Introduction

Among gynecological malignancies, epithelial ovarian cancer (EOC) is the most difficult to cure. Maximum debulking surgery followed by chemotherapy consisting of taxane and platinum is the standard treatment for advanced EOC [1]. Over 70 % of patients respond to chemotherapy initially, but the majority will relapse. Recurrent tumors can acquire resistance to chemotherapy during treatment, and chemotherapy becomes ineffective in the end (Fig. 1). Despite the emergence of a variety of cytotoxic anti-cancer agents and targeted therapy such as bevacizumab, the progression of EOC has been difficult to control [2, 3].

Fig. 1
figure 1

Overview of acquired drug resistance Chemoresistance is acquired during treatment of epithelial ovarian cancer as follows. 1. The primary tumor is incompletely removed at the cellular level in debulking surgery. 2. If complete eradication of tumor cells is achieved by chemotherapy, the patient is cured. 3. Cancer cells that have acquired stemness, with tumor re-growth after developing chemotherapy resistance, ultimately result in death of the host

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Drug resistance, namely chemoresistance, is a major cause of therapeutic failure. Many mechanisms of drug resistance have been proposed. More than 20 years ago, the expression of multidrug resistance genes in cancer cells was considered a main cause of chemoresistance. Stem cell theory, tumor heterogeneity, and epithelial-mesenchymal transition have recently been proposed to explain chemoresistance. The importance of the tumor microenvironment has also been suggested. Recent advances in molecular genetic techniques have revealed that genetic and epigenetic factors are involved in the chemoresistance of EOC cells. However, overcoming anticancer drug resistance of EOC has not been achieved because the mechanism is complicated.

Identification of the molecular mechanisms associated with chemoresistance is a crucial step towards improving patient survival. In this review, we will describe recent advances in the understanding of the molecular mechanisms of chemoresistance in EOC and strategies for overcoming them.

Tumor and Microenvironment

Stem Cells

Cancer stem cells (CSCs) are a subgroup of tumor cells in many malignant tumors that possess characteristics of normal stem cells, with the ability to self-renew and differentiate [8]. CSCs are considered to be the cause of metastasis, recurrence and resistance. A CSC-based model of drug-resistant cells with chemotherapy has been put forward to explain chemoresistance relapse in EOC [4]. In other words, CSCs are the putative mediators of chemoresistance [5, 6]. It is thought that CSCs are able to survive conventional chemotherapeutic treatment [7]. It has been shown that primary treatment with chemotherapeutic agents results in increased drug-resistant CSCs, which leads to recurrence [8]. For example, the percentage of side population cells is increased in the ascites of patients with an initial recurrence after platinum-based chemotherapy compared with that of chemo-naive patients [9]. Despite being a critical issue, there is no specific marker for ovarian CSCs. Some proteins that have been identified in other malignancies, such as CD44, CD133, CD117, ALDH1A1 and EpCAM (CD326), are used as markers of “stemness” for EOC [10–15]. Therefore, whether CSCs are present remains controversial. However, therapeutic strategies that target stem cell-like properties of tumors are meaningful for overcoming the resistance of EOC.

Tumor Heterogeneity

Recent evidence showed that tumor heterogeneity leads to chemoresistance. The presence of multiple tumor clones within a patient provides an opportunity for survival selection during chemotherapy. The problem of gene mutation needs to be solved for tumor eradication, as tumor heterogeneity results in major difficulties in implementing targeted therapy in EOC. The clonal evolution model has been proposed to explain tumor heterogeneity. Surviving cells that were naturally selected by treatment are responsible for tumor progression. The proliferating cells that have acquired additional mutations will have new characteristics [16]. Therefore, in the clonal evolution model, any cancer cell has the potential to become resistant to chemotherapy.

Khaique et al. [17] analyzed the genetic alterations of primary EOC tumors and suggested that monoclonal proliferating cells are selected from a genetically distinct mixed population [18]. Another study of recurrent serous carcinoma confirmed that chemotherapy-resistant clones arise from minor clones that are present in primary tumors. Castellarin et al. [19] investigated the change of p53 mutations of cells that were collected from the ascites of high-grade serous carcinoma (HGSC) patients receiving chemotherapy, using whole exome sequencing. The vast majority of somatic variants found in recurrent tumors were present in primary tumors. These results demonstrated that cancer cells could be resistant to the effects of chemotherapy. Elucidation of the molecular events that control tumor heterogeneity may lead to the development of strategies for overcoming chemoresistance.

Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) converts epithelial cancer cells into mesenchymal cancer cells with migratory capability and the capacity to invade and metastasize. EMT is characterized by the loss of epithelial polarity and differentiation markers such as E-cadherin and β-catenin, and the gain of mesenchymal markers such as N-cadherin and vimentin [20, 21]. Some signaling pathways involved in EMT include Wnt/β-catenin, TGF-β, Notch and Hedgehog [22]. Among them, Wnt/β-catenin pathway is one of the major signaling pathways thought to be involved in EMT. Wnt/β-catenin signaling plays an important role in the transcription of multidrug resistance genes such as ABCB1/MDR-1 [23]. Furthermore, this pathway is an important pathway in cell survival and has been implicated in the mechanism of chemoresistance of ovarian CSCs [24]. Thus, Wnt/β-catenin pathway may be a potential target for chemosensitization in EOC.

Recently, chemoresistance has been reported to be associated with EMT in EOC cells [25–27]. Clinical studies have implicated a link between EMT-related gene expression and relapse after platinum-based treatment [28], as well as a link between EMT and innate resistance to platinum-based chemotherapy [29] in EOC. Through platinum-based treatment, EOC cells acquire not only resistance to platinum, but also to mesenchymal phenotypes [30–32]. A latest study confirmed that metastasizing EOC cells taken from patients have a different molecular structure from primary tumor cells and display genetic signatures consistent with EMT [33]. Miow et al. [34] assessed the cellular responses to cisplatin using expression microarray analyses of EOC cell lines. Their results show that epithelial-like and mesenchymal-like EOC cells exhibit distinct responses following cisplatin administration. This distinction may suggest a need for differential therapeutic regimens in the treatment of EOCs based on the EMT status of cancer cells.

Microenvironment

Recently, substantial evidence supports the key role of a heterogeneous tumor microenvironment in carcinogenesis and cancer progression, including various stromal compartments such as fibroblasts, endothelial cells, leukocytes, pericytes and extracellular matrix [35, 36]. There is also growing evidence that highlights the importance of tumor microenvironment-mediated chemoresistance mechanisms in EOC [37]. Accumulating evidence of tumor microenvironment-mediated chemoresistance in EOC suggests that targeting relevant molecules and signaling pathways is critical for overcoming chemoresistance in EOC [38]. Direct cell-to-cell contact, trogocytosis and efflux pumps of drugs are known mechanisms of chemoresistance that involve cancer-associated mesenchymal stem cells (CA-MSCs). In addition, a recent report demonstrated that CA-MSCs protected an EOC cell line from carboplatin-induced apoptosis through inhibiting the inactivation of X-linked inhibitor of apoptosis protein (XIAP) [39]. Lim et al. [40] found that tumor-associated macrophages (TAMs) in the tumor microenvironment produced high levels of VEGF-C and transduced signals to cancer cells through VEGF-C interactions with VEGFR-3, a primary receptor of VEGF-C on tumor cells. Upregulation of the ATP-binding cassette genes by insulin-like growth factor (IGF)-I via the PI3K, MEK and JAK2/STAT3 signaling pathways was another mechanism of chemoresistance. Benabbou et al. [41] demonstrated that the OVCAR-3 cell line showed significant drug resistance to paclitaxel or carboplatin when co-cultured with Hospicells. Triulzi et al. [42] showed that the overexpression of maspin, a member of the serpin protease inhibitor family, led to doxorubicin resistance through the maspin-induced, collagen-enriched microenvironment.

Genetic Factors

Drug Resistance Genes

Adenosine 5’-triphosphate (ATP)-binding cassette (ABC) and solute carrier (SLC) transporters are known to lower intracellular drug concentrations and are important multidrug resistance factors. The most important ABC transporters in the context of EOC drug resistance are ABCB1 (also known as P glycoprotein [P-gp]), ABCC2 (multidrug resistance-associated protein-2 [MRP2]), and breast cancer resistance protein (BCRP), which are encoded by the ABCB1 (multidrug resistance protein 1 [MDR1]), ABCC2 and ABCG2 genes, respectively [43, 44]. P-gp is able to actively expel nearly 20 cytostatics, including paclitaxel and doxorubicin. A recent in vitro study confirmed that the main mechanisms of drug resistance were due to P-gp expression in the doxorubicin-resistant, vincristine-resistant and paclitaxel-resistant cell lines, and BCRP expression in the topotecan-resistant cell line [45]. Clinically, Naniwa et al. [46] demonstrated that the expression of the MDR-1 gene in EOC tumors was significantly higher in nonresponders of chemotherapy, which consisted of paclitaxel and carboplatin.

Currently, microarray-based multigene assays are available. Comprehensive analyses of genetic alterations of drug resistance genes in ovarian cancer cells are being carried out, but the detailed mechanism of drug resistance remains unknown [47–49]. These approaches still require extensive validation before they can be considered as putative biomarkers [50]. Furthermore, inhibitors of drug transporters have been evaluated. The results of most of the trials of these agents were disappointing, as the inhibitors lacked specificity and exhibited high systemic toxicity [51].

Transcriptions

Multiple studies have reported the involvement of transcription factors in gynecological cancer progression. The tumor suppressor gene TP53 encodes a DNA-binding transcription factor that induces cell growth arrest, senescence and cell death by apoptosis upon cellular stress [52]. Once activated by DNA damage detection or UV radiation, p53 induces the expression of many well-known apoptosis inducers and other tumor suppressors such as p21, BAX, PTEN and TSP-1. Mutation of TP53, the gene encoding p53, is very common in EOC [53]. At least 50 % of all ovarian tumors have p53 mutations. Serous carcinoma is classified as high-grade or low-grade (HGSC or LGSC). HGCS accounts for 67 % of all EOCs and often leads to chemoresistance. The majority of HGCS have an inactive p53 because of genetic mutation [54].

P53 protein aggregation is associated with p53 inactivation and platinum resistance. A recent study showed that overexpression p14ARF, a p53-positive regulator, inhibited MDM2-mediated p53 degradation. The authors of the study also demonstrated that inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment in vitro and in vivo. Furthermore, they discovered that the aggregated p53 might function by interacting with proteins that are critical for cancer cell survival and tumor progression by using two-dimensional gel electrophoresis and mass spectrometry. These findings suggested that p53 aggregation is a new marker for chemoresistance of HGCS. In addition, this indicated that inhibition of p53 aggregation can reactivate the pro-apoptotic function of p53 [55•]. Therefore, p53 aggregation is a promising therapeutic target for overcoming resistance to chemotherapy.

TWIST1 has been shown to be important in the regulation of programmed cell death and inflammation [56, 57]. Recently, it was found that TWIST1 is involved in the process of tumor metastasis via modulation of epithelial-mesenchymal transition (EMT). Some studies have reported that TWIST1 is overexpressed in EOC [58, 59]. Emerging evidence suggests that TWIST1 plays an important role in the chemoresistance of cancer cells. Kajiyama et al. [60] found that TWIST1 expression predicts poor clinical outcomes in patients with clear cell carcinoma (CCC) of the ovary. They also found that positive TWIST1 was an independent prognostic factor for survival of EOC patients [61].

TWIST2 is a novel zinc finger transcription factor that has been shown to be an important inducer of EMT. A recent study demonstrated that TWIST2 also plays a crucial role in the chemoresistance of EOC. Downregulation of TWIST2 expression facilitated apoptosis and previously chemoresistant EOC regained sensitivity through the AKT/GSK-3β pathway [62].

Many other transcription factors are being studied as potential targets in general cancer treatment, such as STATs, NF-κB and Notch1 [63–65]. However, clinical treatments that target transcription factors have not been realized.

Epigenetic Factors

Epigenetics is a phenotypical change in gene expression without any alteration of the DNA sequence. The epigenetic change in tumors generates the diverse gene expression involved in drug resistance. This allows cancer cells to acquire chemoresistance. Furthermore, this phenomenon may complicate the selection of chemotherapy that is based on mutation biomarkers.

DNA Methylation

DNA methylation is the most frequent epigenetic phenomenon. In cancer cells, DNA hypermethylation is associated with gene silencing, while DNA hypomethylation is associated with gene expression. DNA methyltransferase (DNMT), an enzyme that catalyzes the transfer of a methyl group to DNA, is essential for this process [66]. DNA methylation has attracted attention as a target for overcoming chemoresistance. The efficacy of some demethylating agents in treating EOC has been examined in clinical trials [67, 68]. Human mutL homolog 1 (hMLH1) is a promising target. Hypermethylation of hMLH1 inhibits the apoptotic response to platinum chemotherapy. This is considered a major molecular cause of acquired resistance to platinum chemotherapy in EOC [69]. In addition, the presence of methylated hMLH1 DNA in plasma after chemotherapy predicts poor survival for EOC patients [70]. In EOC cells, histone deacetylation at the RGS10-1 promoter correlates with suppression of RGS10 and chemoresistance [71]. This data suggest the possibility of using histone biomarkers to determine the appropriate selection of therapy in cases of EOC chemoresistance [71, 72]. Recent preclinical research on resensitizing platinum-resistant cells using a novel DNMT inhibitor SGI-110 is noteworthy. This study demonstrated that ALDH+ EOC cells possessing stem cell characteristics are enriched in platinum-resistant EOC cell lines, human tumors and residual xenografts after platinum therapy. SGI-110 inhibited ALDH+ cell viability, sphere formation and tumor-initiating capacity; repressed stem cell-associated gene transcription; and resensitized platinum-resistant EOC cells to platinum [73•].

Histone Modification

Histone modifications also play important roles in epigenetic regulation. Histones are dynamic proteins that can become methylated or acetylated at specific amino acid residues, which correlate with active or repressive transcription [74, 75]. By tightly winding and condensing chromatin or loosening up the structure of chromatin, transcription factors and other proteins are denied or permitted access to DNA for transcription. Histone deacetylases (HDAC) cause repression of gene expression by regulating the condensation of chromatin. Aberrant expression of HDACs in gynecological cancers is associated with chemoresistance. The use of HDAC inhibitors such as valproic acid, belinostat and vorinostat in combination therapy for overcoming chemoresistance has been proposed. The results of clinical trials of combination therapy with belinostat and carboplatin have been reported for platinum-resistant EOC [76, 77]. Clinical trials of EOC epigenetic therapeutics are ongoing.

MicroRNAs

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MiRNAs play an important role in carcinogenesis and cancer progression. Certain miRNAs have also recently emerged as important epigenetic modulators of autophagy in cancer cells [78]. Two major approaches in targeting transcription factors are post-transcriptional silencing by siRNAs or miRNAs, or blocking the binding of transcription factors to DNA. Multiple studies have focused on the roles of miRNAs in overcoming resistance to chemotherapy for EOC.

It has been reported that many miRNAs are expressed differentially between chemosensitive and chemoresistant ovarian cell lines such as miR-30c, miR-130a and miR-335 [79]. MiR-214 induced cell survival and cisplatin resistance through direct targeting of PTEN and inactivation of the AKT pathway [80]. Vecchione et al. [81] analyzed miR signatures associated with chemoresistance in 198 samples of serous ovarian cancer. They demonstrated that the presence miR-217, miR-484 and miR-617 could predict chemoresistance. MiR-199a-5p was shown to increase chemoresistance by simultaneously promoting autophagy and suppressing apoptosis. By downregulating Beclin-1 expression, miR-30a and miR-376b downregulate not only autophagy but also apoptosis, since the level of free antiapoptotic BCL-2 protein decreased in the cell. MiR-30a was found to be deregulated in stage I ovarian cancer patients, together with other miRNAs. In particular, it was downregulated in samples from patients with relapse [82, 83]. MiR-27a increases MDR1/P-glycoprotein expression in EOC cells by targeting HIPK2 [84]. Similarly, miR-451 and miR-21 facilitate MDR1/P-glycoprotein overexpression, leading to paclitaxel resistance in EOC cells [85, 86]. Let-7a is a potential biomarker for the selection of chemotherapy in EOC. Patients with low let-7 showed a good response to platinum–paclitaxel combination therapy [87]. The down-regulation of let-7i is associated with resistance of EOC cells to cisplatin [88]. Let-7 g downregulates the multiple drug resistance 1 (MDR1) gene, one of the major factors causing paclitaxel resistance in EOC [89]. Lower expression of miR-31 and higher expression of MET significantly correlated with PTX resistance and poor prognosis in ovarian cancer patients [90]. Epigenetic silencing of miR-199b-5p is associated with chemoresistance in EOC through the activation of JAG1/Notch1 signaling [91].

As noted above, many of the miRNAs are involved in chemoresistance. Further research is needed before miRNAs can be used in clinical treatment.

Related Signaling Pathways

Ubiquitin–Proteasome Pathway

The ubiquitin–proteasome pathway is responsible for maintaining cellular homeostasis by regulating the degradation of proteins. Disruption of this pathway can result in cell cycle arrest and apoptosis as a result of incompatible regulatory protein accumulation within the cell [92]. Cancer cells generally have higher levels of proteasome activity and are more sensitive to the proapoptotic effects of proteasome inhibition than normal cells [93]. Bortezomib is a reversible proteasome inhibitor that targets the chymotrypsin-like and caspase-like active sites of the proteasome complex [94]. By inhibiting the proteasome, bortezomib acts through several mechanisms to suppress tumor survival pathways and to arrest tumor growth, metastasis and angiogenesis. These mechanisms of action underlie the rationale for the combined use of bortezomib with other chemotherapeutic and targeted agents, some of which have been evaluated in EOC clinical trials [95–97]. A recent study demonstrated that proteasome inhibition affects microtubule stabilization in a manner similar to taxanes, and increases sensitivity to paclitaxel [98].

Toll-like Receptor 4 Signaling

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that is required for Toll-like receptor 4 (TLR4) signaling. The activation of the TLR4/MyD88 signal pathway can induce the activation of the Akt survival pathway and enhance the expression of the antiapoptotic protein XIAP. Furthermore, knockout of Myd88 reduces MRP1, which is a pump involved in chemoresistance. For the above reasons, MyD88 is involved in resistance to chemotherapy. In particular, EOC cells expressing MyD88 induces resistance to cisplatin and paclitaxel [99, 100]. MyD88 is expected to be a new therapeutic target.

Conclusions

Chemoresistance limits the survival of advanced cancer patients receiving chemotherapy. Many drugs have been developed so far, however, the improvement of the prognosis of EOC patients is insufficient. Chemoresistance occurs as a result of a complex interplay of the various factors described above. We can only confirm the status of the tumor clinically. Thus, it is important to identify a simple clinical biomarker that indicates the mechanisms of chemotherapy resistance of a recurrent tumor. There is a need for detailed validation studies of tumor and plasma at the time of relapse and clinically acquired resistance, for optimizing tailored therapy. A new treatment paradigm for overcoming the resistance of ovarian carcinoma is urgently needed.