Epithelial ovarian cancer (EOC), which is the leading cause of death in women with gynecological malignancies, is very difficult to diagnose and treat due to its asymptomatic presentation and insufficient knowledge on factors that initiate tumorigenesis (1–3). It comprises over 95% of existing ovarian cancers (4) and women have a one in 78 chance of developing EOC (5). Women with EOC have poor prognosis with 61% of cases being detected at advanced stages with a 5-year survival rate of only 27% (1, 2). EOC includes a high-grade serous, low-grade serous, mucinous, an endometrioid, and a clear-cell subtype (6). The endometrioid or clear cell carcinoma represents the main types of endometriosis-associated ovarian cancer (EAOC) that may develop from precursor endometriotic lesions in the ovary. EAOC has an odds ratio of 1.42 for the progression to ovarian cancer in the presence of endometriosis (7, 8). This odds ratio greater than one clearly demonstrates that exposure to endometriosis is a major risk factor for developing EOC. However, the reason for this increased risk of developing ovarian cancer from the progression of endometriosis currently remains unclear.

Endometriosis is an estrogen-dependent chronic inflammatory gynecological disorder that often leads to debilitating symptoms including chronic pelvic pain, menstrual irregularities, dysmenorrhea, and dyspareunia. Approximately 10% of women of childbearing age suffer from endometriosis (9). Similar to EOC, 20–25% of patients remain asymptomatic which contributes to a delay in diagnosis and treatment (10). There are various theories for the etiology and pathogenesis of endometriosis with the most widely accepted hypothesis being Sampson’s retrograde menstruation which is characterized by the development of ectopic endometrial cell implants in the peritoneum (11, 12). Endometrial cells can also escape from immune clearance, attach and invade the peritoneal epithelium, and play a role in angiogenesis (13). While endometriosis is often considered a benign condition, endometrial implants exhibit several molecular and histopathological characteristics similar to those demonstrated by neoplastic cells (14). These characteristics include metastasis to distant sites, invasion and migration, and the establishment of neurovascularity. In addition to this pathogenesis, researchers have found increased levels of inflammatory peritoneal fluid in women suffering from endometriosis (13) which we propose is a main contributor for malignant transformation to EOC.

The peritoneal fluid of women with endometriosis is characterized by increased levels of cytokines, chemokines, growth factors, pain-inducing molecules, and inflammatory cells. Increased amounts of prostaglandins, activated macrophages, IL-1, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) have been found in the PF of patients with endometriosis when compared to the PF of control groups (15–17). Other studies have shown that the increased levels of PF contribute to sustained peritoneal inflammation which regulates both the growth and proliferation of endometrial lesions (18). In patients with endometriosis, the NK cells present in the PF has a defect in its cytotoxic function, hence these cells are unable to remove the endometriotic implants leading to endometriosis. There is also a proposed lack of tissue clearance due to an increase in immunosuppressive regulatory T cells (T_regs) present in the endometrial PF (18). These increased levels of FoxP3+ Tregs decrease immune recognition and clearance of endometrial antigens leading to increased endometrial implants at ectopic sites (19, 20). The escape of endometrial antigens from immune surveillance and increased implantation is important as some of these endometriotic implants may navigate towards the ovaries where they can undergo malignant transformation under the environment of the endometriotic milieu or remain in the peritoneal environment and undergo transformation. In ovarian cancer, increased expression of FoxP3 has been shown to correlate with reduced survival time and disease progression (21). However, some other studies have shown that when FoxP3 is upregulated, this inhibited disease progression for EOC (22). Hence, a better understanding of this pathway in EOAC is essential.

Dissemination of EOC follows a non-traditional invasion–migration cascade by forming loosely attached outgrowths that transit through the peritoneal fluid and attach to new sites. Studies have suggested that the metastasis of ovarian carcinoma is less complicated compared to other types of malignancies as the cancer cells break off individually or in clusters from the original site and passively disseminate to other locations in the peritoneum (23). The ovarian cancer cells invade the mesothelium covering all structures in the peritoneum as its primary microenvironment (23). This invasion migration cascade is very similar to the one seen in endometriosis representing common features present in both conditions.

Endometriosis and EOC may have some relation since they both pertain to pelvic tissues that grow uncontrollably. We propose that this connection between endometriosis and its malignant progression to EOC may be the result of these PF microenvironmental changes which induce cancer-related genetic alterations. Recently, our laboratory has shown that in endometriosis, the PF regulates epigenetic pathways to induce the growth of these lesions (24). We showed that when PF from women with endometriosis was added to endometrial cells, there was an increase in the PRC2 complex, specifically EZH2, and its target H3K27me3. Additionally, there was an increase in miR-155/FoxP3 expression which may be working in coordination with the PRC2 complex to drive the progression of endometriosis. While we believe that these mechanisms may also be behind the transformation of endometriosis to EOC, there is insufficient knowledge that the PF milieu in EOC has similar properties as the PF milieu in endometriosis. Based on these findings, we hypothesize that the endometriotic PF microenvironment would induce inflammatory and epigenetic pathways resulting in transformative changes in the ovarian or endometrial cells leading to the development of EAOC. In our present study, we used a human ovarian clear cell carcinoma line, TOV-21G, treated with PF from women with endometriosis compared to women without endometriosis to discover the effects of the endometrial PF milieu on ovarian cell growth and proliferation.

Through these studies presented here, we provided evidence that endometriotic PF increases proliferation, induces inflammatory genes (including FoxP3 mediated signaling) and modulates EZH2/H3K27me3 in human clear-cell ovarian carcinoma cell lines (TOV-21G) compared to normal ovarian epithelial cells (IOSE). This provides insights into the possible mechanisms by which endometriotic peritoneal milieu by modulating epigenetic pathways may promote endometriosis associated cancers.

Inflammation is a key player in both endometriosis (28–30) and ovarian cancer (31–33). Patients with endometriosis, have an impaired endometrium and an inflamed peritoneal microenvironment. In endometriosis, peritoneal fluid (PF) is a highly dynamic microenvironment that is in a constant state of inflammation (34–37). There is a similar increase in PF accumulation in patients with ovarian cancer. Cytologic examinations of peritoneal washings are a common prognostic (as well as epigenetic) tool in ovarian cancer (38, 39). An increased presence of immune cells including monocyte-macrophages, and T_regs is quite common in the peritoneal microenvironment (40–43). We have earlier shown increased presence of inflammatory and redox markers in the PF of women with endometriosis compared to control women (44–50). We also showed that endometrial cells treated with PF from women with endometriosis induced colony stimulatory factor-1 (44) and monocyte chemotactic protein-1 (47).

Forkhead box protein P3 (FoxP3 T_regs) cells are essential for maintenance of immune tolerance (51, 52). However, the expression and function of FoxP3 in cancer cells is contradictory, since in pancreatic cancer and melanoma it is expressed in the tumor cells whereas it is present in the normal epithelial cells in human breast and prostate cancer (53–56). Furthermore, FoxP3 can act both as a tumor suppressor as seen in breast cancer (57) but also enables cancer cells to prevent T-cell responses directed towards them and hence results in tumor progression and poor prognosis (58, 59). A higher percentage of FoxP3 T_regs, and immunosuppressive cytokines, interleukin-10 (IL-10), transforming growth factor beta (TGF-β) levels are present in the PF and increased expression in endometriotic tissue of patients with endometriosis compared to control women (43, 60). In endometriosis, increased FoxP3 T_reg response is suggested to lead to cancer progression (61). A recent study showed that endometriotic PF increased the recruitment of FoxP3-Tregs in the peritoneal microenvironment (19) We observed an induction of FoxP3 levels in ovarian cells treated with endometriotic PF, thus, it is plausible that though it is an important component of T_reg cells and essential in immune homeostasis, it may also enable cancer cells to escape T cell responses thus resulting in tumor progression and poor prognosis. There are suggestions in the literature that in endometriosis, increased FoxP3 T_reg response may lead to cancer progression (19, 61, 62).

Our interest in FoxP3 is also related to its ability to associate with the polycomb proteins EZH2/PRC2 complex and play a role in epigenetic regulation (63–67) and modulating the expression of tumor associated genes The polycomb group of proteins (PRC1 and PRC2) plays important roles in cell growth and proliferation (68, 69). EZH2/PRC2 complex catalyzes trimethylation of lysine 27 of histone H3 (H3K27me3) which then interacts with the chromatin complex resulting in gene repression (70–72). EZH2 is overexpressed in patients with EOC (68, 73–76) and promotes proliferation, inhibits apoptosis, and enhances angiogenesis in ovarian cancer (77). Inhibition of EZH2, inhibits the growth of ovarian cancer (77–80). In most cancers, there is a reduction in FoxP3 and increased EZH2, however, we recently showed that endometriotic PF promoted a cross-talk between EZH2 and FoxP3 (24). We found that endo PF increased the expression of EZH2/H3K27me3 in TOV-21G cells, suggesting a plausible interaction with the inflammatory modulators such as FoxP3.

Toll like receptors (TLR2 and TLR4), signal through the adaptor molecule, myeloid differentiation primary response gene 88 (MyD88) leading to nuclear translocation of NF-kB and upregulation of pro-inflammatory genes such as IL-6, CCL4 (Treg released cytokine) and CCL2 (MCP-1). The TLR pathway interacts with FoxP3-Treg in immune response (81, 82) and plays a role in chemoresistance (83, 84). Our study showed endometriotic PF induced several of these inflammatory genes involved in cancer pathways, including the TLR pathway (63, 64, 67). Figure 4 highlights some of the mechanistic pathways (TLR pathway, NFkB activation, and Cox-2 activated prostaglandin mediated proliferation) that are possible targets of FoxP3-EZH2 crosstalk and that may be at play in endometriosis associated ovarian cancer. More studies are needed to understand the importance of these pathways in the etiology of EAOC, in order to develop early diagnostic or treatment options for this condition.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Marshall University IRB. The patients/participants provided their written informed consent to participate in this study.

Conceptualization, NS. Data curation, SB and NS. Formal analysis, SB and NS. Funding acquisition, NS. Investigation, SB and NS. Methodology, SB, LC and NS. Project administration, NS. Resources, BM, NZ and NS. Supervision, NS. Validation, SB, TF and NS. Writing—original draft, SB, LC and NS. Writing—review and editing, NS. All authors contributed to the article and approved the submitted version.

Funding for SB was provided by PhRMA Grant 218218 Pre-Doctoral Fellowship for Pharmacology/Toxicology. NS was partially supported by intramural funding from the Edwards Comprehensive Cancer Center, JCESOM, Marshall University and by the NIGMS under grant number 3P20GM103434-21S2 (WV-INBRE).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.