Fei et al. Cell Communication and Signaling (2017) 15:56 DOI 10.1186/s12964-017-0211-0 RESEARCH Open Access A functional role of LEFTY during progesterone therapy for endometrial carcinoma Wu Fei1,2†, Daiki Kijima1†, Mami Hashimoto1†, Miki Hashimura1, Yasuko Oguri1, Sabine Kajita1, Toshihide Matsumoto1, Ako Yokoi1 and Makoto Saegusa1* Abstract Background: The left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas. Methods: Regulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3β, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas. Results: In clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3β showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3β, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3β resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3β suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively. Conclusion: Upregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2. Keywords: Lefty, Endometrial carcinoma, Progesterone therapy, TGF-β, Smad, Cyclin a Background factors including marked obesity, diabetes mellitus, and Endometrial carcinoma (Em Ca) is the most common hypertension [3, 4]. In these younger patients who want to gynecologic malignancy. Although most patients are post- have children in the future, hormone therapy using high- menopausal, approximately 5% of women with this disease dose progesterone is frequently performed for the preser- are diagnosed before 40 years of age [1, 2]. This paradigm vation of fertility [3–5]. shift to younger ages may be due to an increase in the risk Transforming growth factor β (TGF-β)isamemberof the large family of structurally related cytokines that play an important role in controlling cell proliferation, differen- * Correspondence: [email protected] † tiation, migration, and apoptosis [6–9]. Upon ligand bind- Equal contributors 1Department of Pathology, Kitasato University School of Medicine, 1-15-1 ing, TGF-β receptor I recruits and subsequently induces Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan an intracellular downstream signaling cascade involving Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fei et al. Cell Communication and Signaling (2017) 15:56 Page 2 of 15 Smad proteins which in turn act as transcription factors acetate (MPA) via daily oral administration. Biopsy sam- [6–9]. In human endometrium, TGF-β1expressionis ples were taken by diagnostic curettage or hysteroscopy up-regulated during periods of increasing plasma pro- before and during progesterone therapy, and all con- gesterone concentrations and down-regulated during tained sufficient tumor elements to allow examination of its withdrawal, along with significant correlations to sequential morphological changes. manifestation of various abnormalities including endomet- Ninety-six biopsy specimens of normal endometrial riosis, abnormal uterine bleeding, and Em Ca [10–15]. tissues including 24 in the proliferative phase, 52 in the The left-right determination factor (LEFTY) is a novel secretory phase (10 early and 20 middle and late), and member of the TGF-β superfamily, consisting of 20 in the menstrual phase were also investigated. All LEFTY1 and LEFTY2. They are both 366 amino acids tissues were routinely fixed in 10% formalin and proc- long, and differ at only 16 residues, thus they are 97% essed for embedding in paraffin. In addition, 40 fresh identical and share 350 identical residues [16, 17]. Em Ca samples (20 G1, 7 G2, and 13 G3), as well as 22 LEFTY1/2 serve as repressors for TGF-β signaling by normal endometrial tissues were applied. inhibiting the phosphorylation of Smad2 after activation Histopathological analysis of endometrial tumors dur- of the TGF-β receptor [18]. LEFTY2, also known as endo- ing progesterone therapy. metrial bleeding-associated factor (ebaf), is controlled by Evaluation of morphological changes that occurred dur- ovarian steroids, and its expression correlates chronologic- ing progesterone therapy was performed in accordance ally and spatially with that of bleeding-associated matrix with methods described previously [24, 25]. Briefly, the metalloproteinases, indicating that LEFTY2 is a good can- sections obtained from tumors were examined in terms of didate to locally modulate progesterone action on these the following four parameters: 1) cellularity, 2) nuclear re- proteinases within the endometrium [19–21]. arrangement, 3) eosinophilia in the cytoplasm, and 4) the In this study, we set out to first clarify the expression nuclear / cytoplasmic ratio. Therapeutic efficacy (TE) was and the regulation of LEFTY1/2 in normal and malignant graded by counting the numbers of altered parameters. endometrium. Next, change in LEFTY1/2 expression in Cases were subdivided into two categories, as follows: TE patients with Em Cas undergoing progesterone therapy grade 0, 1, or 2 is defined as poor response; TE grade 3 or was investigated. Finally, we examined the functional roles 4 is defined as good response. of LEFTY1/2 in TGF-β/Smad signaling pathways in Em Ca patients receiving the therapy. Antibodies and reagents Anti-LEFTY, anti-Smad2, and anti-phospho(p)-Smad2 at Methods serine 255 (pSmad2) antibodies were purchased from Clinical cases Abcam (Cambridge, MA, USA). Anti-p27Kip1 and anti- Histological findings were reviewed in hysterectomy speci- glycogen synthase kinase (GSK)-3β antibodies were bought mens of endometrioid-type Em Cas from the case records from BD Biosciences (San Jose, CA, USA). Anti-p21waf1, of Kitasato University Hospital during the period from anti-cyclin D1, and anti-Ki-67 antibodies were purchased 2007 to 2015, according to the criteria of the 2014 World from Dako (Copenhagen, Denmark). Anti-cyclin A2, anti- Health Organization classification. [22] Each case was also estrogen receptor α (ERα), and anti-progesterone receptor staged according to the 2009 International Federation of (PR) antibodies were from Novocastra (Newcastle, UK). Gynecology and Obstetrics (FIGO) staging system. [23] A Anti-pGSK-3β at Ser9 (pGSK-3β), anti-Akt, anti-pAkt at total of 120 cases of Em Cas, including 49 of grade (G)1, serine 473 (pAkt), and anti-ubiquitin antibodies were from 14 of G2, and 57 of G3, as well as 22 samples of complex Cell Signaling Technology (Danvers, MA, USA). Anti-β- hyperplasia with or without atypia, were investigated. For actin antibody, nocodazole, 17β-estradiol (E2), medroxypro- the carcinoma cases, the mean age of the patients was gesterone 17-acetate (MPA), MG132, and lithium chloride 54.5 years (range from 30 to 84), and 53 were post- (LiCl) were purchased from Sigma-Aldrich Chemicals (St. menopausal. In addition, our cases included 74 subcate- Louis, MO, USA). Rapamycin and aphidicolin were gorized as clinical FIGO stage I and 29 as stage II-IV, 27 obtained from Calbiochem (Cambridge, MA, USA). with upper (≦1/2) myometrial invasion and 39 with lower Recombinant TGF-β1 was purchased from R&D Systems (>1/2) myometrial invasion, as well as 18 that were posi- (Minneapolis, MN, USA). tive for nodal metastasis and 67 that were negative. A total of 32 patients with G1 Em Cas who received Immunohistochemistry (IHC) progesterone therapy were also selected (Table 1). These IHC was performed using a combination of microwave- patients were diagnosed as stage I based on the findings oven heating and Histofine Simple Stain MAX-PO of both physical and radiographic examinations, and (MULTI) (Nichirei Biosciences, Tokyo, Japan) methods. their mean age was 30.3 years (range from 22 to 38). All For evaluation of IHC findings, scoring of nuclear and/ patients received 400 to 800 mg of medroxyprogesterone or cytoplasmic immunoreactivity for LEFTY,