Targeting fibrotic signaling pathways by EGCG as a therapeutic strategy for uterine fibroids.

Fibrosis is characterized by excessive accumulation of extracellular matrix, which is a key feature of uterine fibroids. Our prior research supports the tenet that inhibition of fibrotic processes may restrict fibroid growth. Epigallocatechin gallate (EGCG), a green tea compound with powerful antioxidant properties, is an investigational drug for uterine fibroids. An early phase clinical trial showed that EGCG was effective in reducing fibroid size and its associated symptoms; however, its mechanism of action(s) has not been completely elucidated. Here, we probed effects of EGCG on key signaling pathways involved in fibroid cell fibrosis. Viability of myometrial and fibroid cells was not greatly affected by EGCG treatment (1-200 µM). Cyclin D1, a protein involved in cell cycle progression, was increased in fibroid cells and was significantly reduced by EGCG. EGCG treatment significantly reduced mRNA or protein levels of key fibrotic proteins, including fibronectin (FN1), collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and actin alpha 2, smooth muscle (ACTA2) in fibroid cells, suggesting antifibrotic effects. EGCG treatment altered the activation of YAP, ß-catenin, JNK and AKT, but not Smad 2/3 signaling pathways involved in mediating fibrotic process. Finally, we conducted a comparative study to evaluate the ability of EGCG to regulate fibrosis with synthetic inhibitors. We observed that EGCG displayed greater efficacy than ICG-001 (ß-catenin), SP600125 (JNK) and MK-2206 (AKT) inhibitors, and its effects were equivalent to verteporfin (YAP) or SB525334 (Smad) for regulating expression of key fibrotic mediators. These data indicate that EGCG exhibits anti-fibrotic effects in fibroid cells. These results provide insight into mechanisms behind the observed clinical efficacy of EGCG against uterine fibroids.

Extracellular matrix and Hippo signaling as therapeutic targets of antifibrotic compounds for uterine fibroids.

BACKGROUND: Uterine fibroids are highly prevalent, collagen-rich, mechanically stiff, fibrotic tumors for which new therapeutic options are needed. Increased extracellular matrix (ECM) stiffness activates mechanical signaling and Hippo/YAP promoting fibroid growth, but no prior studies have tested either as a therapeutic target. We tested the hypothesis that injection of a purified form of collagenase Clostridium histolyticum (CCH) that selectively digests type I and type III collagens would alter ECM stiffness, Hippo signaling, and selectively reduce fibroid cell growth. We also used two FDA-approved drugs, verteporfin and nintedanib, to elucidate the role of Hippo/YAP signaling in uterine fibroid and myometrial cells. METHODS: The clinical trial was registered (NCT02889848). Stiffness of samples was measured by rheometry. Protein expression in surgical samples was analyzed via immunofluorescence. Protein and gene expression in uterine fibroid or myometrial cell lines were measured by real time PCR and western blot, and immunofluorescence. RESULTS: Injection of CCH at high doses (0.1-0.2 mg/cm3 ) into fibroids resulted in a 46% reduction in stiffness in injected fibroids compared to controls after 60 days. Levels of the cell proliferation marker proliferative cell nuclear antigen (PCNA) were decreased in fibroids 60 days after injection at high doses of CCH. Key Hippo signaling factors, specifically the transcriptionally inactive phosphorylated YAP (p-YAP), was increased at high CCH doses, supporting the role of YAP in fibroid growth. Furthermore, inhibition of YAP via verteporfin (YAP inhibitor) decreased cell proliferation, gene and protein expression of key factors promoting fibrosis and mechanotransduction in fibroid cells. Additionally, the anti-fibrotic drug, nintedanib, inhibited YAP and showed anti-fibrotic effects. CONCLUSIONS: This is the first report that in vivo injection of collagenase into uterine fibroids led to a reduction in Hippo/YAP signaling and crucial genes and pathways involved in fibroid growth. These results indicate that targeting ECM stiffness and Hippo signaling might be an effective strategy for uterine fibroids.

Diet and Nutrition in Gynecological Disorders: A Focus on Clinical Studies.

A healthy lifestyle and a balanced diet play a paramount role in promoting and maintaining homeostatic functions and preventing an array of chronic and debilitating diseases. Based upon observational and epidemiological investigations, it is clear that nutritional factors and dietary habits play a significant role in gynecological disease development, including uterine leiomyoma, endometriosis, polycystic ovary syndrome, and gynecological malignancies. Diets rich in fruits and vegetables, Mediterranean diets, green tea, vitamin D, and plant-derived natural compounds may have a long-term positive impact on gynecological diseases, while fats, red meat, alcohol, and coffee may contribute to their development. Data regarding the association between dietary habits and gynecological disorders are, at times, conflicting, with potential confounding factors, including food pollutants, reduced physical activity, ethnic background, and environmental factors limiting overall conclusions. This review provides a synopsis of the current clinical data and biological basis of the association between available dietary and nutritional data, along with their impact on the biology and pathophysiology of different gynecological disorders, as well as an outlook on future directions that will guide further investigational research.

A-Kinase Anchoring Protein 13 (AKAP13) Augments Progesterone Signaling in Uterine Fibroid Cells.

Context: Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood. Objective: Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids. Methods and Results: Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus. Conclusion: These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.