Objective
To explore the role of estrogen receptor-α36 (ER-α36) in epidermal growth factor receptor (EGFR)-related carcinogenesis in endometrial cancer.
Study Design
The expression of ER-α36, EGFR, and phospho-extracellular signal-regulated kinase was analyzed using immunohistochemistry in endometrial cancer samples. The cellular localization of ER-α36 and EGFR was determined using immunofluorescence in the endometrial cancer Hec1A cells. The level of phospho-extracellular signal-regulated kinase of Hec1A cells was determined using Western blotting after treatment with epidermal growth factor.
Results
Positive rate of ER-α36 was increased in high-stage ( P = .03) and high-grade ( P = .224) endometrial cancer; expression of ER-α36 and EGFR exhibited a significant positive correlation (r = 0.334, P = .025) and they showed substantial colocalization on the plasma membrane of glandular cells; phospho-extracellular signal-regulated kinase positive rate in ER-α36 positive group and EGFR positive group was higher than that of ER-α36 negative group ( P = .014) and EGFR negative group ( P = .016); finally, ER-α36 mediated epidermal growth factor-stimulated extracellular signal-regulated kinase activation in Hec1A cells.
Conclusion
ER-α36 mediates EGFR-related extracellular signal-regulated kinase activation in endometrial cancer.
Endometrial cancer is one of the most common gynecologic malignancies and is the seventh most common cancer in women worldwide. Multiple factors are involved in development and progression of endometrial cancer, such as estrogen, testosterone, and epidermal growth factor (EGF).
Recently, a novel variant of estrogen receptor α, termed estrogen receptor-α36 (ER-α36), was cloned. ER-α36, which is generated from a promoter located in the first intron of the ER-α66 (the classic estrogen receptor) gene, lacks transcriptional activation domain (AF1 and AF2) of ER-α66, but retains the DNA-binding domain, partial dimerization and ligand-binding domains. ER-α36 possesses unique 27-aa domain to replace the last 138-aa encoded by extrons 7 and 8 of ER-α66 gene. Current research found that ER-α36 is mainly localized on the membrane and modulates nongenomic signaling pathways, such as PI3K/Akt, MAPK/ERK, and PKC pathway, which participate in the development and progression of many types of cancers. Clinical studies reported that overexpression of ER-α36 was associated with poorer disease-free survival and disease-specific survival in patients with ER-α66-positive breast cancer who received tamoxifen treatment. Breast cancer cell line MCF-7 that constitutively express high levels of recombinant ER-α36 exhibited insensitivity to tamoxifen treatment. However, up to now, there was no related clinical data about ER-α36 and endometrial cancer.
Epidermal growth factor receptor (EGFR), the prototypic member of the ErbB/HER receptor tyrosine kinase family, is associated with cancer in general, and the up-regulation of EGFR contributes to the resistance and progression of cancer. Overexpression of EGFR plays an important role in activating the growth factor signaling. Increasing evidence has shown that EGFR is involved in the development of endometrial cancer in particular. Studies have found that EGF could stimulate the mitogen-activated protein kinase (MAPK) pathway, causing an increase in the level of phosphorylation of AF1 domain of ER-α66, which results in the expression of estrogen-response genes. Taken together, estrogen receptor and EGFR may act coordinately in the development of gynecologic neoplasm.
As ER-α36 lacks the transcriptional activation domains of ER-α66, we thus hypothesized that ER-α36 might be associated with the EGFR-related carcinogenesis through a different mechanism from ER-α66. In this study, we aimed to establish the evidence that ER-α36 plays a role in the EGFR-related endometrial cancer and preliminarily explore its potential mechanism, hoping to provide a new insight to the complicated genesis and development of endometrial cancer.
Materials and Methods
Materials and reagents
Anti-ERK1/2 antibody, anti-phospho-ERK1/2 antibody (Thr 202 /Tyr 204 ), and EGFR antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ER-α36 specific antibody against the 20 unique amino acids at the C-terminal of ER-α36, was described previously. EGF was obtained from Sigma-Aldrich (St. Louis, MO).
Cell culture and cell lines
Human Hec1A endometrial cancer cells were provided by Dr Li-Hui Wei (Peking University People’s Hospital, Beijing, China). It has been reported that endometrial cancer Hec1A cell is an ER-α66-negative cell line. Hec1A cells were grown at 37°C with 5% CO 2 in DMEM supplemented with 10% fetal calf serum. We established stable Hec1A cell lines transfected with an ER-α36 shRNA expression vector (Hec1A/RNAi) and the empty expression vector (Hec1A/V) as described elsewhere.
Immunofluorescence and confocal microscopy
The cellular colocalization of ER-α36 and EGFR was determined by indirect immunofluorescence. Hec1A cells cultured on sterile glass coverslips were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes. After being permeabilized with 0.4% Triton X-100 for 10 minutes at room temperature, cells were blocked in 4% bovine serum albumin (BSA)-supplemented PBS for 1 hour and incubated overnight at 4°C with anti-ER-α36-specific antibody against the 20 unique amino acids at the C-terminal of ER-α36. After 3 washes in PBS, the cells were labeled with TRITC-conjugated secondary antibody. The cells were again blocked in 1% BSA-supplemented PBS for 1 hour at room temperature, followed by staining with EGFR antibody. After 3 washes in PBS, the cells were labeled with FITC-conjugated secondary antibody. The DNA dye Hoechst 33258 was used for nuclear staining. Microscopic analyses were performed using a Confocal Laser-Scanning Microscope (Zeiss LSM 510 META; Carl Zeiss, LLC, Berlin, Germany).
Western blotting analysis
Cells were grown in phenol-red-free medium with 2.5% dextran charcoal-stripped FCS (Biochrom AG, Berlin, Germany) for 48-72 hours and then switched to medium without serum 12 hours before stimulation by the agents indicated. The cells were collected in ice-cold PBS, and the cell extracts were prepared in RIPA buffer with proteinase inhibitor cocktail from Sigma-Aldrich (St. Louis, MO). Cell lysates were boiled with gel-loading buffer for 5 minutes at 100°C, resolved on 10% SDS-PAGE, transferred to polyvinylidene fluoride membrane. After transfer, the membranes were blocked in TBST (TBS containing 0.1% Tween 20) containing 5% skimmed milk for 2 hours, followed by incubation overnight at 4°C with appropriate primary antibodies. After washing 3 times in TBST, 10 minutes each, the membranes were incubated for 1 hour at 37°C with 1:2000 horseradish peroxidase-conjugated appropriate secondary antibodies. Finally, the membranes were processed and visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotechnology, Piscataway, NJ).
Immunohistochemistry
Endometrial tissues were collected for immunohistochemistry when patients with endometrial cancer underwent hysterectomy in Peking University Third Hospital. The study was approved by the Ethics Committee of Peking University Third Hospital and all written informed consent documents were signed by all patients. The mean age of the patients was 56.94 ± 1.48 years old. All the samples were type I endometrial cancers with histology of endometrioid cancer. We used International Federation of Gynecology and Obstetrics 2000 to define the stage. Stage Ia (low-stage) was defined as the cancer confined in the endometrium without metastasized to myometrium or other position else. Stage above Ia (high-stage) was defined as cancer metastasized outward endometria. Tissue slides were deparaffinized with xylene and rehydrated through a graded alcohol series. Antigen retrieval was carried out by immersing the slides in natrium citrate buffer and boiling in a water bath at 100°C for 30 minutes. The endogenous peroxidase activity was blocked by incubation in a 3% hydrogen peroxide buffer for 10 minutes. The slides were rinsed in PBS and incubated with normal goat serum to block nonspecific staining. The slides were then incubated with the primary antibody overnight at 4°C in a humidified chamber. The sections were incubated with the second antibody for 30 minutes. Diaminobenzidine was used as a chromogen, and sections were counterstained with hematoxylin. The staining intensity in the plasma membrane was evaluated. Duplicate sections were immunostained without exposure to primary antibodies and were used as negative controls. Samples were evaluated by 3 observers who were unaware of the identity of the sections. Scoring for staining was graded as follows: no staining or staining observed in less than 10% of glandular cells was scored as 0; faint/barely perceptible staining detected in ≥10% of glandular cells was scored as 1+; a moderate or strong complete staining observed in ≥10% of glandular cells was scored as 2+ or 3+, respectively. Scores of 0 were considered negative, whereas 1+, 2+, and 3+ were considered positive. The χ 2 tests were used to analyze the data.
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