We speculate that this ED effect on gene expression could be attributed to the inhibition of topoisomerase II-beta by both agents, as we have described previously in the case of
RAR-β induction [
20]. Pathway analysis showed that ED treatment regulated genes related to inflammation, growth arrest, and differentiation (Fig.
1). ED downregulated MYC and E2F targets and genes related to progression through the G2M checkpoint (Fig.
1). Recently, Topper et al. [
30] showed that the combination of HDAC and DNA methyltransferase inhibitors decreased MYC-driven cell proliferation in lung cancer. In line with this finding related to the regulation of cell cycle arrest and death genes, we observed that entinostat treatment sensitized TNBC cells to doxorubicin-induced G2 cell cycle arrest (Fig.
2). Retinoic acid [
3], doxorubicin [
31], and HDACi [
13] as single agents were also shown to induce cell arrest. Previously, we showed that ED and EAD increased apoptosis and EAD is the most effective to induce necrosis [
20]. Collectively, these data showed that the increase in G2 cell arrest by ED and EAD (Fig.
2) led to cancer cell apoptosis and necrosis [
20] and contributed to the decrease of tumor volume [
20].
Among the inflammatory genes, ED increased the expression of interferon (IFN) genes, which correlated to the higher levels of infiltrated immune cells in patient breast tumors (Fig.
3). These findings suggest that the induction of IFN genes by the epigenetic treatment may favor immune cell recruitment to the tumor site. Type I IFN possesses the potent ability to activate several immune cell types [
32,
33]. The presence of lymphocytic infiltration in early-stage breast cancer was associated with good prognosis and high response rates to neoadjuvant chemotherapy [
34], especially in ER
−/HER2
− tumors [
35]. Interestingly,
CXCL10,
IRF1, and
STAT1, shown to be expressed in breast cancers of patients who did not relapse [
26], were also induced by EAD (Fig.
3). In addition to the regulation of interferon responsive genes, such as members of the tripartite motif (TRIM) family (Fig.
3), ED treatment also significantly induced the expression of members of the cancer/testis antigens (CTA) family in TNBC cells (Fig.
4). Epigenetic modifications including promoter hypomethylation and histone deacetylation have important roles in CTA gene activation [
36]. CTAs are protein antigens normally expressed in a wide variety of malignant tumors but not in normal adult tissues, except for testis, and therefore have been viewed as attractive targets for cancer immunotherapy [
37]. Similarly, 5-azacitidine regulates interferon signaling, antigen processing and presentation, cytokines/chemokines, and CTA genes [
30,
38,
39]. In addition, we showed that several inflammatory genes related to immune cell activation and migration, including immune checkpoints, are also epigenetically regulated and induced by entinostat and further potentiated by the ED combination (Fig.
4). In fact, HDACi have been shown to enhance immunogenicity of cancer cells. Several groups have reported the upregulation of natural killer (NK) cell-activating ligands, MHC class I and II molecules, components of the machinery for antigen presentation, and costimulatory molecules on the surface of cancer cells exposed to HDACi [
40‐
42]. Entinostat potentiates the effect of immune checkpoint-blocking antibodies, and the combination decreased regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSC) in mice [
43], and in patients [
44]. Treatment with epigenetic therapy prevented exhaustion of CD8
+ T cells and increased their expansion after immune-checkpoint blockade [
45]. Chemotherapy was also shown to augment tumor immunity [
46]. Previously we observed that EAD induced almost two times higher levels of necrosis compared to ED in vitro, and in tumor xenografts [
20]. The increase in the number of dying cells in tumors of EAD-treated mice [
1] may release damage-associated molecular patterns, release antigens, and initiate an immune response [
47]. We found that entinostat in combination with doxorubicin (ED) most effectively upregulated interferon response, tumor antigens, cytokines, costimulatory molecules, and other inflammatory genes (Figs.
1,
3, and
4), and therefore may improve immunotherapies. Although we observed a similar effect of ED and EAD on the regulation of inflammatory genes, in-vivo EAD treatment was the most effective to increase the recruitment of immune cells and edema at the tumor site of nude mice (Fig.
5), the least immune-deficient mouse model [
28].
ATRA is critical in maintaining immune homeostasis [
4,
48] to differentiate myeloid-derived suppressor cells (MDSCs) into dendritic cells (DCs) and to improve their immunostimulatory capacity [
49,
50]. Treatment of renal cell carcinoma patients with ATRA substantially decreased the presence of MDSCs in peripheral blood [
51]. A recent study demonstrated that in lung cancer patients, p53 vaccine-generated immune responses were improved if patients received a short course of ATRA [
52]. Some of the genes induced by EAD in comparison to ED, such as
ELF3 [
53,
54],
DHRS3,
IL-1β,
CCL26 [
55],
TNF-α,
CD14, and
IL-1α (Fig.
5 and Additional file
12: Figure S4), play a role in inflammation.
DHRS3 is involved in maintaining the cellular supply of retinol metabolites [
29] and was described to be induced by the retinoid X receptor (RXR) rexinoid ligand bexarotene in MMTV-erbB2 mice [
56]. We also observed a correlation of the epigenetic therapy-induced genes, related to growth arrest and inflammation, with survival in patients with TNBC. Collectively, these data suggested that ED and EAD treatment likely potentiates tumor immune surveillance.