Extensive exposure to estrogen is generally acknowledged as a risk factor for endometrial cancer. Given that the accumulation of adipocytes also contributes to the increased production of estrogen, in the present study, we evaluated the expression of the fat mass and obesity-associated (
Endometrial cancer is one of the most common gynecologic malignancies worldwide (
FTO was identified as an oncoprotein frequently over-expressed in several types of cancer, including endometrial, breast and pancreatic cancer (
Although less than 0.1% of the total cellular protein, kinase and phosphatase enzymes play a pivotal role in conducting signals to control cell growth or invasion, abundant signaling pathways have been reported to be involved in estrogen-driven endometrial cancer. Inhibition of the PI3K/AKT pathway was found to lead to a decrease in proliferative and invasive activities. Blocking MAPK signaling also resulted in similar effects. These results are consistent with our previous study (
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
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
In our previous study, increasing nuclear expression of FTO was observed after estrogen stimulation in endometrial cancer cells (
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
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
To understand FTO nuclear localization in endometrial cancer proliferation, we carried out an MTT assay. As shown in
In the present study, we found that the fat mass and obesity-associated (FTO) gene was overexpressed in endometrial carcinoma tissues and estrogen induced FTO nuclear localization, which facilitated endometrial cancer proliferation through the mTOR signaling pathway.
Most endometrial adenocarcinomas are characterized by positive nuclear estrogen receptor (ER) expression and responsiveness to hormone stimulation. Increasing evidence indicates that prolonged estrogen exposure is associated with initiation of type 1 endometrioid cancers (
An investigation with a larger sample size confirmed that the obesity-associated polymorphism FTO rs9939609 is strongly associated with endometrial cancer risk in non-Hispanic white women (
A previous study demonstrated that mTOR signaling is involved in estrogen-driven endometrial cancer development (
Given that ERα mediated estrogen-induced multiple functions, we further investigated the role of ERα in FTO nuclear localization. In the present study, we found that knockdown of ERα had a slight effect on FTO protein nuclear localization. However, deletion of ERα blocked estrogen-induced FTO nuclear accumulation. We considered that ERα has no effect on FTO expression, but estrogen-mediated function is ERα-dependent. Therefore, depletion of ERα resulted in the inability of estrogen to bind with ERα, in turn leading to estrogen-driven FTO nuclear accumulation failure. These data imply that ERα is required for FTO nuclear localization. Although we gathered significant data in the present study, to the best of my knowledge, ERα was also located in the nucleus and served as a transcription factor. Yet, we still raised the question of how ERα mediates FTO nuclear accumulation. We hypothesized that ERα may recruit some molecules and construct a protein complex, which in turn helps FTO to transfer into the nucleus. Various proteins have different functions when they present in different cellular localizations, such as caveolin. In the present study, we found that FTO nuclear localization promoted cell proliferation, whereas blocking the nuclear accumulation by ICI or Rap pretreatment, even by direct deletion of FTO by siRNA, resulted in attenuated proliferative activity. The decreased proliferation could not be rescued by estrogen stimulation.
In conclusion, the present study suggests that overexpression of FTO in endometrial carcinoma may be a poor prognostic marker. Importantly, FTO nuclear accumulation may be an essential step for estrogen-driven endometrial tumor formation and progression. Our findings may provide new insight into the mechanisms underlying E2-induced proliferation. Additionally, the present study further supports the possibility of using FTO as a target for the treatment of endometrial cancer.
The present study was supported by grants from the National Natural Science Foundation of China (NSFC no. 81372795).
Type I endometrial cancer exhibits elevated FTO expression. (A) The expression levels of FTO in normal endometrial tissues and cancers were assessed using IHC staining. (B) FTO IHC scores in normal endometrial samples and cancers. (C and D) The overall survival and disease-free survival curves of endometrial cancer patients with different expression of FTO.
Estrogen promotes FTO nuclear localization. Western blotting was used to determine the FTO expression pattern after treatment with different doses of estrogen (A) and for different times (B). The upper panels are representative bands of the western blotting. Lower panels show statistical analysis of changes in FTO protein after the above indicated treatments. *P<0.05, compared with the control. (C) After treatment with estrogen for 48 h, western blotting was used to detect the alteration of FTO expression in the nuclear and cytosolic fractions. GAPDH and lamin B1 served as loading controls. Ishikawa cells were used in the present study.
Estrogen controls FTO nuclear localization through the mTOR signaling pathway. (A) Activation of mTOR signaling was determined after estrogen treatment at different doses. The upper panels are representative bands of the western blotting. Lower panels show statistical analysis of changes in mTOR signaling (p-mTOR/mTOR) after treatment with different dose of estrogen. *P<0.05, compared with the control. (B) Effects of rapamycin (Rap) (100 nM) on estrogen-induced (10−9 M) activation of mTOR signaling. Effects of rapamycin (100 nM) on estrogen-induced (10−9 M) FTO nuclear localization were evaluated by western blotting (C) and immunocytochemical assay (D).
ERα knockdown is required for estrogen-induced FTO nuclear localization. (A) Transfection efficiency of siERα was determined by western blotting. (B) The effect of knockdown of ERα on FTO nuclear localization after estrogen stimulation. (C) Immunocytochemical assay was used to determine the decrease in FTO nuclear accumulation resulting from ICI (1
Estrogen-induced FTO nuclear localization promotes endometrial cancer cell proliferation. After treatment with ICI (1