine male germ cells. Under the light of this precedent, we hypothesize that KIF17 might be involved in regulating nuclear transport of ESRRA, and hence, its activity. Our goal is to reveal the functional significance of KIF17-ESRRA interaction in breast cancer cells and we will do this by first, determining the mechanisms by which KIF17 controls the intracellular distribution of ESRRA (nuclear vs cytoplasmic) in presence of EGF. We will measure alterations of ESRRA localization and determine the nuclear:cytoplasmic ratio by immunocytochemistry and fluorescence microscopy. Knowing the mechanisms underlying ESRRA regulation is important because its activity is related to aggressive tumor behavior and poor prognoses in breast cancer patients. Introduction Figure 1. Kinesin structure. Kinesins are motor proteins that transport a variety of cargos. The N-term is the motor domain that binds to MT, the coiled-coil domain regulates homodimerization and the C-term is the cargo binding domain Materials and methods Cell lines―the cells used were MCF-7 and MDA-MB-231. These are mammary epithelial cells Estrogen receptor positive and negative, respectively. Cell culture―all cells were grown in 10 cm dishes with DMEM medium supplemented with 10% Fetal Bovine Serum (10% FBS) and 20mM HEPES, incubated at 37 o C and 5% CO 2. Fixation and immunocytochemistry― all cells were rinsed with Phosphate buffered saline (PBS) with Ca 2+ and Mg 2+, fixed during 2 minutes in paraformal- Goals We want to determine the mechanism by which KIF17: References Massarweh, S. and Schiff, R. Resistance to endocrine therapy in breast cancer: exploiting estrogen receptor/growth factor signaling crosstalk. Endocrine-Related Cancer (2006) 13, S15-S24 Ciguere, V. and Barry, J. Epidermal Growth Factor-induced signaling in breast cancer cells results in selective target gene activation by orphan nuclear receptor Estrogen-Related α. Cancer Res 2005; 65: (14) Kotaja, N., Macho, B. and Sassone-Corsi, P. Microtubule-independent and Protein Kinase A-mediated function of KIF17b controls the intracellular transport of activator of CREM in testis (ACT). J. Biol. Chem. 280, Stein, R.A. and McDonnell, D.P. Estrogen-related receptor α as a therapeutic target in cancer. Endocrine-Related Cancer (2006) 13 S25-S32 Results dehyde 2% and permeabilized with -20 o C methanol for 15 secs. Endo- ESRRA was stained using anti human ESRRA mouse monoclonal primary antibody and Cy5 anti mouse secondary antibody. DAPI was used as a nuclear stain. Fluorescence microscopy―Nikon Eclipse TE2000-U microscope was used to take the pictures and they were analyzed using MetaMorph version 6.3r1. EGF treatment―MCF-7 and MDA-MB-231 cells were starved in DMEM Serum Free Medium (Fatty Acid Free BSA) for 36 hours at 37 o C and 5% CO 2. Cells were treated with 100 ng/mL EGF for the final 30 and 60 minutes of the incubation. Phase contrast Endogenous ESRRA DAPI T 0 (control) T 30 (30 mins, +EGF) T 60 (60 mins, +EGF) Figure 5. Intracellular distribution of endogenous ESRRA in MCF-7 cells after EGF treatment Summary Endogenous ESRRA (MCF-7 control) is mainly localized in the nucleus, however after 30 minutes of treating the cells with EGF, ESRRA begins accumulating in the outer periphery of the nucleus. After 60 minutes of treatment, ESRRA is localized within the nucleus and the cytoplasm in MCF-7 cells. In MDA-MB-231, ESRRA is mostly nuclear (Figure 4). Comparing both cell lines, ESRRA localization in MCF-7 (ER positive) cells starts in the nucleus and after the treatment, the distribution becomes both nuclear and cytoplasmic, while MDA-MB-231 is mostly nuclear throughout the whole treatment. Our findings suggest that EGF might be altering ESRRA intracellular localization. Overall, a complete understanding of the mechanism by which EGF and KIF17 are involved in the subcellular translocation of ESRRA is essential and may foster the development of new treatments for breast cancer patients. MCF-7 cells  ER (+) Phase contrast Endogenous ESRRA DAPI T 0 (control) T 30 (30 mins, +EGF) T 60 (60 mins, +EGF) MDA-MB-231 cells  ER (-) Figure 6. Intracellular distribution of endogenous ESRRA in MDA-MB-231 cells after EGF treatment Figure 2. KIF17 colocalizes with microtubules. GFP-KIF17-FL (red) and microtubules (green) 3. regulates interactions of transcriptional co- activators with ESRRA 2. can modulate transcription of ESRRA target genes 1. controls the intracellular distribution of ESRRA (nuclear vs cytoplasmic) Figure 7. Over expression of GFP-ESRRA FL and RFP-KIF17 mut GE in MCF-7 cells Measure nuclear: cytoplasmic ratio to see if there’s a significant change in ESRRA distribution after EGF treatment Look at up/down regulation of transcription of ESRRA target genes (+/- EGF, +/- KIF17) Do live, time-lapse imaging of the movement of ESRRA into the nucleus after EGF treatment (+/- KIF17 GE or tail domain) Abstract Introduction Goals Materials and methods Results Summary What’s next? References ACT MT N CREM Figure 3. KIF17 binds to ACT and transports it into the nucleus, were activates transcription of CREM dependent genes in murine male germ cells. This is a novel regulatory function of KIF17. ESRRA MT N Figure 4. KIF17 might regulate ESRRA activity by controlling its transport into the nucleus. ESRRA is an orphan nuclear receptor that is constitutively active and does not bind to any known physiological ligand. Even though ESRRA is widely expressed in normal tissues, studies have shown that ESRRA is an unfavorable biomarker for breast cancer.