Effective cytotoxic drug treatment requires a sufficient intracellular drug concentration in tumor cells. Drug concentration is coordinated by transporters mediating the influx and efflux of drugs and enzymes mediating drug metabolism. The effectiveness of chemotherapeutic and targeted drug delivery into cancer cells is determined by drug inflow and efflux transporters. The most known such transporters are the ATP-binding cassette (ABC) transporter family members, namely, ABCB1 (MDR1), ABCC2 (MRP-2), and ABCG2 (BCRP/MXR1), which have been widely shown to be associated with drug resistance (54, 55). The epigenetic control of ABC transporter-induced OC resistance has recently progressed significantly ( Figure 3 ). ABCB1 is the first ABC transporter identified and plays a crucial role in determining drug sensitivity. The epigenetic regulation of ABCB1 is related to drug transportation in OC cells. For instance, paclitaxel therapy increased histone H3 acetylation and targeted the ABCB1 promoter in conjunction with the androgen receptor (AR), resulting in ABCB1 gene expression and the establishment of the paclitaxel resistance phenotype (56). Wu et al. found that overexpression of miR-873 improved the susceptibility of OC cells to paclitaxel and cisplatin by targeting ABCB1 (57). Besides, Tian et al. demonstrated that miR-490-3p enhances the sensitivity of OC cells to cisplatin by downregulating ABCC2 expression (58). Nevertheless, hnRNPA2B1 was shown to bind to the 5’UTR of ABCC2 mRNA and promote its translation, leading to cisplatin resistance in OC (59). Furthermore, ABCG2 is strongly expressed in cisplatin- or paclitaxel-resistant OC and OC stem cells, indicating the key role of ABCG2 in drug resistance and stemness acquisition in OC (60). Calcagno and colleagues discovered that elevated acetylation of histone H3 in the ABCG2 promoter is a cellular response to the treatment of doxorubicin, which underlies its doxorubicin resistance (61). Some lncRNAs and miRNAs found in extracellular vesicles (EVs) generated by drug-resistant cells control the expression of ABCG2, hence impacting tumor drug resistance (62–65). These investigations indicate that epigenetic regulation plays an essential role in OC drug resistance by modulating the ABC transporter family.

Drug metabolism regulates bioactivation, catabolism, conjugation, and excretion, determining drug clinical efficacy and toxicity (66). Drug metabolism enzymes can be affected by alterations in the expression level of affect drug metabolism. For instance, epigenetic alterations regulate the expression of the cytochrome P450 (CYP) enzyme family, which is the most well-known drug-metabolizing enzyme, thus affecting the metabolism of several anti-cancer medications. A recent study showed that histone modification enzyme G9a regulates the expression of CYP450 by affecting histone 3 lysine 4 and histone 3 lysine 27 methylation, suggesting G9a alters drug sensitivity (67). Luo and colleagues emphasized an unconventional epigenetic regulation mechanism of CYP gene expression; that is, miR-148a stimulates CYP2B6 expression by binding to the 3’UTR region to improve mRNA stability (68). Furthermore, miR-543 overexpression increases the production of CYP3A4, which is involved in the metabolism of ruxolitinib (69). Other drug-metabolizing enzymes are controlled by epigenetic alterations, which must be investigated further.

Over the past decade, targeted agents have been steadily introduced into clinical trials for treating recurrent OC, namely, the anti-VEGFR agent bevacizumab, PARP inhibitors (olaparib, niraparib, and rucaparib), the anti-MEK inhibitor trametinib, and anti-HER2 pertuzumab. However, chemotherapy resistance resulting from epigenetic alterations limits the effectiveness of these targeted treatments. Resistance to targeted therapy arises primarily through reduced expression of drug targets and activated bypass signaling pathways during treatment with targeted drugs (70–72). Here, we summarize the epigenetic modification-mediated drug resistance in OC, caused by the deregulated expression of drug targets, mutation of drug targets, and the persistent activation of bypass signaling pathways.

Angiogenesis is critical in the etiology of OC, which is associated with the generation of vascular endothelial growth factor (VEGF), linked to the progression of malignant ascites and OC (73, 74). In clinical trials, bevacizumab, an anti-VEGF monoclonal antibody, has been proven to improve outcomes in OC patients in clinical trials (75, 76). However, epigenetic modifications may diminish VEGF expression. Vesna and colleagues found twenty significant CpG sites in promoter regions, suggesting that the concentration of VEGF-A can be regulated by DNA methylation (77). Based on The Cancer Genome Atlas (TCGA) databases, Zhou and colleagues discovered that overexpression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was related to cancer progression and poor survival of patients. Mechanistically, IGF2BP3 can bind with the mRNA of VEGF, where IGF2BP3 serves as a reader to recognize the m6A modification on VEGF mRNA. This effect controls both the production and stability of VEGF mRNA, which may affect drug sensitivity (78). Besides, VEGFA is the direct target of miR-652-5p, and miR-652-5p is down-regulated by hypermethylation at the upstream CpG site. Increasing VEGF synthesis is linked to tumor progression and metastasis (79). Moreover, it has been shown that cells with methylated BRCA1 have defective homologous recombination (HR) activity, thus being highly responsive to PARP inhibitors (olaparib, niraparib, and rucaparib), implying that BRCA1 inactivated by epigenetic mechanisms contributes to drug sensitivity (80–82).

In addition to alterations in drug targets, epigenetic modifications are implicated in the activation of bypass signaling pathways to regulate OC drug resistance. Although the Notch pathway is closely related to the growth of OC tumors, its clinical significance and molecular mechanisms remain unclear. Hu et al. found that alterations in the Notch pathway are prevalent and closely related to poor clinical outcomes in patients with OC (83, 84). For instance, epigenetic regulation of multiple Notch target genes (such as PPARG, CCND1, and RUNX1) can regulate the activation of the Notch pathway, which is associated with the prognosis of OC patients (85). Liu and colleagues found that Fas deficiency inhibits the release of miR-29b, thereby increasing intracellular miR-29b levels and subsequently downregulating DNA methyltransferase 1 (DNMT1) expression, which results in hypomethylation of the Notch1 promoter region and activation of Notch signaling (86). Hirsch et al. discovered that inhibition of histone deacetylase might alleviate abnormalities in the Notch and Eph axis in prion protein PrP deficient and prion-infected cells (87). Besides, studies have demonstrated that lncRNA HOTAIR induces OC drug resistance to cisplatin through activating the Wnt/β-catenin pathway (88). Further studies of epigenetic modifications that regulate drug targets and bypass signaling pathways may increase our understanding of the development of potential strategies to reverse drug resistance.

Most chemotherapeutic drugs induce cell death through apoptosis due to DNA damage (89, 90). After treatment with cytotoxic agents, eukaryotic cells usually undergo damage repair to avoid apoptosis, leading to drug resistance (91). Therefore, components of the DNA repair system, such as O6-methylguanine-DNA methyltransferase (MGMT), can promote chemotherapeutic resistance. MGMT mediates the direct removal of O6-methylguanine (O6-MEG), a mark of DNA damage induced by temozolomide (TMZ), thereby facilitating DNA repair in melanoma cells. Other TMZ-induced lesions are mostly repaired by base excision repair (BER) or direct removal mechanisms catalyzed by DNA demethylase ALKBH2/3 (91, 92). Poly-ADP-ribose polymerase inhibitors (PARPis) are the most effective therapies approved for treating OC, and poly-ADP-ribose polymerase (PAPR) inhibitors (olaparib, niraparib, and rucaparib) are already being investigated in OC clinical trials. Indeed, all PARPis exhibit radio- or chemo-potentiation effects in vitro and in vivo, which is consistent with their ability to inhibit DNA damage repair (93, 94). According to a recent study by Nephew and colleagues, platinum-induced DNA damage contributes to the activation of the NF-κB pathway by the lncRNA HOTAIR and cellular senescence. Furthermore, DNA damage response activated NF-κB, which in turn triggered HOTAIR and created a positive-feedback loop, resulting in sustained NF-κB activation and persistent DNA damage signaling (40). Mutations in p53 are observed in 42% of human tumors (95). The regulation of RNA m6A on TP53 has been proven to overcome drug resistance by controlling downstream pathways and DNA damage repair, suggesting that it is a viable therapeutic approach (96). Meng et al. discovered that AZD1775, not only carboplatin, can increase sensitivity to gemcitabine and olaparib in TP53-mutated gynecologic cancer cells (97). WEE1 is a tyrosine kinase that blocks the CDK1/cyclin B complex by inducing CDK1 phosphorylation at tyrosine 15 (Y15), inducing cell cycle arrest and allowing DNA repair (98, 99). AZD1775 is a first-in-class, powerful, and selective WEE1 inhibitor that has shown a considerable anti-tumor effect in OC patients with TP53 mutations when combined with carboplatin (100). In conclusion, uncovering the mechanisms that influence epigenetic modification function in DNA damage repair may develop new strategies for the sensitization of OC cells to DNA damage inducers.

The interaction of anti-cancer drugs with corresponding cellular targets can induce cell death. However, a variety of adaptive cellular responses governed by epigenetic modifications are engaged to support the survival of tumor cells, which is the major target of cancer therapy. These include inhibition of apoptosis, initiation of autophagy, ferroptosis, and activation of pro-survival signals (101–103).

Cancer stem cells (CSCs) are a sub-population of tumor cells that are responsible for driving tumor growth, metastasis, and therapy resistance (52, 138, 139). Emerging results indicate that CSCs contribute to chemoresistance and poor clinical outcomes in various malignancies, including OC (140). Accumulating evidence has shown that CSCs display an innate predisposition to be chemotherapy-resistant and result in tumor relapse (141–143). Furthermore, ovarian cancer stem cells (OCSCs) contribute to resistance to chemotherapy (144). Therefore, solving this thorny problem might lead to the development of new strategies to tackle drug-resistant OC. Because epigenetic regulation plays an integral role in the control of normal stem cell differentiation, strategies to target cancer stem cells can be developed through epigenetics. Epigenetic modifications enable cancer cells to self-renew and generate CSCs, leading to drug resistance (140).

Wang and colleagues hypothesized that hypomethylation drugs might target resistant OCSCs to cure tumors along with chemotherapy drugs. In an orthotopic mouse model, they identified and analyzed ALDH (+) OCSC from OC cell lines and clinical samples, finding that ALDH (+) cells were more chemoresistant than ALDH (−) cells. Treatment with SGI-110, a second-generation DNA methyltransferase inhibitor (DNMTi), re-sensitizes OCSCs to platinum (140). Xu et al. demonstrated that STON2 could negatively regulate the stemness in OC cells by DNMT1-MUC1. STON2 plays a role in OCSC biology and could be used as a therapeutic target for OC treatment (145). Studies have shown that MYPT1 encoding myosin phosphatase target subunit 1 is down-regulated in OC, leading to resistance to platinum-based therapy (146). Similarly, miR-30b could target MYPT1 to lead to enhanced CSC-like properties in OC cells and activate the Hippo pathway. Moreover, inhibition of YAP sensitizes cells to platinum-based therapy (50). Taken together, these studies suggest that epigenetic modifications of these signaling pathways play an essential role in the development of drug resistance in OC.

A tumor microenvironment (TME) refers to the ecological niche in which tumor cells interact with the host stroma, including various immune cells, endothelial cells, fibroblasts, tumor cells, and metabolites. It is now generally accepted that TME significantly influences the efficacy of anti-cancer drugs (51). TME reprogramming caused by epigenetic dysregulations has recently been recognized as an important factor in the progression and drug resistance of OC (2, 147). Here, we systematically generalize the epigenetic modifications of non-malignant OC-related microenvironment cells (namely, cancer-associated fibroblasts, mesenchymal stem cells, and tumor-associated macrophages), which is conducive to evaluating the therapeutic potential of epigenetic regulation of TME-associated cells ( Figure 4 ).

As one of the most well-known epigenetic modifications, DNA methylation plays an essential role in DNA repair, apoptosis, angiogenesis, gene expression regulation, and drug resistance. To date, aberrant DNA hypermethylation has been found in drug-resistant cancer cells, and drug-induced DNA hypermethylation has been proposed as a potential mechanism and biomarker of drug resistance. DNA methylation is mediated by three DNA methyltransferases (DNMTs), primarily by DNMT1, which mediates maintenance (one strand) methylation, and by DNMT3A and DNMT3B, which catalyze de novo methylation (180). The activity of DNMTs can be blocked by DNMT inhibitors (DNMTis), which are analogs of deoxycytosine and effectively block methyl transfer (181). 5-aza-2-deoxy-cytidine (decitabine) and 5-azacytidine were first successfully studied in hematological malignancies and myelodysplastic syndrome (MDS) and were approved by the FDA in 2006 for treating MDS, where they exhibit anti-tumor activity through the inhibition of DNA methylation (182).

To date, DNMTis has been successfully used for treating chemotherapy-resistant OC, restoring platinum sensitivity in refractory to standard chemotherapy of patients. DNA hypomethylation caused by DNMTi decitabine renders OC patients more susceptible to platinum treatment and is associated with a better prognosis. Matei et al. found that platinum resistance is mostly induced by epigenetic abnormalities, including abnormal DNA methylation. The regimen consists of a modest dosage of decitabine 5 days before the administration of carboplatin, which decreases toxicity and boosts the demethylation effect of decitabine, leading to the recovery of carboplatin sensitivity in patients with advanced OC (183). Decitabine has been shown in studies to be more effective than 5-azocytidine for treating platinum-resistant OC. It is capable of regulating the methylation status of the tumor antigen NY-ESO-1 to improve immunotherapy efficacy (184). Furthermore, decitabine improves responses of OC patients to platinum therapy by influencing signaling pathways that promote tumor progression, such as the TGF-β signaling pathway (185). Taken together, DNMTis plays a promising role in reversing or preventing chemotherapeutic and molecular-targeted drug resistance in OC patients.

Histone deacetylase (HDAC) enzymes, which remove acetyl groups from histone and non-histone proteins, downregulate the transcription of genes (186). In cancer cells, HDAC inhibitors (HDACis) can restore transcriptional inhibition of tumor suppressor genes and generate an anti-cancer environment. Meng et al. showed that a combination of proteasome and HDAC inhibitors can inhibit gynecologic cancer growth (187). Inhibitors of enzyme-catalyzed histone modifications, among which HDACis are the most rapidly developed, have been investigated in solid tumors (188).

Fukumoto et al. found that ARID1A mutations confer sensitivity to pan-HDAC inhibitors such as SAHA in OC, which is associated with more significant effects of growth inhibition owing to suppression of HDAC2 activity (189). By suppressing HDAC6 with the small molecule ACY1215, Bitler et al. revealed that mice with ARID1A mutant tumors had dramatically increased survival. Mechanistically, HDAC6 deacetylates Lys120 on p53, thereby inactivating the pro-apoptotic function of p53 (190). However, not all HDAC subtypes are abnormally expressed in all malignancies, so pan-inhibition of HDAC is not an effective way to treat cancer (191). Besides, because of its poor activity as a single agent, HDACis have been investigated along with radiotherapy, chemotherapy, and other epigenetic drugs. For instance, belinostat re-sensitized drug-resistant OC cells to platinum, and the combination of decitabine and belinostat was more effective in re-sensitizing platinum than belinostat alone (192, 193). Furthermore, there is mounting evidence showing that HDACs-mediated deacetylation of non-histones is involved in many key cellular processes associated with drug resistance, such as apoptosis, suggesting that enhanced acetylation of non-histones using HDACis is promising for overcoming the drug resistance of OC. Taken together, targeting HDACs is an appealing strategy for OC treatment.

Multiple ncRNAs, including miRNAs and lncRNAs, are increasingly implicated in the treatment resistance in OC (194, 195). Therefore, targeting tumor-specific miRNAs and lncRNAs is expected to overcome drug resistance of OC. Recently, miRNAs have received increasing attention as biomarkers and therapeutic targets for OC. For instance, Belur Nagaraj and colleagues found that miR-181a is an activator of Wnt/β-catenin signaling, driving stemness and chemotherapy resistance in high-grade serous ovarian cancer (HGSOC), thus being a potential target for treating recurrent OC (194). Using next-generation sequencing (NGS) techniques, Au Yeung et al. identified miR-21 in exosomes and tissue lysates isolated from cancer-associated adipocytes and fibroblasts, which is significantly higher than OC cells. They discovered that miR21 is transferred from CAAs or CAFs to OC cells, suppresses apoptosis and confers chemotherapeutic resistance in OC cells by binding with APAF1 (154). Additionally, Vescarelli et al. have demonstrated that miR-200c significantly enhanced the anti-cancer effect of the PARP inhibitor olaparib in drug-resistant OC cells (196). This finding indicates that combining olaparib with miRNA-based therapy is a potential therapeutic option for drug-resistant OC. Sun et al. found that miR-9 increases the sensitivity of OC cells to DNA damage by down-regulating BRCA1, thus improving the efficacy of chemotherapy (197).

In addition to miRNAs, LncRNAs are considered promising therapeutic targets for OC. For instance, the non-coding RNA HOTAIR has been shown to be a potential target for overcoming carboplatin resistance in OC (195). Furthermore, it has been discovered that HOTAIR is overexpressed in cisplatin-resistant OC cells, and knocking down HOTAIR enhances apoptosis in cisplatin-resistant OC cells by down-regulating HOXA7, thereby restoring cisplatin sensitivity (198, 199). Additionally, HOTAIR-mediated platinum resistance in OC can also be attributed to the upregulation of HOXA7 and activation of the Wnt/β-catenin pathway (88). These findings point to it as a possible target for re-sensitizing OC cells to platinum therapy. Besides, Wu et al. demonstrated that the lncRNA WDFY3-AS2 modulates the hsa-miR-139-5p/SDC4 axis and may play an essential role in the platinum resistance of OC (200). Taken together, the above studies show that it is promising to prevent or overcome OC resistance by targeting ncRNAs.

Antibodies against inhibitory immune receptors, namely, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4/CD152), programmed death protein 1 (PD-1/CD279), and programmed death ligand 1 (PD-L1/B7H1/CD274), have become effective standard immunotherapy for many advanced malignancies (177, 201). However, its effectiveness has yet to be successfully applied to the treatment of OC (202). Although a small fraction of OC patients respond well to immunotherapy, most patients fail to respond or develop secondary resistance to immunotherapy. Therefore, a combinatorial strategy is needed to enhance the efficacy of immunotherapy for OC, which can be achieved by drug repurposing or de novo drug development that stimulates the immune response.

Initial findings showed that epigenetic repressive processes were linked to a “cold” immunological environment in OC. Based on them, epigenetic modifiers were used in several of preclinical and clinical trials to explore immune targeting techniques (188). For instance, Stone and colleagues have demonstrated that DNMTi 5-azacytidine (AZA) activates the type I interferon signaling pathway, boosts IFNγ+ T cells and natural killer (NK) cells, and reduces the percentage of macrophages in the TME. They also discovered that clinically relevant dosages of DNMTi and HDACi increased the responsiveness of OC to immune checkpoint therapy by reducing the immunosuppressive milieu via the type I IFN signaling pathway (203). The triple combination of DNMTi/HDACi with the immune checkpoint inhibitor α-PD-1 showed significantly better anti-tumor efficacy than DNMTi 5-azacytidine (AZA) alone or AZA along with HDACi, which may be a therapeutic option for treating OC. In a follow-up study, Travers et al. employed a hypomethylating agent (HMA) in conjunction with 2-difluoromethylornithine (DFMO) to rewire TME in OC, leading to a longer-lasting anti-tumor response. They found that AZA and DFMO, either alone or in combination, significantly recruited CD4⁺/CD8⁺ T and NK cells, decreased tumor burden, and improved the survival of OC patients (204). Another trial used decitabine in conjunction with paclitaxel and carboplatin to treat 55 patients with recurrent OC and found that the triple combination treatment group had a 58% overall response rate, which is superior to other groups (205). Therefore, combinational therapy with epigenetic modifiers may be able to avoid the loss of tumor antigens and expand the T cell pool by recognizing the initially targeted antigens and epitopes, showing a new and intriguing promise for vaccine and cell transfer platforms.