Abstract A31: mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Purpose of the Study: Phosphorylation of estrogen receptor α (ERα) Ser167 is associated with development of endocrine resistance, and is a prognostic marker for the disease. Hereby we dissect the signaling pathways responsible for Ser167 phosphorylation. Experimental Procedures: MCF7 cells were grown in standard media. For experiments, cells were deprived of serum for 24h, and were pre‐treated with 20ng/mL rapamycin for 30 min and/or 10 µM U0126 for 2h, and then stimulated with 100nM insulin or 100ng/mL PMA. Cell extracts were prepared, and proteins were resolved on SDS‐PAGE, followed by immunoblots. For the siRNA studies, double‐stranded RNAs for RSK1 and RSK2 were transfected using Lipofectamine2000 (Invitrogen). At 72h post‐transfection, cells were lysed using 1X Passive Lysis Buffer (Promega). For luciferase reporter assays, cells were transfected with plasmids encoding for firefly luciferase under control of three ERE, and control renilla luciferase. At 48h post‐transfection, cells were harvested and relative luciferase activity was measured using the Dual Luciferase Reporter Assay System and Glomax 20/20 luminometer (Promega). Data: mTOR/S6K1 and MAPK/RSK contribute non‐overlapping inputs into ERα activation via Ser167 phosphorylation. The degree of input of each pathway depends on the nature of the activating signal. In unsynchronized cells that are growing in the presence of serum or in cells acutely stimulated with insulin, which preferentially activated the mTOR/S6K1 pathway, we observed that the MAPK/RSK pathway has a minor contribution to Ser167 phosphorylation. However, the MAPK/RSK contribution is more evident following acute stimulation with PMA, which activates both the mTOR/S6K1 and MAPK/RSK pathways. In response to PMA we observed significant rapamycin‐insensitive Ser167 phosphorylation, which was U0126‐sensitive. Using RNAi against RSK1/2 isoforms we demonstrated that this rapamycin‐insensitive phosphorylation is mediated by RSK. In addition to being sensitive to different stimuli, the mTOR and MAPK pathways are also activated with different kinetics. S6K1 and RSK perform a non‐redundant role with regard to ERα Ser167 phosphorylation, and are responsible for a biphasic phosphorylation pattern. The early‐activated RSK acts in a priming role, while the late‐responding S6K1 is responsible for the maintenance of high‐level phosphorylation. Estrogen receptor is an important clinical target in treatment of breast cancer. However, resistance to anti‐estrogen therapy often develops. One of the mechanisms of resistance is through mitogenic estrogen‐independent activation of ERα. Thus, a question arises whether dual inhibition of the mTOR and MAPK pathways may result in a better clinical response. We investigated the effect of a combination of rapamycin and U0126 on transcriptional activity mediated by ERα. We demonstrated that the combination produces a greater inhibition of ERα activity than each agent alone. This strategy may be effective for combating resistance to anti‐estrogen or endocrine therapy. Conclusions: The data presented here reveal that the effect of rapamycin therapy in breast cancer could be augmented by combination with MEK inhibitors, which would target two major pathways leading to ERα activation and stimulation of breast cancer cell proliferation. Citation Information: Cancer Res 2009;69(23 Suppl):A31.