A phosphotyrosine switch determines the antitumor activity of ERβ
Bin Yuan, Long Cheng, Huai-Chin Chiang, Xiaojie Xu, Yongjian Han, Hang Su, Lingxue Wang, Bo Zhang, Jing Lin, Xiaobing Li, Xiangyang Xie, Tao Wang, Rajeshwar R. Tekmal, Tyler J. Curiel, Zhi-Min Yuan, Richard Elledge, Yanfen Hu, Qinong Ye, Rong Li
Bin Yuan, Long Cheng, Huai-Chin Chiang, Xiaojie Xu, Yongjian Han, Hang Su, Lingxue Wang, Bo Zhang, Jing Lin, Xiaobing Li, Xiangyang Xie, Tao Wang, Rajeshwar R. Tekmal, Tyler J. Curiel, Zhi-Min Yuan, Richard Elledge, Yanfen Hu, Qinong Ye, Rong Li
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Research Article Oncology

A phosphotyrosine switch determines the antitumor activity of ERβ

Abstract

Estrogen receptors ERα and ERβ share considerable sequence homology yet exert opposite effects on breast cancer cell proliferation. While the proliferative role of ERα in breast tumors is well characterized, it is not clear whether the antitumor activity of ERβ can be mobilized in breast cancer cells. Here, we have shown that phosphorylation of a tyrosine residue (Y36) present in ERβ, but not in ERα, dictates ERβ-specific activation of transcription and is required for ERβ-dependent inhibition of cancer cell growth in culture and in murine xenografts. Additionally, the c-ABL tyrosine kinase and EYA2 phosphatase directly and diametrically controlled the phosphorylation status of Y36 and subsequent ERβ function. A nonphosphorylatable, transcriptionally active ERβ mutant retained antitumor activity but circumvented control by upstream regulators. Phosphorylation of Y36 was required for ERβ-mediated coactivator recruitment to ERβ target promoters. In human breast cancer samples, elevated phosphorylation of Y36 in ERβ correlated with high levels of c-ABL but low EYA2 levels. Furthermore, compared with total ERβ, the presence of phosphorylated Y36–specific ERβ was strongly associated with both disease-free and overall survival in patients with stage II and III disease. Together, these data identify a signaling circuitry that regulates ERβ-specific antitumor activity and has potential as both a prognostic tool and a molecular target for cancer therapy.

Authors

Bin Yuan, Long Cheng, Huai-Chin Chiang, Xiaojie Xu, Yongjian Han, Hang Su, Lingxue Wang, Bo Zhang, Jing Lin, Xiaobing Li, Xiangyang Xie, Tao Wang, Rajeshwar R. Tekmal, Tyler J. Curiel, Zhi-Min Yuan, Richard Elledge, Yanfen Hu, Qinong Ye, Rong Li

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Figure 2

EYA2 inhibits ERβ transcriptional activity by directly dephosphorylating p-Y36.

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EYA2 inhibits ERβ transcriptional activity by directly dephosphorylating...
(A) Sequences of mammalian ERα and ERβ orthologs surrounding the Y36 residue of human ERβ. ClustalW was used for the sequence alignment. (B) The anti–p-Y36 antibody recognized WT ERβ, but not the Y36F mutant or ERα, in IP-Western blotting of FLAG-ER proteins from HEK293T cells. (C) IP-Western blot of FLAG-ERβ in HEK293T cells indicated that EYA2 reduced the p-Y36 signal. (D) EYA2 knockdown in HEK293T cells increased the p-Y36 signal of FLAG-ERβ. (E) Ligand-stimulated p-Y36 signal of endogenous ERβ in MDA-MB-231 cells was reduced by EYA2. EYA2-transfected cells were treated with vehicle, E2, or DPN for 2 hours. The lysates were used in an ERβ-specific IP, followed by immunoblotting with the anti–p-Y36 or anti–total ERβ antibody. (F) Recombinant WT EYA2, but not phosphatase-deficient mutant proteins, efficiently dephosphorylated IP FLAG-ERβ in vitro. (G) Real-time RT-PCR compared WT ERβ and the mutants in activation of the ERβ target genes MDA7 and MSMB in MDA-MB-231 cells. Error bars represent SEM. *P < 0.05; **P < 0.01.