Purpose. Wnt signaling regulates the fine balance between stemness and differentiation. Here, the role of Wnt signaling to maintain the balance between estrogen-induced proliferation and progesterone-induced differentiation during the menstrual cycle, as well as during the induction of hyperplasia and carcinogenesis of the endometrium, was investigated. Experimental Design: Endometrial gene expression profiles from estradiol (E 2 ) and E 2 + medroxyprogesterone acetate-treated postmenopausal patients were combined with profiles obtained during the menstrual cycle (PubMed; GEO DataSets). Ishikawa cells were transfected with progesterone receptors and Wnt inhibitors dickkopf homologue 1 (DKK1) and forkhead box O1 (FOXO1), measuring Wnt activation. Expression of DKK1 and FOXO1 was inhibited by use of sequence-specific short hairpins. Furthermore, patient samples (hormone-treated endometria, hyperplasia, and endometrial cancer) were stained for Wnt activation using nuclear β-catenin and CD44. Results: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E 2 and progesterone. In Wnt-activated Ishikawa cells, progesterone inhibits Wnt signaling by induction of DKK1 and FOXO1. Furthermore, using siRNA-mediated knockdown of both DKK1 and FOXO1, progesterone inhibition of Wnt signaling was partly circumvented. Subsequently, immunohistochemical analysis of the Wnt target gene CD44 showed that progesterone acted as an inhibitor of Wnt signaling in hyperplasia and in well-differentiated endometrial cancer. The female sex hormones estradiol (E 2 ) and progesterone play rate-limiting roles in the cyclical renewal of the inner layer of the uterus (endometrium). In the first half of the regular menstrual cycle, the proliferation phase, E 2 is required to expand the endometrial layer by inducing cell proliferation. In the second half of the menstrual cycle, the secretory phase, progesterone levels increase, which antagonizes the proliferative activity of E 2 by inducing differentiation of epithelial and stromal cells of the endometrium (1). Thus, inhibition of E 2 -induced proliferation by progesterone is crucial for the maintenance of homeostasis in the endometrium.Increased estrogen signaling often underlies endometrial hyperplasia and is a well-established risk factor for endometrial cancer (2). Because progesterone inhibits estrogen-induced endometrial proliferation, progesterone has been used in its synthetic form [i.e., medroxyprogesterone acetate (MPA)] in palliative treatment of advanced and recurrent endometrial cancer with modest though significant response rates (15-25%; ref. 3). Progesterone has also been used as a primary treatment for endometrial carcinoma confined to the endometrial layer of the uterus, for example, in premenopausal women determined to preserve fertility. Response rates in these women can be up to 60% (4, 5), indicating that progesterone signaling in well-differentiated endometrial cancer is a potent inhibitor of endometrial carcinogene...

In many type I endometrial cancers, the PTEN gene is inactivated, which ultimately leads to constitutively active Akt and the inhibition of Forkhead box O1 (FOXO1), a member of the FOXO subfamily of Forkhead/winged helix family of transcription factors. The expression, regulation, and function of FOXO1 in endometrial cancer were investigated in this study. Immunohistochemical analysis of 49 endometrial tumor tissues revealed a decrease of FOXO1 expression in 95.9% of the cases compared with the expression in normal endometrium. In four different endometrial cancer cell lines (ECC1, Hec1B, Ishikawa, and RL95), FOXO1 mRNA was expressed at similar levels; however, protein levels were low or undetectable in Ecc1, Ishikawa, and RL95 cells. Using small interfering RNA technology, we demonstrated that the low levels of FOXO1 protein were due to the involvement of Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, given that silencing Skp2 increased FOXO1 protein expression in Ishikawa cells. Inhibition of Akt in Ishikawa cells also increased nuclear FOXO1 protein levels. Additionally, progestins increased FOXO1 protein levels, specifically through progesterone receptor B (PRB) as determined by using stably transfected PRA-specific and PRB-specific Ishikawa cell lines. Finally, overexpression of triple mutant (Tm) FOXO1 in the PR-specific Ishikawa cell lines caused cell cycle arrest and significantly decreased proliferation in the presence and absence of the progestin, R5020. Furthermore, TmFOXO1 overexpression induced apoptosis in PRB-specific cells in the presence and absence of ligand. Taken together, these data provide insight into the phosphoinositide-3-kinase/Akt/FOXO pathway for the determination of progestin responsiveness and the development of alternate therapies for endometrial cancer.

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Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.

Tibolone, a tissue-selective compound with a combination of estrogenic, progestagenic, and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares the endometrial gene expression profiles after short-term (21 days) treatment with tibolone to the profiles after treatment with estradiol-only (E 2 ) and E 2 + medroxyprogesterone acetate (E 2 + MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolapse. The impact of E 2 treatment on endometrial gene expression (799 genes) was much higher than the effect of tibolone (173 genes) or E 2 + MPA treatment (174 genes). Furthermore, endometrial gene expression profiles after tibolone treatment show a weak similarity to the profiles after E 2 treatment (overlap 72 J Mol Med (2007) genes) and even less profile similarity to E 2 + MPA treatment (overlap 17 genes). Interestingly, 95 tibolonespecific genes were identified. Translation of profile similarity into biological processes and pathways showed that ER-mediated downstream processes, such as cell cycle and cell proliferation, are not affected by E2 + MPA, slightly by tibolone, but are significantly affected by E 2 . In conclusion, tibolone treatment results in a tibolone-specific gene expression profile in the human endometrium, which shares only limited resemblance to E 2 and even less resemblance to E2 + MPA induced profiles.