The Study Of A Positive Feedback Loop Of 17β-estradiol In Situ Biosynthesis Involving IL-6 And Aromatase In Endometrial Carcinoma

Endometrial carcinoma is the most common gynecologic malignancy, with an estimated 49560 diagnosed cases and 8190 deaths in 2014 in the United States. The standard therapy is a total abdominal hysterectomy, but for women with well-differentiated endometrial cancer, in order to preserve their fertility, conservative management may be a therapeutic option. However, progestin is the only endocrine therapy currently used for conservative treatment and more than 30% of patients do not respond to progestin due to de novo or acquired progestin resistance. Therefore, a more effective or combined endocrine therapeutic strategy is desirable.Existing evidence indicates that unopposed estrogens contribute to the tumorigenesis and promotion of endometrial carcinoma. The conversion of androstenedione and testosterone into estrogen is catalyzed by the cytochrome P450 aromatase enzyme. While postmenopausal women have low levels of circulating plasma estrogens, the intratumoral production of estrogens can lead to higher estrogen levels in tumors. Several studies have indicated that, in normal and abnormal human endometrium, aromatase expression and activity are associated with malignancy and only aromatase expression in stromal cells, but not in epithelial cells, is positively correlated with poor survival. Additionally, indirect coculture model confirmed that tumor-stromal communication contributed to upregulation of aromatase activities. Therapeutic strategies that focus on tumor-stroma interactions require the identification of molecular targets that regulate intercellular interactions. To the best of our knowledge, however, there have been no exact stimulators reported to induce aromatase expression in endometrial carcinoma microenvironment.Interleukin(IL)-6 is a pleiotropic cytokine that plays an important role in multiple pathological and physiological processes. Several studies have demonstrated that IL-6 activation is associated with cancer development including endometrial carcinoma. IL-6 is also regarded as an inducible factor of the aromatase gene in breast cancer, cervical cancer and non-small cell lung carcinoma. Whether IL-6 can also mediate aromatase expression in endometrial cancer is unknown. We previously reported that IL-6 is upregulated by 17β-estradiol(E2) through G protein-coupled receptor 30(GPR30) in endometrial cancer, whereas there is also evidence showing that IL-6 is downregulated by E2 through estrogen receptor(ER) pathway, suggesting that IL-6 is an important estrogen target gene.Therefore, we investigated the relationship between E2, IL-6 and aromatase in endometrial cancer and tested the hypothesis that a positive feedback loop was presence in which E2-induced IL-6 production in cancer cells stimulates aromatase expression in stromal cells in a paracrine manner, thereby promoting intratumoral E2 biosynthesis. This feedback loop also involves ERα, nuclear factor-kappa B(NF-κB). A monoclonal antibody to IL-6 receptor(IL-6R Ab) is currently available as an approach to selectively block IL-6 signaling. Then we examined the effect of IL-6R antibody on the intratumoral E2 biosynthesis using cell coculture systems and an orthotopic endometrial carcinoma model in vivo. These studies define a novel mechanism for local E2 biosynthesis and suggest an effective therapeutic strategy to overcome intratumoral E2 elevation.Part I.The expression of IL-6 and aromatase in normal human endometrium and endometrial carcinoma tissuesObjective To investigate the expression of IL-6 and aromatase in various uterine endometrial tissues and the correlation between them.Methods We detected the expression and localization of IL-6 and aromatase in various uterine endometrial tissues using immunohistochemistry and immunofluorescence assay.Results In our study, immunohistochemical staining showed that expression of IL-6 and aromatase was upregulated in endometrial carcinoma tissues compared with normal tissues. Only positive staining for aromatase in intratumoral stroma, but not in tumor epithelium, correlated positively with IL-6 expression.Immunofluorescence assay showed colocalization of IL-6 and aromatase in cancer tissues.Conclusion Aromatase expression in intratumoral stroma, but not in tumor epithelium, correlated significantly with that of IL-6, which suggested that they promoted the endometrial cancer carcinogenesis and progression by the tumor microenvironment.Part Ⅱ.Regulation of IL-6 and aromatase expression in endometrial carcinoma cells and stromal cellsObjective To investigate the mechanism involved in IL-6 expression in endometrial carcinoma cells and aromatase expression in stromal cells.Methods Primary stromal cells employed in this study were isolated from human endometrial carcinoma tissues. We detected the expression of IL-6 and aromatase using real-time RT-PCR,western blot,ELISA,aromatase assay,chemiluminescent technology and immunoprecipitation.Results Treatment of endometrial cancer cell lines(Ishikawa, RL95-2 and HEC-1B) with E2 resulted in enhanced IL-6 production through cooperation between ERα and NF-κB(P < 0.01). IL-6 significantly increased aromatase m RNA and protein production(P < 0.01). Furthermore, IL-6R Ab reduced the enhancement.Conclusion These studies suggested the existence of a positive feedback loop, that is, IL-6 stimulated by E2 in endometrial cancer cells induced aromatase expression in stromal cells, promoting enhanced intratumoral E2 synthesis in situ.Part Ⅲ. A positive feedback loop of E2 biosynthesis involving IL-6 and aromatase in cell coculture model and orthotopic endometrial carcinoma modelObjective To investigate the activation of a positive feedback loop of E2 biosynthesis involving IL-6 and aromatase in cells coculture model and an orthotopic endometrial carcinoma model.Methods We detected the expression of aromatase and E2 concentration in various coculture conditions and the effect of IL-6R Ab in an orthotopic endometrial carcinoma model.Results We found that aromatase m RNA in stromal cells and E2 concentration were significantly increased in coculture system previously treated with E2 compared with the control group. The mean tumor volume in E2-treated mice increased significantly compared with the control group(P = 0.007), and IL-6R Ab treatment reduced the enhancement(P = 0.001).Conclusion These studies elucidated the activation of a positive feedback loop involving IL-6 and aromatase. IL-6R Ab had the therapeutic effect to overcome in situ estrogen biosynthesis in endometrial carcinoma.