Increased activation of ERK signaling has been reported in breast cancer models of acquired tamoxifen resistance. Here, we examined the expression of Mitogen-Activated Protein Kinase Phosphatases (MKPs) 1 and 2 following tamoxifen treatment and the effects of MKP-1/MKP-2 overexpression on tamoxifen sensitivity. Treatment of MCF7 breast cancer cells with tamoxifen increased MKP-2, but not MKP-1, protein levels. Overexpression of MKP-1 or MKP-2 inhibited estrogen-induced MCF7 cell proliferation compared to vector controls. MCF7-MKP-2 cells displayed significantly increased sensitivity to tamoxifen as compared to vector control or MCF7-MKP-1 cells. MKP-1 or MKP-2 overexpression eliminated ERK1/2 phosphorylation, suggesting that decreases in estrogen-induced proliferation of MKP-1 and MKP-2 overexpressing cells are due to ERK1/2 dephosphorylation. JNK1/2 activation was not detectable in any of these cells. These data suggest that tamoxifen-induced death of these cells is not dependent upon JNK signaling, but rather that ERK is the major MAPK driving their proliferation. MCF7-TAMR cells express higher levels of MKP-2 mRNA and protein than MCF7 cells. MKP-2 and phospho-ERK1/2 proteins are constitutively expressed in MCF7-TAMR cells, and activated JNK1/2 is not detectable. These data suggest that MKP-2 rather than MKP-1 is tamoxifen-regulated and that the elevated expression of MKP-2 in MCF7-TAMR cells potentially functions to restore tamoxifen sensitivity.

The majority of patients develop resistance against suppression of HER2-signaling mediated by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Using gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further restricted to markers significantly associated with worse survival. Functional significance of the two strongest predictive markers was evaluated in vitro by gene silencing in HER2 overexpressing, trastuzumab resistant BC cell lines SKTR and JIMT-1. The strongest predictive MKPs were DUSP4/MKP-2 (AUC=0.75, p=0.0096) and DUSP6/MKP-3 (AUC=0.77, p=5.29E-05). Higher expression for these correlated to worse survival (DUSP4: HR=2.05, p=0.009 and DUSP6: HR=2, p=0.0015). Silencing of DUSP4 had significant sensitization effects - viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected controls (SKTR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Our findings suggest that DUSP4 may represent a new potential target to overcome trastuzumab resistance.