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Supplemental Materials and Methods and Supplemental References

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β. The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β. Notably, EAD was the most effective combination in inducing differentiation of breast tumor–initiating cells in vivo. Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013–24. ©2016 AACR.

Supplemental Figures: Supplementary Figure 1 - Paclitaxel or carboplatin is less effective than doxorubicin in combination with entinostat and ATRA on induction of cell death. Supplementary Figure 2 - Paclitaxel or carboplatin in the combination is less effective than doxorubicin on induction of RAR-β expression; depletion of RAR-β decreases sensitivity to EAD; and transcriptional regulation of RAR-β by TopoII-β Supplementary Figure 3 - EAD decreases the number of CD44+/CD24-/Epcam+ cells and induces expression of differentiation markers Supplementary Figure 4 - EAD potentiates differentiation in vivo. Supplementary Figure 5 - ELF-3 is increased in differentiated stem cell and mediates the entinostat-differentiation effect. Supplementary Figure 6 - EAD targets metastatic samples.

Supplemental Tables: Supplementary Table S1A. Estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapies. Supplementary Table S1B. Comparison of the estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapies. Supplementary Table S2A. Estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapy. Supplementary Table S2B. Comparison of the estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and doxorubicin as single and combined therapy. Supplementary Table S3A. Estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S3B. Comparison of the estimated tumor volume of MDA-MB-231 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapies. Supplementary Table S4A. Estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S4B. Comparison of the estimated tumor volume of SUM-159 xenografts treated with entinostat, ATRA, and carboplatin as single and combined therapy. Supplementary Table S5. Necrosis scores in mouse xenografts. Supplementary Table S6. EAD treatment shows reduced tumor incidence at limiting dilution. Supplementary Table S7. Characteristics of primary patient samples.

Supplemental Figure S1 Legend Supplemental Figure S2 Legend Supplemental Figure S3 Legend Supplemental Figure S4 Legend Supplemental Figure S5 Legend Supplemental Figure S6 Legend

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-β. The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-β expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-β (TopoII-β) and relieved TopoII-β-mediated transcriptional silencing of RAR-β. Notably, EAD was the most effective combination in inducing differentiation of breast tumor–initiating cells in vivo. Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013–24. ©2016 AACR.