DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine
Jia Yu, … , Matthew P. Goetz, Liewei Wang
Jia Yu, … , Matthew P. Goetz, Liewei Wang
Published April 30, 2018
Citation Information: J Clin Invest. 2018;128(6):2376-2388. https://doi.org/10.1172/JCI97924.
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Research Article Oncology Therapeutics

DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis that lacks targeted therapies, especially in patients with chemotherapy-resistant disease. Since DNA methylation-induced silencing of tumor suppressors is common in cancer, reversal of promoter DNA hypermethylation by 5-aza-2′-deoxycytidine (decitabine), an FDA-approved DNA methyltransferase (DNMT) inhibitor, has proven effective in treating hematological neoplasms. However, its antitumor effect varies in solid tumors, stressing the importance of identifying biomarkers predictive of therapeutic response. Here, we focused on the identification of biomarkers to select decitabine-sensitive TNBC through increasing our understanding of the mechanism of decitabine action. We showed that protein levels of DNMTs correlated with response to decitabine in patient-derived xenograft (PDX) organoids originating from chemotherapy-sensitive and -resistant TNBCs, suggesting DNMT levels as potential biomarkers of response. Furthermore, all 3 methytransferases, DNMT1, DNMT3A, and DNMT3B, were degraded following low-concentration, long-term decitabine treatment both in vitro and in vivo. The DNMT proteins could be ubiquitinated by the E3 ligase, TNF receptor–associated factor 6 (TRAF6), leading to lysosome-dependent protein degradation. Depletion of TRAF6 blocked decitabine-induced DNMT degradation, conferring resistance to decitabine. Our study suggests a potential mechanism of regulating DNMT protein degradation and DNMT levels as response biomarkers for DNMT inhibitors in TNBCs.

Authors

Jia Yu, Bo Qin, Ann M. Moyer, Somaira Nowsheen, Tongzheng Liu, Sisi Qin, Yongxian Zhuang, Duan Liu, Shijia W. Lu, Krishna R. Kalari, Daniel W. Visscher, John A. Copland, Sarah A. McLaughlin, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Zhenkun Lou, Vera J. Suman, Richard Weinshilboum, Judy C. Boughey, Matthew P. Goetz, Liewei Wang

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Figure 3

Decitabine treatment induced lysosome-dependent degradation of DNMTs.

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Decitabine treatment induced lysosome-dependent degradation of DNMTs. (A...
(A) DNMT-specific Western blot using whole-tissue lysate from the indicated PDX tumors treated with either vehicle (n = 3 mice) or decitabine (n = 3 mice). Protein was quantified using ImageJ software (NIH), and relative protein levels were corrected for the vehicle group. Data represent mean ± SD from n = 3 different samples. **P < 0.01, ***P < 0.001, 2-tailed t tests. (B) Relative mRNA levels of DNMT1, DNMT13A and DNMT13B genes in tumor samples after decitabine treatment compared with samples treated with vehicle control group (n = 3 independent experiments). (C) Breast cancer cells were treated with 100 nM decitabine (DAC) for 7 days, and Western blot analysis was performed using the indicated antibodies. (D) Breast cancer cells were treated with 100 nM decitabine for 7 days, and mRNA levels were quantified by qRT-PCR (n = 3 independent experiments). (E) Representative well from clonogenic assay of 3 different breast cancer cell lines 14 days after decitabine treatment. Average colony number was quantified for each well, and each experiment was conducted as n = 3 independent experiments in triplicate. ***P < 0.001, 2-tailed t test. (F) MDA-MB-231 cells were treated with decitabine for 7 days and treated with concanamycin A, calpetin, Z-VAD-FAM, and MG132 for an additional 24 hours. The protein levels of DNMT1, DNMT3A, and DNMT3B were analyzed by Western blot and quantified by ImageJ software (n = 3). (G) MDA-MB-231 cells were treated with decitabine for 7 days, and the ubiquitination of DNMTs was determined by pulling down DNMTs with the indicated antibodies followed by Western blot analysis.