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. 2018 Dec 3;128(12):5603-5619.
doi: 10.1172/JCI121679. Epub 2018 Nov 12.

Fbxo22-mediated KDM4B Degradation Determines Selective Estrogen Receptor Modulator Activity in Breast Cancer

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Free PMC article

Fbxo22-mediated KDM4B Degradation Determines Selective Estrogen Receptor Modulator Activity in Breast Cancer

Yoshikazu Johmura et al. J Clin Invest. .
Free PMC article

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.

Keywords: Breast cancer; Endocrinology; Molecular diagnosis; Ubiquitin-proteosome system.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Proteasome-dependent protein degradation is required for the antagonistic activity of TAM.
(A) Experimental outline (top). MCF7 cells starved of E2 for 72 hours were cultured in medium containing E2 (10 nM) for 6 hours and then incubated in medium containing 4-OHT (100 nM) with or without MG132 (10 μg/ml). Total RNA from the treated cells collected at the indicated time points was subjected to qRT-PCR analysis using the indicated primers. Data are presented as the mean ± SD of 3 independent experiments. ****P < 0.001 and ***P < 0.005, by 2-tailed Student’s t test. (B) Nuclear extracts (NEs) of cells treated as in A were collected at 12 hours and immunoprecipitated using the indicated antibodies and then subjected to immunoblotting. (C) MCF7 cells starved of E2 for 72 hours were cultured with medium containing E2 (10 nM) for 18 hours (E2), or for 6 hours with or without MG132 (10 μg/ml) and then starved of E2 (E2-dep) for 12 hours. Total RNA from the treated cells was subjected to qRT-PCR analysis using the indicated primers. Data are presented as the mean ± SD of 3 independent experiments. *P < 0.05, by 2-tailed Student’s t test. (D) Nuclear extracts of cells treated as described in C were immunoprecipitated using the indicated antibodies and subjected to immunoblotting. (E) MCF7 cells expressing the indicated doxycycline-inducible shRNAs (Dox-shRNA-MCF7 cells) were starved of E2 in the presence of doxycycline (1 μg/ml) for 72 hours and then treated with E2 (10 nM) for 6 hours. Nuclear extracts were immunoprecipitated using the indicated antibodies and subjected to immunoblotting.
Figure 2
Figure 2. Fbxo22 forms a ternary complex with ER and KDM4B in a ligand type–dependent manner in MCF7 cells.
(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.
Figure 3
Figure 3. SCFFbxo22 preferentially ubiquitylates KDM4B complexed with unliganded or 4-OHT–bound ER.
(A) Lysates from MCF7 cells expressing Fbxo22 and/or ER were subjected to immunoblotting using the indicated antibodies. Numbers shown at the bottom of the KDM4B blot indicate relative signal intensities. (B) Fbxo22-KO HeLa cells were transfected with the indicated plasmids, treated with MG132, and lysed under denaturing conditions. WCEs were subjected to StrepTactin (Strep) pulldown, followed by immunoblotting. (C) Fbxo22-KO HeLa cells expressing the same genes as indicated in B were treated in the presence or absence of E2 and 4-OHT. WCEs were subjected to StrepTactin pulldown, followed by immunoblotting. St2-KDM4B, tandem strep-II–tagged KDM4B.
Figure 4
Figure 4. Antagonistic activity of 4-OHT requires Fbxo22 via AF1 activity in MCF7 cells.
(A) Experimental outline (top). Dox–shFbxo22–MCF7 cells were starved of E2 in the presence or absence of doxycycline (1 μg/ml) for 72 hours, treated with E2 (10 nM) for 6 hours, and then treated with 4-OHT (100 nM). Total RNA from the treated cells at the indicated time points was subjected to qRT-PCR analysis. Data are presented as the mean ± SD of 3 independent experiments. *P < 0.05 and ****P < 0.001, by 2-tailed Student’s t test. (B) Nuclear extracts from the cells treated as in A at 12 hours were immunoprecipitated using the indicated antibodies and subjected to immunoblotting. (C) U2OS cells expressing WT ER or its Δ44 mutant were treated and analyzed as in A. ****P < 0.001, by 2-tailed Student’s t test.
Figure 5
Figure 5. Essential role of Fbxo22 in 4-OHT–induced dissociation of SRC-1 from ER in living cells.
(A) The indicated Dox-shRNAs-U2OS-LacO-I-SceI-TetO cells expressing FLAG-KDM4B were transfected with YFP–SRC-1 and CFP-ERα-Lac plasmids, followed by treatment with doxycycline (1 μg/ml) in E2-depleted medium for 72 hours. These cells were then treated with or without E2 (10 nM) and/or 4-OHT (100 nM) for 2 hours and fixed with 4% formaldehyde. The resultant cells were subjected to immunostaining with anti-FLAG antibody. Representative images are shown. Scale bars: 10 μm. (B) Cells positive for triple colocalization of CFP-ERα-Lac, YFP–SRC-1, and FLAG-KDM4B foci were counted. Data are presented as the mean ± SD of 3 independent experiments. **P < 0.01, by 2-tailed Student’s t test.
Figure 6
Figure 6. Genome-wide analysis revealed that TAM-mediated dissociation of SRC-3 from ER requires Fbxo22 on almost all ER- and SRC-3–binding sites.
(A) ChIP binding site counts and proximity relationships. Venn diagram of binding sites for ER with sequence centers within 0.1 kb of each other were identified as a shared peak within 4 data sets (MCF7 cells with E2 and E2 plus 4-OHT, and Fbxo22-depleted MCF7 cells with E2 and E2 plus 4-OHT) and those of SRC-3 were identified as a shared peak within 2 data sets (MCF7 cells with E2 and Fbxo22-depleted MCF7 cells with E2). (B) Tag counts of SRC-3 binding within the 410 shared SRC-3 peaks shown in A. ****P < 0.001, by 1-way ANOVA. (C) Heatmap visualization of ER and SRC-3 in the indicated MCF7 cells. (D) Genome browser snapshot of ChIP-Seq samples for ER (black) and SRC-3 (green) in GREB1 and IGFBP4 gene loci in the indicated MCF7 cells.
Figure 7
Figure 7. Fbxo22 is required for 4-OHT–mediated inhibition of breast cancer cell growth both in vitro and in vivo.
(A) The indicated Dox-shRNA-MCF7 cells with or without doxycycline-inducible FLAG-Fbxo22 were starved of E2 in the presence of doxycycline (1 μg/ml) for 72 hours, treated with E2 (10 nM) in the presence or absence of 4-OHT (100 nM) for 6 hours, and then subjected to a quantitative colony formation assay. Representative images and results (mean ± SD) are from 3 independent experiments. (B) MCF7 cells expressing the indicated doxycycline-inducible shRNAs were treated as in A and subjected to a quantitative colony formation assay. Results are shown as the mean ± SD of 3 independent experiments. (C) Tumor growth of the control (n = 5) or Fbxo22-KO (n = 5) T47D cells transplanted into the mammary fat pads of NOD/Scid mice was measured over a 2-week period with E2 pellet supplementation and then over a 4-week period with TAM pellet supplementation. *P < 0.05, by 2-tailed Welch’s t test. (D) Mice as in C were sacrificed 6 weeks after transplantation. The tumors were then excised and weighed. *P < 0.05, by 2-tailed Welch’s t test. (E) Images of tumors excised from mice as in D are shown. The weight of each tumor is indicated below the images. (F) Paraffin-embedded tumor sections from 5 mice harboring WT or Fbxo22-KO T47D cells were subjected to immunohistochemical analyses using anti–Ki-67 antibodies and antibodies against cleaved caspase 3. Ki-67–positive and cleaved caspase 3–positive cells were counted, and their numbers were normalized to that of cell nuclei in each section. *P < 0.05 and ***P < 0.05, by2-tailed Student’s t test.
Figure 8
Figure 8. Fbxo22 loss predicts a poorer outcome in patients with ER-positive/HER2-negative breast cancers.
Representative images of normal mammary gland tissue (A) and positive (left) and negative (right) immunohistochemical staining for Fbxo22 in human breast cancer tissue (B). Scale bars: 20 μm. RFS was stratified by Fbxo22 protein expression in all T2 ER-positive/HER2-negative breast cancers (C); luminal A–like (low Ki-67) breast cancers (D); node-negative breast cancers (E); TAM-treated breast cancers (F); and in another cohort of T2 ER-positive/HER2-negative breast cancers stained with Fo-22, a monoclonal antibody established in the current study (clinicopathological variables are listed in Supplemental Table 3) (G). Kaplan-Meier survival curves are shown for patients whose cells were positive for Fbxo22 staining (red lines) and for patients whose cells were negative for Fbxo22 staining (blue lines). P values and HRs were calculated using a log-rank test.

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