Endometrial stromal cell responsiveness to progesterone and plasminogen activator inhibitor 1 in endometriosis, a precursor to ovarian oncogenesis
Introduction
Endometriosis is an invasive disease characterized by the growth of endometrial-like lesions, composed of epithelial and stromal cells, in the abdominal cavity. The ovaries are the major target for endometriosis lesion formation, called endometriomas. Despite the highly invasive nature of endometriomas, they are not malignant. On the other hand, endometriomas have been associated with subsets of ovarian cancer, including clear cell carcinomas. Whether endometriosis lesions are the premalignant precursors of these ovarian cancer subtypes is still not clear. Several studies suggested that ovarian cancer have origins from Müllerian tissue, which is also the progenitor tissue for the endometrium, but they mainly focused on the target of oncogenesis, mainly the epithelial cells. Few studies, if any, examined the contribution of endometrial stromal cells (ESCs) to the initiation of endometriosis-associated ovarian cancer. Given that obesity is a risk factor for ovarian cancer and contributes to the progression of endometriosis, we examined the effects of a major adipokine secreted by adipocytes, the plasminogen activator inhibitor (PAI-1) on ESC phenotype. In addition, we examined the hormonal response, mainly due to progesterone, in conjunction with elevated PAI-1 exposure by primary ESCs obtained from the endometrial lining. We hypothesize that ESCs in endometriosis lose inhibitory responsiveness to P4 and induce inflammatory markers that can induce ovarian cancer development.
Methods
To investigate the combinatorial effects of PAI-1 and hormonal influence on ESCs, we targeted the decidualization stage of the menstrual cycle, which involves a sensitive window of endometrial stromal differentiation. Disruption of this phase can lead to de-differentiated cells that contribute to oncogenesis when seeding the ovaries during endometrioma formation. To stimulate a decidualization program, ESCs were treated with a decidualization hormonal regimen: estrogen (E2), progestogen (P4), and cAMP, and in the presence or absence of PAI-1 to test the effects of this adipokine. We then examined cell morphology and proliferation using the Incucyte automated cell monitoring system, as well as the levels of decidualization and inflammatory markers in the culture supernatant using Luminex multiplex assay and RNA levels using qRT-PCR.
Results
While decidualization hormone treatment of ESCs elicited a decidualization epithelioid like morphology, addition of PAI-1 resulted in the maintenance of a fibroblastic morphology of ESCs. Induction of inflammatory cytokine IL-6, angiogenic marker VEGFA, and anti-apoptotic factor IL-8 was observed in culture of cells treated with decidualization hormones and PAI-1. However, little effect is seen on the decidualization marker IGFBP1. An induction of progesterone receptor RNA levels is seen in the control group versus endometriosis group while estrogen receptor RNA levels increased in endometriosis group but not control group. Comparing the proliferation effects of hormonal exposure on ESCs from women with and without endometriosis, ESCs showed that while P4 exposure inhibited proliferation of ESCs from women without endometriosis, ESCs from women with endometriosis were resistant to this hormonal effect.
Conclusion
Endometrial stromal cells are sensitive to P4 induced decidualization. However, PAI-1 exposure alters this program and results in the induction of inflammatory and anti-apoptotic markers, which can contribute to ovarian oncogenesis by invading stromal endometriomas. In addition, ESCs from women with endometriosis exhibit a refractory response to growth inhibition by P4, suggesting that these cells are primed to colonize the ovaries during endometrioma formation. Given these changes in the ESC phenotypic output, future studies will examine potential oncogenic effects of endometriosis ESCs on ovarian surface epithelial cells.