Iron Overload and Ferroptosis in Endometriosis and Endometriosis-associated Ovarian Cancer

Abstract

Introduction: Women with endometriosis (EM) are at increased risk of developing ovarian endometrioid and clear cell carcinoma (OCCC). Retrograde menstruation, localized bleeding, and inflammation within EM lesions generate excessive iron and free radicals that can promote ferroptosis, a process of non-apoptotic programmed cell death due to iron-dependent lipid peroxidation. Glutathione peroxidase (GPX) enzymes scavenge free radicals and are the key enzymes that protect cells from ferroptosis and lipid peroxidation. Methods: Small molecule erastin was used to induce ferroptosis in a panel of immortalized normal human reproductive cell lines and ovarian cancer cell lines. Cell death was measured by MTT assay. The effect of iron overload was tested by adding ferric ammonium citrate (FAC) and knockdown of ferroportin (Fpn), the only known iron exporter in human cells. Immunohistochemistry (IHC) was performed using healthy eutopic endometrium (n=6), EM (n=6), and OCCC tissues (n=4). Results: Compared with ovarian cancer cell lines, normal human cell lines, especially human endometrial epithelial cells (IC50=0.8μM), were more sensitive to erastin-induced ferroptosis. Within ovarian cancer cell lines, EM-associated endometrioid cancer cells were killed by 5μM erastin, and addition of FAC significantly sensitized the OCCC cell line RMG1 to erastin. Fpn knockdown cell lines were similarly more sensitive to erastin compared with control cell lines. Interestingly, the iron overload and erastin sensitivity relationship was not observed in high-grade serous ovarian cancer cell lines. IHC showed that staining for the lipid peroxidation product 4-hydroxynonenal (4-HNE) was significantly higher in EM vs normal endometrium (P=0.005, Mann-Whitney), while 4-HNE staining was completely absent in OCCC. Intriguingly, GPX4, the key intracellular GPX that protects cells against ferroptosis in other tissues, was not detected in the IHC. Instead, GPX3 expression was significantly different in normal endometrium, EM, and OCCC (P=0.002, KruskalWallis test), with an increase in OCCC vs endometrium (P=0.014) and EM (P=0.042, Mann-Whitney). Conclusion: Normal human endometrial epithelial cells and EMassociated ovarian cancer cells are more vulnerable than other cells to iron overload-mediated ferroptotic cell death. Increased 4-HNE staining in EM suggests that this lipid peroxidation product may be involved in the pathophysiology of EM. GPX3 rather than GPX4 appears to regulate ferroptotic stress in OCCCs and may play an important role for EM and cancer cells to evade ferroptotic cell death.