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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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 4

The E3 ubiquitin-protein ligase TRAF6 interacts with and ubiquitinates DNMTs.

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The E3 ubiquitin-protein ligase TRAF6 interacts with and ubiquitinates D...
(A) DNMT1, DNMT3A, and DNMT3B proteins contain a potential TRAF6-binding site. The consensus TRAF6-binding site motif has been described previously (78). (B) Endogenous interactions of TRAF6 with DNMT1, DNMT3A, and DNMT3B. MDA-MB-231 cells were used to perform endogenous immunoprecipitation with TRAF6 antibody, followed by Western blot with indicated DNMT antibodies. (C–E) In vitro binding assays of TRAF6 and DNMTs. Myc-TRAF6 expressed in E. coli was purified and incubated with GST-DNMT1, His-DNMT3B, and Flag-DNMT3A with agarose beads. The precipitated samples were analyzed by Western blot using indicated antibodies. (F) Schematic representation of WT TRAF6 and its different deletion mutants. (G) FLAG-tagged TRAF6 constructs were transfected into 293T cells, and FLAG beads were used for immunoprecipitation. Precipitates were subjected to Western blot analysis with indicated antibodies. WCL, whole-cell lysates. (H) MDA-MB-231 cells were transiently transfected with negative control or TRAF6-specific siRNAs for 72 hours. DNMT protein from the cell lysates was immunoprecipitated with corresponding anti-DNMT antibody. The ubiquitinated DNMTs were detected by Western blot using anti-ub antibody at high molecular weight ladders. (I) Crispr/Cas9 TRAF6-KO cell line was generated and WT and Crispr/Cas9 TRAF6-KO cell lines were subjected to immunoprecipitation using anti-DNMT antibodies. The ubiquitinated DNMTs were detected by Western blot analysis. (J) MDA-MB-231 TRAF6 Crispr/Cas9-KO cells (KO1) were transfected with FLAG-tagged WT TRAF6 (WT) and catalytic-dead CA mutant (C70A). DNMTs from the cell lysates were immunoprecipitated with anti-DNMT1, anti-DNMT13A, and anti-DNMT13B antibodies. The ubiquitinated DNMTs were detected by Western blot analysis.