Epigenetic silencing of tumor suppressor Par-4 promotes chemoresistance in recurrent breast cancer
Nathaniel W. Mabe, … , J. Will Thompson, James V. Alvarez
Nathaniel W. Mabe, … , J. Will Thompson, James V. Alvarez
Published August 27, 2018
Citation Information: J Clin Invest. 2018;128(10):4413-4428. https://doi.org/10.1172/JCI99481.
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Research Article Oncology

Epigenetic silencing of tumor suppressor Par-4 promotes chemoresistance in recurrent breast cancer

Abstract

Tumor relapse is the leading cause of death in breast cancer, largely due to the fact that recurrent tumors are frequently resistant to chemotherapy. We previously reported that downregulation of the proapoptotic protein Par-4 promotes tumor recurrence in genetically engineered mouse models of breast cancer recurrence. In the present study, we examined the mechanism and functional significance of Par-4 downregulation in recurrent tumors. We found that epithelial-to-mesenchymal transition (EMT) promotes epigenetic silencing of Par-4 in recurrent tumors. Par-4 silencing proceeded through binding of the EMT transcription factor Twist to the Par-4 promoter, where Twist induced a unique bivalent chromatin domain. This bivalent configuration conferred plasticity at the Par-4 promoter, and Par-4 silencing could be reversed with pharmacologic inhibitors of Ezh2 and HDAC1/2. Using an epigenome editing approach to reexpress Par-4 by specifically reversing the histone modifications found in recurrent tumors, we found that Par-4 reexpression sensitized recurrent tumors to chemotherapy in vitro and in vivo. Upon reexpression, Par-4 bound to the protein phosphatase PP1, caused widespread changes in phosphorylation of cytoskeletal proteins, and cooperated with microtubule-targeting drugs to induce mitotic defects. These results identify Twist-induced epigenetic silencing of Par-4 as a targetable axis that promotes chemoresistance in recurrent breast cancer.

Authors

Nathaniel W. Mabe, Douglas B. Fox, Ryan Lupo, Amy E. Decker, Stephanie N. Phelps, J. Will Thompson, James V. Alvarez

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Figure 7

Epigenome editing to reverse Par-4 silencing prolongs survival of mice with recurrent tumors.

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Epigenome editing to reverse Par-4 silencing prolongs survival of mice w...
(A and B) Western blot (A) and qPCR (B) analysis of Par-4 expression in recurrent tumor cells expressing dCas9-p300 and either a control sgRNA (NT) or 1 of 2 sgRNAs targeting the Par-4 promoter. (C) ChIP-qPCR showing H3K9ac and H3K27ac enrichment at the Par-4 promoter in recurrent tumor cells expressing dCas9-p300 and a nontargeting sgRNA or 1 of 2 sgRNAs targeting the Par-4 promoter. (D) Schema of AC+T treatment in the orthotopic model. A total of 12 mice were injected bilaterally into the fourth mammary glands. Once tumors reached approximately 75 mm3, mice were randomized into either untreated (n = 3 mice, 6 tumors per cohort) or AC+T (n = 9 mice, 18 tumors per cohort) groups. Mice were sacrificed once tumors reached at least 1,800 mm3 in size. (E) Mean growth rate measurements for treatment-naive recurrent tumors expressing control sgRNA (NT) or 1 of 2 sgRNAs targeting the Par-4 promoter. P values were calculated by 1-way ANOVA and Tukey’s post hoc test. (F) Area under the curve measurements for chemotherapy-treated recurrent tumors expressing control sgRNA (NT) or 1 of 2 sgRNAs targeting the Par-4 promoter. P values were calculated by 1-way ANOVA and Tukey’s post hoc test. Differences between experimental and control tumors are shown. (G) Kaplan-Meier plots showing endpoint survival of recurrent tumors expressing a control sgRNA (NT) or sgRNAs targeting the Par-4 promoter. P values, hazards ratios, and 95% confidence intervals are indicated as compared with sgNT. Statistical significance was determined by Mantel-Cox log rank test. Note that the survival curve for the control cohort (NT) represents the same tumors as shown in the black dashed line in Figure 6A. (H) qPCR analysis of Par-4 expression in untreated and treated orthotopic recurrent tumors expressing sgNT, sgPar-4/1, or sgPar-4/2. Significance was determined by 1-way ANOVA and Tukey’s post hoc test. Error bars denote mean ± SEM.