In mice carrying xenograft GDC 0199 tumors composed of HER2-overexpressing MCF-7 cells,

tumor growth was stimulated by tamoxifen treatment (Arpino et al., 2007). Clinical studies also showed that the response rate to TAM was reduced from 50% in ER-positive cases with normal HER2 expression to 17% in ER-positive cases with HER2 overexpression (Chung et al., 2002). The HER2 transmembrane protein (185 kDa), which is encoded by the HER2 gene, consists of an extracellular domain for homo- and hetero-dimerization at the N-terminus, a single membrane spanning region and an intracellular domain for tyrosine kinase activity at the C-terminus (Klapper et al., 1999). HER2 is considered to be an orphan receptor, unlike other HER family members, because HER2 is activated without binding a ligand. HER2 is favored as a dimerization partner within the HER family. HER2 dimerization results in autophosphorylation of the intracellular tyrosine kinase domain and regulates cell growth, differentiation and potentiation of intracellular signaling mainly for the initiation GSK1210151A of cancer formation (Carpenter and Cohen, 1990). The

determinant for the HER2 homo- or hetero-dimerization process with other HER family members is HER2 overexpression (Tzahar et al., 1996). Breast cancer cells that overexpress epithelial-specific ETS transcription factor (ESX/ESE-1/Elf-3) exhibited HER2 gene amplification (Eckel et al., 2003 and Schedin et al., 2004). HER2 overexpression requires the binding of ESX to the HER2 promoter Phosphatidylinositol diacylglycerol-lyase (Chang et al., 1997)

in addition to the binding of DRIP130/Sur2, a metazoan-specific subunit of the human mediator complex, to the transactivation domain of ESX (Asada et al., 2002). The 8 amino acid helical region of ESX mediates its interaction with Sur2; during this process, small organic molecules may interfere with the ESX–Sur2 interaction (Asada et al., 2003). The small molecules reported previously to suppress HER2 expression include adamanolol (Asada et al., 2003), wrenchnolol (Shimogawa et al., 2004), amphipathic isoxazolidine (Lee et al., 2009) and fluoroquinophenoxazine derivatives (Kim et al., 2012). In the present study, we focused on the development of small molecules that were able to down-regulate HER2 expression via inhibition of the ESX–Sur2 interaction. We found that CHO10, a dithiiranylmethyloxy azaxanthone derivative (Fig. 1A), potently inhibited the ESX–Sur2 interaction, which caused the down-regulation of HER2 expression, inhibition of the HER2-mediated signal pathway and apoptosis in HER2-overexpressing breast cancer cells. The inhibitory activity of CHO10 against the HER2-mediated signal pathway sensitized TAM-resistant cancer cells to TAM. HER2, Phospho-HER2 (Tyr877), Phospho-HER2 (Tyr1221/1222), Phospho-HER2 (Tyr1248), EGFR, Phospho-EGFR (Tyr1068), MAPK (Erk1/2), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Thr204), Akt, Phospho-Akt (Ser473), caspase-3, PARP, α-tubulin and Anti-IgG secondary antibody were purchased from Cell Signaling Technology Inc.