Induction of cell cycle arrest and inflammatory genes by combined treatment with epigenetic, differentiating, and chemotherapeutic agents in triple-negative breast cancer

Abstract

Background: A combination of entinostat, all-trans retinoic acid, and doxorubicin (EAD) induces cell death and differentiation and causes significant regression of xenografts of triple-negative breast cancer (TNBC).

Methods: We investigated the mechanisms underlying the antitumor effects of each component of the EAD combination therapy by high-throughput gene expression profiling of drug-treated cells.

Results: Microarray analysis showed that entinostat and doxorubicin (ED) altered expression of genes related to growth arrest, inflammation, and differentiation. ED downregulated MYC, E2F, and G2M cell cycle genes. Accordingly, entinostat sensitized the cells to doxorubicin-induced growth arrest at G2. ED induced interferon genes, which correlated with breast tumors containing a higher proportion of tumor-infiltrating lymphocytes. ED also increased the expression of immune checkpoint agonists and cancer testis antigens. Analysis of TNBC xenografts showed that EAD enhanced the inflammation score in nude mice. Among the genes differentially regulated between the EAD and ED groups, an all-trans retinoic acid (ATRA)-regulated gene, DHRS3, was induced in EAD-treated xenografts. DHRS3 was expressed at lower levels in human TNBC metastases compared to normal breast or primary tumors. High expression of ED-induced growth arrest and inflammatory genes was associated with better prognosis in TNBC patients.

Conclusions: Entinostat potentiated doxorubicin-mediated cell death and the combination induced inflammatory signatures. The ED-induced immunomodulation may improve immunotherapy. Addition of ATRA to ED may potentiate inflammation and contribute to TNBC regression.

Keywords: Breast cancer; Entinostat; Epigenetic; Immunotherapy.

Fig. 1

ED regulates inflammatory, arrest, and differentiation genes. a Heatmap depicting unsupervised hierarchical clustering of top 10% differentially expressed genes in MDA-MB-231 cells following different treatments (2.5 μM entinostat, 1 μM ATRA, 0.2 μM doxorubicin). Color scale indicates log2 expression values. b Total number of genes regulated in MDA-MB-231 cells by indicated treatments. c Gene set variation analysis (GSVA) scores of gene set analysis (GSEA) hallmark gene sets for samples in the study. Heatmap colors from blue to red represent low to high enrichment; red color bar (right) shows false discovery rate (FDR)-adjusted p value from one-way analysis of variance (ANOVA) test comparing GSVA enrichment scores across treatment groups. d Volcano plot (log2 fold-change (FC) vs – log10 FDR) of genes upregulated and downregulated in MDA-MB-231 cells following treatment with ED in comparison to cells treated singly with E or D. Orange dots represent genes with at least 2-fold increase or decrease in gene expression and FDR < 0.05. e Heatmap of disease and biological functions analysis using Ingenuity® Pathway Analysis software on ED genes. Size and color of the boxes represent – log p value and z score, respectively. E entinostat (Ent), A all-trans retinoic acid (ATRA), D doxorubicin (Dox), Veh vehicle

Fig. 2

ED and EAD induce growth arrest. a qRT-PCR of few genes, related to cell growth arrest and death (identified by array analysis), in MDA-MB-231 cells treated with entinostat (2.5 μM), ATRA (1 μM), and doxorubicin (0.2 μM) singly, and combinations, for 48 h. b Western blot analysis of cyclin A and cyclin D1 on MDA-MB-231 cells, treated as described in text. Flow cytometry determination of percentage of cell cycle distribution of (c) MDA-MB-231 and (d) SUM-149 cells treated with different groups containing doxorubicin 6.25–200 nM (doxorubicin 12.5 nM highlighted on right), for 48 h. RPL39 and GAPDH used as controls for qRT-PCR and western blot, respectively. Student’s t test performed, mean (± SEM) of triplicate results shown. *Compared to entinostat in qRT-PCR and doxorubicinin flow cytometry; #compared to entinostat in flow cytometry: *p < 0.05, **/##p < 0.01, ***p < 0.001. E entinostat, A all-trans retinoic acid, D doxorubicin (Dox), Veh vehicle, GAPDH glyceraldehyde 3-phosphate dehydrogenase

Fig. 3

ED and EAD induce expression of interferon-alpha genes. (A) Hierarchical supervised clustering of expression of interferon (IFN)-α genes against signatures of MDA-MB-231 cells following treatments. (B) qRT-PCR of (a) type 1 IFN genes (CXCL10, STAT1 and IRF1) and (b) interferon-responsive genes (TRIM48 and TRIM51) in MDA-MB-231 cells and (c) CXCL10 and TRIM48 in xenografts of treated mice. t test used to compare mean level of expression (± SEM) in qRT-PCR after RPL39 normalization. *Compared to entinostat or doxorubicin: *p < 0.05, **p < 0.01, ***p < 0.001. Mann–Whitney test performed, median of CXCL10 and TRIM48 expression in xenografts shown. (C) (a) Hierarchical supervised clustering of expression of IFN-α genes against The Cancer Genome Atlas (TCGA) RNA-seq breast cancer patient dataset (bars above identify different tumor subtypes (PAM50) and inflammatory cell content (immune, low–high)); (b) one-way ANOVA showed significant difference across one or more groups (#1 low, #2 medium, #3 high immune cells) and post-hoc pairwise Student t test with Bonferroni correction revealed statistically significant differences across all groups (p < 0.05); (c) IFN-α score correlation with immune infiltration. E entinostat (Ent), A all-trans retinoic acid (ATRA), D doxorubicin (Dox), Veh vehicle, Her2 human epidermal growth factor receptor 2, Lum luminal

Fig. 4

ED and EAD induce expression of proinflammatory genes. qRT-PCR of (a) genes associated with immune cell activation and recruitment, (b) cancer testis antigens (CTA), and (c) immune checkpoints. t test used to compare mean level of expression (± SEM) in qRT-PCR after RPL39 normalization. *Compared to entinostat; #compared to doxorubicin: */#p < 0.05, **/##p < 0.01, ***p < 0.001. d Western blot analysis of GITRL and IL13RA2 in MDA-MB-231 cells treated as described in text. Loading control: β-actin. E entinostat, A all-trans retinoic acid, D doxorubicin, Veh vehicle, ns not significant

Fig. 5

EAD induces inflammatory features and DHRS3 expression. (A) Quantification of inflammation from tumor xenografts (n = 7–10/group) of treated mice (score 0–3) by pathologist blinded to nature of treatment. (B) Volcano plot (log2 fold-change (FC) vs – log10 p value) of genes upregulated and downregulated in MDA-MB-231 cells following EAD in comparison to ED treatments. (C) qRT-PCR detection of (a) CCL26 and (b) DHRS3 in MDA-MB-231 cells treated with entinostat (2.5 μM), ATRA (1 μM), and doxorubicin (200 and 12.5 nM) singly, and combinations, for 48 h; (c) western blot analysis of DHRS3 in MDA-MB-231 cells treated as described in text. Loading control: β-actin. qRT-PCR detection of DHRS3 mRNA levels in (D) tumor xenografts treated as indicated and (E) normal breast organoids (ORG), primary, and metastatic samples from TNBC patients. Mann–Whitney test performed, median of DHRS3 expression in xenograft and primary samples shown. Student’s t test performed, mean (± SEM) of CCL26 and DHRS3 expression in MDA-MB-231 cells shown. RPL39 mRNA used as control in q-RT-PCR. *p < 0.05, **p < 0.01, ***p < 0.001. E entinostat, A all-trans retinoic acid, D doxorubicin, Veh vehicle, NS not significant, NA not available

Fig. 6

ED-induced genes correlate with better prognosis in TNBC patients. a Kaplan–Meier curves of overall survival (OS) showing correlation of ED-induced gene expression and prognosis in basal/TNBC patients, over a period of 12 years. b Scheme summarizing conclusions of this study on effect of EAD combination therapy in TNBCs. Ent entinostat, ATRA all-trans retinoic acid, Dox doxorubicin, HDAC histone deacetylase, RAR retinoic acid receptor, RXR retinoid X receptor, TopoIIβ topoisomerase II beta, NCOR nuclear receptor co-repressor