Fbxo22-mediated KDM4B degradation determines selective estrogen receptor modulator activity in breast cancer
Yoshikazu Johmura, … , Tomohiko Ohta, Makoto Nakanishi
Yoshikazu Johmura, … , Tomohiko Ohta, Makoto Nakanishi
Published November 12, 2018
Citation Information: J Clin Invest. 2018;128(12):5603-5619. https://doi.org/10.1172/JCI121679.
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Research Article Endocrinology

Fbxo22-mediated KDM4B degradation determines selective estrogen receptor modulator activity in breast cancer

Abstract

The agonistic/antagonistic biocharacter of selective estrogen receptor modulators (SERMs) can have therapeutic advantages, particularly in the case of premenopausal breast cancers. Although the contradictory effects of these modulators have been studied in terms of crosstalk between the estrogen receptor α (ER) and coactivator dynamics and growth factor signaling, the molecular basis of these mechanisms is still obscure. We identify a series of regulatory mechanisms controlling cofactor dynamics on ER and SERM function, whose activities require F-box protein 22 (Fbxo22). Skp1, Cullin1, F-box–containing complex (SCFFbxo22) ubiquitylated lysine demethylase 4B (KDM4B) complexed with tamoxifen-bound (TAM-bound) ER, whose degradation released steroid receptor coactivator (SRC) from ER. Depletion of Fbxo22 resulted in ER-dependent transcriptional activation via transactivation function 1 (AF1) function, even in the presence of SERMs. In living cells, TAM released SRC and KDM4B from ER in a Fbxo22-dependent manner. SRC release by TAM required Fbxo22 on almost all ER-SRC–bound enhancers and promoters. TAM failed to prevent the growth of Fbxo22-depleted, ER-positive breast cancers both in vitro and in vivo. Clinically, a low level of Fbxo22 in tumor tissues predicted a poorer outcome in ER-positive/human epidermal growth factor receptor type 2–negative (HER2-negative) breast cancers with high hazard ratios, independently of other markers such as Ki-67 and node status. We propose that the level of Fbxo22 in tumor tissues defines a new subclass of ER-positive breast cancers for which SCFFbxo22-mediated KDM4B degradation in patients can be a therapeutic target for the next generation of SERMs.

Authors

Yoshikazu Johmura, Ichiro Maeda, Narumi Suzuki, Wenwen Wu, Atsushi Goda, Mariko Morita, Kiyoshi Yamaguchi, Mizuki Yamamoto, Satoi Nagasawa, Yasuyuki Kojima, Koichiro Tsugawa, Natsuko Inoue, Yasuo Miyoshi, Tomo Osako, Futoshi Akiyama, Reo Maruyama, Jun-ichiro Inoue, Yoichi Furukawa, Tomohiko Ohta, Makoto Nakanishi

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Figure 2

Fbxo22 forms a ternary complex with ER and KDM4B in a ligand type–dependent manner in MCF7 cells.

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Fbxo22 forms a ternary complex with ER and KDM4B in a ligand type–depend...
(A) MCF7 cells expressing the indicated doxycycline-inducible shRNAs (Dox-shRNA-MCF7 cells) were treated with doxycycline (1 μg/ml). At the indicated time points, the lysates were subjected to immunoblotting. (B) The indicated Dox-shRNA-MCF7 cells, in the presence of doxycycline (1 μg/ml) for 24 hours, were treated with 50 μg/ml cycloheximide (CHX) and analyzed as in A. The relative KDM4B intensities were determined using ImageJ software. Data are presented as the mean ± SD of 3 independent experiments. ****P < 0.001, by 2-tailed Student’s t test. (C) Dox-FLAG-HA-Fbxo22-MCF7 cells, in the presence or absence of doxycycline (1 μg/ml) for 48 hours, were treated with MG132 (10 μg/ml) for 4 hours. The whole-cell extracts (WCEs) were sequentially immunoprecipitated using anti-FLAG M2 gel and anti-HA gel and then subjected to immunoblotting. (D) MCF7 cells were treated with MG132 (10 μg/ml) for 4 hours. WCEs were immunoprecipitated and subjected to immunoblotting using the indicated antibodies. (E and F) The indicated Dox-shRNA-MCF7 cells were incubated with doxycycline (1 μg/ml) for 48 hours and analyzed as in D. (G) MCF7 cells, Dox-WT FLAG-Fbxo22 (WT) cells, or mutant cells lacking FIST-N (ΔFN) or FIST-C (ΔFC) were treated as in C. WCEs were immunoprecipitated using anti-FLAG M2 affinity gel and subjected to immunoblotting. (H) Dox-FLAG-Fbxo22-MCF7 cells were treated as in C. WCEs were sequentially immunoprecipitated with anti-FLAG M2 gel and anti-ER antibodies and subjected to immunoblotting. Dox-FLAG-Fbxo22-MCF7 cells were starved in E2-depleted medium with or without doxycycline (1 μg/ml) for 72 hours and treated with or without 0.1 nM, 1 nM, or 10 nM E2 (I) or E2 (10 nM) and/or 1 nM, 10 nM, or 100 nM 4-OHT (J) in the presence of MG132 (10 μg/ml) for 6 hours. WCEs were immunoprecipitated using the indicated antibodies and subjected to immunoblotting.