The MAPK inhibitor, MKP3, is an estrogen receptor α coactivator associated with tamoxifen resistance

4364 Tamoxifen (Tam) is the most frequently prescribed antiestrogen for the prevention and treatment of ER positive breast cancers, but while Tam is initially useful in many breast cancer patients, tamoxifen resistance (TR) eventually will develop. It has been shown recently that enhanced growth factor signaling is associated with TR, but the mechanisms are still undefined. To examine acquired resistance, we performed a microarray expression profiling study to identify genes associated with clinical TR with two groups of tumors: Tam Sensitive (TS, n=4) were primary tumors from patients who were treated with Tam and had not experienced a recurrence, and TR tumors (n=5) were metastatic breast tumors from patients who were treated with Tam and whose metastatic lesions recurred while on treatment. We found that the major cellular inhibitor of MAPK, MAPK Phosphatase 3 (MKP3, also called DUSP6), was significantly overexpressed in the TR group of tumors. Since we observed that there were two “LXXLL” motifs, called NR boxes, in MKP3 with similarity to nuclear receptor interaction domains, we hypothesized that MKP3 could interact with ER, and function as an ERα coactivator protein. Through transient transactivation assay, we demonstrated that MKP3 could potentiate ligand-induced ERα activity in ERα-positive breast cancer cell lines in a dose-dependent manner. We also found that MKP3 expression enhances ligand-dependent transcriptional activity of other nuclear receptors, such as androgen receptor, progesterone receptor, and retinoic acid receptor, but not a TATA-box driven promoter, demonstrating specificity for nuclear receptors. We next engineered MCF-7 cells to stably overexpress MKP3 to study its effects on estrogen-induced growth and tamoxifen response. MKP3 overexpression led to significantly increased growth in soft agar in the presence of tamoxifen which could be inhibited by the pure steroidal antiestrogen fulvestrant. A preliminary experiment demonstrated that MKP3 overexpression did not affect estrogen-stimulated xenograft growth compared with vector-control tumors. However, in the Tam-treated groups, the MKP3 expressing xenografted tumors continued to grow (p=0.047), suggesting that MKP3 overexpression might confer TR in ERα-positive breast cancer cells. In estrogen-treated MKP3 transfectants, MAPK was inhibited. But we observed increased phospho-ERK1/2 levels, and phosphorylation at S118 ERα in tamoxifen-treated MKP3-overexpressing cells, suggesting that the interaction of ERα and MKP3 might represent a novel mechanism independent of its effects on MAPK. Alternatively, MKP3 overexpression overexpression may lead to a paradoxical activation of MAPK in tamoxifen-treated tumors. In summary, our data suggest that MKP3 is a new ERα coactivator which may be involved in the evolution of clinical resistance.