Forty-nine samples were obtained from the Tissue Bank of the Department of Obstetrics and Gynecology of the Shanghai First People's Hospital affiliated to Shanghai Jiao Tong University, which were comprised of 18 normal endometrial tissues and 31 cases of type I endometrial carcinoma. None of the patients in the study had a history of prior radiotherapy or chemotherapy. Any patient with a known history of hormone replacement was excluded. The use of these specimens was approved by the Ethics Committee of the Medical College of Shanghai Jiao Tong University, China.

IHC analysis of FTO protein expression was performed as previously described and assessed using a semi-quantitative method. Briefly, specimens were deparaffinized in xylene and rehydrated in a graded series of ethanol and subsequently endogenous peroxidase activity was blocked by a 10-min treatment with 3.0% hydrogen peroxide. Subsequently, the sections were subjected to antigen retrieval by boiling in citrate buffer (pH 6.0) and incubated for 30 min with 0.01% Trixon and then incubated for 20 min with 5% bovine serum albumin (BSA). The sections were incubated overnight with a rabbit anti-human FTO primary antibody at 4°C in a humidity chamber, followed by a 50-min incubation with a biotinylated secondary antibody (Dako, Carpinteria, CA, USA). Omitted primary antibodies served as negative controls. Expression of FTO protein was assessed using a semi-quantitative method: the slides were evaluated for the percentage of positively stained cells (0–4) and the intensity of the staining (0–3). The index of FTO expression was calculated as the percentage x intensity of the staining. Therefore, a score of 0 is negative (−), 1–4 is weakly positive (+), 5–8 is positive (++), and 9–12 is strongly positive (+++).

To investigate the mechanism of estrogen-induced FTO nuclear localization, the Ishikawa cell line was used in the present study, which is an estrogen-responsive cell line derived from a well-differentiated endometrioid carcinoma. The cells were maintained in our laboratory after being generously provided by Dr Masato Nishida, Tsukuba University, Tsukuba City, Japan. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) medium with 10% fetal bovine serum (FBS) (both from Gibco, Gaithersburg, MD, USA), 100 U/ml penicillin, sodium pyruvate and L-glutamine in a humidified atmosphere of 5% CO₂ at 37°C.

Immunblot analysis was performed as previously described. Briefly, the harvested cells were lysed and the supernatant was collected. Then, the protein was loaded onto SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk for 1 h and incubated overnight with the primary antibodies, followed by 1 h of incubation with the appropriate secondary antibody (1:5,000). The anti-GAPDH or anti-lamin B1 rabbit monoclonal antibody was diluted to 1:1,000 for use as a sample loading control. The antibodies for FTO, GAPDH, lamin B1, p-mTOR, mTOR and ERα were purchased from Abcam (Cambridge, UK).

Ishikawa cells treated with estrogen, rapamycin or transfected with siERα were harvested and lysed with cytoplasmic extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 1 mM dithiothreitol, 0.2% Nonidet P-40, 1 mM NaF, 1 mM Na₃VO₄ and protease inhibitor cocktail). After being centrifuged at 14,000 rpm at 4°C for 5 min, the cytoplasmic fraction was collected. Then, the pellet was re-suspended in nuclear extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 0.1 mM EDTA pH 8.0, 1.5 mM MgCl₂, 1 mM dithiothreitol, 0.2% Triton-X 100, 1 mM NaF, 1 mM Na₃VO₄ and protease inhibitor cocktail), and the nuclear fraction was collected after a 5-min centrifugation.

Ishikawa cells treated with 10⁻⁹ M E2 for 48 h were cultured on coverslips before fixation with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) (10 min), permeabilization with 0.2% Triton X-100 (10 min), blocking in 3% BSA (1 h) and then incubation with an anti-human FTO primary antibody (1:100, overnight). After incubation with a FITC-labeled secondary antibody (1 h at room temperature), the cells were photographed.

The acute knockdown of siERα was performed as previously described. Briefly, Ishikawa cells were seeded in 5 ml of growth medium in 6-cm dishes without antibiotics, and grown to 30–50% confluency 24 h prior to transfection with 200 pmol ERα siRNA (Shanghai GenePharma Co., Ltd.) using DharmaFECT (Thermo Scientific). siRNA-treated and untreated Ishikawa cells were exposed to 1 nM E2 for a further 48 h before being collected for western blot analysis. The FTO knockdown was performed in a 96-well with the incubated Ishikawa cells, and the cell proliferation was determined by MTT assay.

To investigate the proliferative activity of endometrial cancer cells after various treatments, the MTT assay was performed. Briefly, Ishikawa cells were plated in a 96-well plate (2,000 cells/well) and incubated for 24 h. The culture medium was then changed to serum-free DMEM/F-12 (1:1) medium for 24 h. In order to determine the effect of ICI or Rap on cellular growth, cells were pre-treated with ICI or Rap for 1 h after stimulation with 1 nM E2 or dimethyl sulfoxide (DMSO) for 48 h. Similarly, prior to 1 nM E2 stimulation, the cells were transiently transfected with FTO siRNA to investigate its role in cell proliferation. MTT solution (20 µl of 5 mg/ml MTT in PBS) was added to the cells. After 4 h of incubation at 37°C, the culture medium was removed, and 150 µl of DMSO was added to dissolve the formazan. Finally, absorbance at 490 nm was measured with a GENios multifunction reader (Tecan, Zurich, Switzerland).

The statistical significance of the differences in the IHC staining in endometrial tissues was calculated using the Chi-square test. The differences in various protein levels and cell proliferation between groups were analyzed using the Student's t-test. A two-sided test with P<0.05 was considered statistically significant. All statistical analyses were performed using SPSS 11.0 (SPSS, Inc., Chicago, IL, USA).

To understand the role of FTO in endometrial cancer development, we first examined the expression of FTO in endometrial carcinoma tissues. As shown in Fig. 1A, little positive staining was detected in the normal endometrial tissues while strong positive staining was observed in type I endometrial carcinomas. The significant difference is summarized in Fig. 1B. Although we reported that FTO expression was not associated with age, stage, grade, invasion and lymph node metastasis, we still found that higher FTO expression correlated with poor prognosis and early relapse (Fig. 1C and D).

In our previous study, increasing nuclear expression of FTO was observed after estrogen stimulation in endometrial cancer cells (7). Consistent with this result, we found that estrogen promoted FTO expression in a dose-dependent manner. The major stimulating peak was observed with 1–10 nM estrogen treatment (Fig. 2A). Moreover, estrogen also induced FTO expression in a time-dependent manner. After treatment with 1 nM estrogen for 48 h, a significant stimulating effect was observed (Fig. 2B). We further isolated the nuclear and cytosolic proteins. Estrogen-induced FTO was detected, however, there was no significant alteration of FTO expression in the cytosol following estrogen treatment (Fig. 2C).

To understand the mechanism of estrogen-induced FTO nuclear localization, we scanned the signaling pathways which may be involve in this event. As shown in Fig. 3A, estrogen increased the phosphorylation of mTOR in a dose-dependent manner. Pretreatment with rapamycin potently blocked the estrogen-activated mTOR signaling pathway (Fig. 3B). After combined treatment with estrogen and rapamycin, we found that estrogen-induced FTO nuclear localization was attenuated. The FTO expression pattern in the nucleus following estrogen plus rapamycin treatment was lower than that with estrogen stimulation alone (Fig. 3C and D).

ERα plays an important role in estrogen-mediated bio-functions. Therefore, we investigated the effect of ERα on estrogen-induced FTO nuclear localization. As shown in Fig. 4A, acute transfection of siERα resulted in a marked decrease in ERα protein, which had no effect on FTO nuclear expression. However, estrogen-induced FTO expression in the nucleus was blocked by deletion of ERα (Fig. 4B). Immunocytochemistry assay demonstrated that estrogen stimulation accumulated FTO protein in the nucleus, however, the increased FTO protein dot in the nucleus was abolished by 1 µM fulvestrant (ICI 182,780; purchased from Sigma-Aldrich) treatment, a selective estrogen receptor antagonist (Fig. 4C). These data suggested that ERα is required for estrogen-induced FTO nuclear localization.

To understand FTO nuclear localization in endometrial cancer proliferation, we carried out an MTT assay. As shown in Fig. 5, pretreatment with ICI, to block the ERα signaling pathway, attenuated Ishikawa cell proliferative activity. Moreover, ICI treatment also blocked estrogen-induced cell growth. Similarly, blocking the mTOR signaling pathway with rapamycin also abolished the estrogen-stimulated cell proliferation. Targeting FTO by siFTO directly resulted in decreases proliferative activity, whereas estrogen treatment could not rescue the cellular growth.