|Year : 2017 | Volume
| Issue : 5 | Page : 817-822
Clinical significance of Twist, E-cadherin, and N-cadherin protein expression in endometrioid adenocarcinoma
Xinping Xie, Xiu Zheng, Jinhua Wang, Lihong Chen
Department of Obstetrics and Gynecology, 1st Affiliated Hospital of Fujian Medicial University, 35005, PR. China
|Date of Web Publication||13-Dec-2017|
Department of Obstetrics and Gynecology, 1st Affiliated Hospital of Fujian Medical University
Source of Support: None, Conflict of Interest: None
Objective: The aim of the study was to investigate the expression of Twist, E-cadherin, and N-cadherin both in normal endometrium and in endometrioid adenocarcinoma tissues (NET and EAT), and further discuss the relationship between the proteins expression and the clinical parameters.
Methods: Seventy-six EAT and 50 NET were collected from endometrioid adenocarcinoma patients and patients who received hysterectomy. We used immunohistochemistry (two steps methods) to detect the expression of Twist, E-cadherin, and N-cadherin proteins in EAT and NET. The Twist, E-cadherin, and N-cadherin protein positive expression rate in EAT and NET were compared by Chi-square test. Moreover, the correlation between patients' clincial characteristics and Twist, E-cadherin, and N-cadherin protein expression was evaluated.
Results: The positive expression of Twist and N-cadherin proteins in EAT was significantly higher than those in NET (u = 14.8, 9.04, P < 0.05), the positive expression of E-cadherin protein in ENT was significantly lower than those in NET (u = 4.14, P < 0.05). The Twist, E-cadherin, and N-cadherin expressions were related with endometrioid adenocarcinoma under different International Federation of Gynecology and Obstetrics (FIGO) clinical stages (P < 0.05), depths of tumor invasion (P < 0.05), and tumor differentiation degrees (P < 0.05). However, these proteins exerted no influence on vessel and lymph metastases (P > 0.05). The Spearman rank correlation analysis showed that the expression of the Twist protein and that of the E-cadherin (r = –0.584, P < 0.05), N-cadherin protein (r = 0.460, P < 0.05) in endometrioid adenocarcinoma was significant correlated with statistical difference.
Conclusion: Twist, E-cadherin, and N-cadherin protein were different expressed in EAT and NET which indicating their potential function for endometrioid adenocarcinoma development. Twist may participate in the occurrence of epithelial–mesenchymal transition, affect the expression of E-cadherin and N-cadherin and may be related to metastasis and progression of endometrioid adenocarcinoma.
Keywords: Endometrioid adenocarcinoma, E-cadherin, immunohistochemistry, N-cadherin, Twist
|How to cite this article:|
Xie X, Zheng X, Wang J, Chen L. Clinical significance of Twist, E-cadherin, and N-cadherin protein expression in endometrioid adenocarcinoma. J Can Res Ther 2017;13:817-22
|How to cite this URL:|
Xie X, Zheng X, Wang J, Chen L. Clinical significance of Twist, E-cadherin, and N-cadherin protein expression in endometrioid adenocarcinoma. J Can Res Ther [serial online] 2017 [cited 2019 Dec 12];13:817-22. Available from: http://www.cancerjournal.net/text.asp?2017/13/5/817/220474
| > Introduction|| |
Endometrial cancer is one of the three major malignancies of the female reproductive system. Its morbidity is about 7% of the female malignancies, which accounts for 20%–30% of the female reproductive system malignancies., Human malignancy occurs and develops because of the interaction between oncogene and the cancer suppressor gene. Oncogene activation and the inactivation of the cancer suppressor gene lead to cell cycle disorder. Consequently, cell proliferation becomes uncontrolled and leads to tumor occurrence. Therefore, the research on oncogenes and cancer suppressor genes has been popular in the discussion of the occurrence, development, invasion, and metastasis mechanism of tumors.
Twist, which is an oncogene, has received considerable attention because of its important role in tumor invasion and metastasis. The present study suggests that the Twist gene is involved in the epithelial–mesenchymal transition (EMT), hereby enabling tumor cells to adapt to the surrounding matrix environment and acquire new epithelial properties to form metastatic lesions.,,, Furthermore, the migration and proliferation abilities of tumor cells in the metastatic lesions are stronger than that of the primary tumor, even with antitumor drugs. E-cadherin, which is an adhesion molecule, is a homogeneously adherent calcium-dependent transmembrane glycoprotein that mainly mediates among cells. The human E-cadherin gene is located near the No. 16 chromosome q22.1, and its molecular weight is 80–124 kD, which is composed of 723–748 amino acids. It is a single-chain type I transmembrane glycoprotein and widely found in epithelial tissues., The absence of E-cadherin is the core of EMT, which can result in the loss of polarity of epithelial cells, adhesion instability among cells, structure changes in the cytoskeleton, and the dissociation of β-catenin from the complex to help tumor cells fall off from the primary lesion.
Recent, publications reported that neuropathic calcium adhesion protein (N-cadherin) is abnormally expressed in some epithelial tumors. Moreover, its ability to enhance the invasion and metastasis of tumor cells is more evident than that of the E-cadherin downregulated expression, which suggests that E-cadherin may be converted to N-cadherin, which is also considered an important EMT mechanism. The most common type of endometrial cancer is endometrioid adenocarcinoma. The expressions of Twist, E-cadherin, and N-cadherin in endometrioid adenocarcinoma tissue (EAT) have been rarely reported. Hence, we used immunohistochemical method to detect expressions of Twist, E-cadherin, and N-cadherin in endometrioid adenocarcinoma and normal endometrium tissues (NETs). The correlation between the Twist, E-cadherin, and N-cadherin expression levels, the changes in the occurrence and development of endometrioid adenocarcinoma, and its relationship with the clinical-pathological characteristics of patients were also explored.
| > Methods|| |
Seventy-six EAT and 50 NET were collected from endometrioid adenocarcinoma patients and patients who received hysterectomy. The inclusion criteria for endometrioid adenocarcinoma patients were as follows (1) The diagnosis of endometrioid adenocarcinoma was confirmed by pathology after surgery; (2) the patients did not received any radiotherapy or chemotherapy before surgery; (3) signed consensus for tissue experiment use was obtained from all the included patients. The inclusion criteria for contol group subjects were as follows: (1) Patients who received surgery for uterine fibroids; (2) no treatment which can affect the endometrium was given before operation; (3) no other malignant tumors of reproductive system were observed. The general characteristics of included 76 endometrioid adenocarcinoma patients were demonstrated in [Table 1].
Reagents and instruments
Twist mouse antihuman monoclonal antibody (Abcam, USA), E-cadherin mouse antihuman monoclonal antibody (Beijing Zhongshan Golden Bridge Company), N-cadherin mouse antihuman monoclonal antibody (Beijing Zhongshan Golden Bridge Company), two-step immunohistochemistry kit (PV-9000, Beijing Zhongshan Golden Bridge Company), paraffin embedding machine (Hisocentre-2 Germany Leica Company), paraffin slicer (LEICA RM2245 Germany Leica Company), microscopic image acquisition and analysis system (DP70 Olympus), and binocular microscope (ECLIPSE80r Nikon) were used in the present study.
All specimen tissues were approximately 2 cm × 1 cm × 0.3 cm in size, rinsed thoroughly with normal saline, and fixed for 24 h in 10% formaldehyde solution for conventional dehydration and paraffin embedding. Each piece of paraffin was sliced with a paraffin slicer into 3 μm thick slices. Adhesive slides were used to adhere the slices to the center of the slides, and four slices were prepared for each paraffin specimens.
Hematoxylin and eosin staining assay
Paraffin sections (3 μm) were dewaxed 2 times with xylene for 8 min, dehydrated with 100%, 95%, 80%, and 75% gradient alcohol for 4 min each, and rinsed 3 times with phosphate buffered saline (PBS) (0.01 mmol/L) for 5 min. These sections were also stained with hematoxylin dye liquor for 5 min and washed with distilled water for 5 min. Color separation was performed with 1% hydrochloric acid alcohol for 30 s. The samples were again washed with water for 1 h, dipped in distilled water for a moment, stained with eosin dye liquor for 5 min, washed with distilled water for 5 min, and dehydrated with 75%, 80%, 95%, and 100% gradient alcohol for 4 min each. Xylene transparency was carried out 2 times for 8 min. Neutral gum was used in sealing.
The conventional 3 μm thick paraffin sections were baked in an oven at 70°C for 4 h. These paraffin sections were dewaxed with xylene I and II for 10 min each (37°C incubator), anhydrous ethanol I and II for 5 min, 95% ethanol for 3 min, and 80% ethanol for 3 min. These sections were rinsed with tap water for 5–10 s and with distilled water for 1 min. Approximately, 2000 ml of alkaline repair solution (1 mM EDTA, pH 9.0) was boiled in an autoclave. Subsequently, the sections were added in the solution until the autoclave jetted air. Soft fire was used for heat preservation for 20 min. The sections were placed in a PBS buffer solution to rinse 3 times for 2 min. The 3% aqueous solution of hydrogen peroxide was placed at room temperature for 8 min for incubation to remove endogenous peroxidase and rinsed with PBS buffer solution. Afterward, the slices were removed from the solution. The surrounding tissues were dried, and a liquid drop (0.1 ml) was added. The sections were incubated for 2 h at room temperature (E-cadherin and N-cadherin) or placed in a 4°C refrigerator overnight (Twist). Subsequently, the sections were rewarmed at room temperature for 30 min. The primary antibody was shaken, and the sections were rinsed with PBS or tris buffered saline (TBS) buffer solution 3 times for 2 min. The buffer solution was removed and 0.1 ml of reagent 1 (polymer adjuvant) in the two-step method kit was added dropwise. The sections were incubated at room temperature for 20 min and rinsed with PBS or TBS buffer solution 3 times for 2 min. The buffer solution was removed, and 0.1 ml of reagent 2 (horseradish enzyme marked goat antirabbit/mouse immunoglobulin G antibody) in the two-step method kit was added. The sections were again incubated at room temperature for 30 min and rinsed with PBS or TBS buffer solution 3 times for 2 min. The buffer solution was again removed, and 0.1 ml of freshly prepared DAB coloring solution was added. The section was observed under the microscope for 5 min until the best color effect was achieved. Tap water was used to rinse the sections sufficiently to complete the reaction. The cell nuclei were counter-stained with hematoxylin for 2 min. After differentiation and returning to blue, a few sections were randomly selected for observation under the microscope to determine whether the nucleus staining intensity was appropriate or not according to the degree of contrast. The sections were dehydrated with gradient alcohol and xylene and sealed with neutral gum.
Protein expression determination
The positive section was used as the positive control, and PBS, instead of the primary antibody, was used as the negative control. Readings were taken at least twice at different times and rechecked by a pathology attending physician. Twist protein expressions were mainly observed in the cytoplasm, and a few were expressed in the cell nucleus and membrane. E-cadherin expressions were mainly observed in the cell membrane, and a few were expressed in the cytoplasm. N-cadherin expressions were observed in the cytoplasm or membrane. This research considered the brown particles of the cytoplasm or cell membrane as positive expressions. According to the semi quantitative integral method, results were determined under the light microscope to observe at random with 10 high-power lens (10 × 40) field of view (FOV), and each FOV counts 100 cells. The scoring is according to the positive cell percentage of each high-power lens FOV (a): <5% = 0, 5%–25% = 1, 26%–50 = 2, 51%–75% = 3, and >75% = 4. Scoring was also performed using the staining intensity (b): Light yellow = 1, yellow = 2, and brown yellow = 3. The product of the two kinds of scores is the positive intensity: 0 is positive (−), 1–4 are weak positives (+), 5–8 are medium positives(++), and 9–12 are strong positives (+++).
| > Results|| |
Twist protein expression in different endometrium tissues
The positive reaction of the Twist protein is indicated by the brown yellow particles in the cytoplasm and cell nucleus [Figure 1]. In the 76 EAT, the positive expression rate of Twist was 86.8% (10 of them were weakly positive, 24 were moderate positive, and 32 were strongly positive expressions). In the 50 NETs, the Twist positive rate was 34.0% (7 were weakly positive and 4 were strongly positive expressions). A significant difference was observed between the two groups (u = 14.8, P < 0.05) [Table 2].
|Figure 1: Twist protein expression in different endometrium tissues. (a) Endometrioid adenocarcinoma tissue ×200; (b) endometrioid adenocarcinoma tissue ×400; (c) normal endometrium tissue ×200; (d) normal endometrium tissue ×400|
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E-cadherin protein expression in different endometrium tissues
The positive reaction of the E-cadherin protein is indicated by the brown yellow particles in the cytoplasm or cytoplasm [Figure 2]. The positive expression rate of E-cadherin protein was 25.0% with 4 weak positive expression, 5 positive expression, and 10 strong positive expression of the 76 EAT. For the 50 NETs, the E-cadherin protein positive expression rate was 86.0% with 4 weak positive expression, 66 moderate positive expression, and 33 strong positive expression. E-cadherin protein positive expression rate was statistical different between the two groups (u = 4.14, P < 0.05) [Table 3].
|Figure 2: E-cadherin protein expression in different endometrium tissues. (a) Endometrioid adenocarcinoma tissue ×200; (b) endometrioid adenocarcinoma tissue ×400; (c) Normal endometrium tissue ×200; (d) normal endometrium tissue ×400|
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N-cadherin protein expression in different endometrium tissues
The positive reaction of the N-cadherin protein is indicated by the brown yellow particles in the cytoplasm or cytoplasm [Figure 3]. The positive expression rate of N-cadherin protein was 88.2% with thirteen weak positive expression, 33 moderate positive expression and 21 strong positive expression of the 76 EAT. For the 50 NETs, the N-cadherin protein positive expression rate was 40.0% with 8 week positive expression, 9 moderate positive expression, and 3 strong positive expression. N-cadherin protein positive expression rate was statistical different between the two groups (u = 9.04, P < 0.05) [Table 4].
|Figure 3: N-cadherin protein expression in different endometrium tissues. (a) Endometrioid adenocarcinoma tissue ×200; (b) endometrioid adenocarcinoma tissue ×400; (c) normal endometrium tissue ×200; (d) normal endometrium tissue ×400|
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Twist, E-cadherin, and N-cadherin positive expression comparison between EAT and NET
Twist and N-cadherin positive expression rate in EAT was higher than those of NET with statistical difference (P < 0.05). However, E-cadherin positive expression rate in EAT was lower than those of NET (P < 0.05) [Table 5].
|Table 5: Twist, E-cadherin, and N-cadherin positive expression rate in different tissues|
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Relationship between Twist, E-cadherin, and N-cadherin expression and endometrioid adenocarcinoma clinical-pathological parameter
The Twist, E-cadherin, and N-cadherin expressions were related with endometrioid adenocarcinoma under different FIGO clinical stages (P < 0.05), depths of tumor invasion (P < 0.05), and tumor differentiation degrees (P < 0.05). However, these proteins exerted no influence on vessel and lymph metastases (P > 0.05) [Table 6].
|Table 6: Association between twist, E-cadherin, N-cadherin expression, and endometrioid adenocarcinoma clinical-pathological parameter|
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Correlation analysis of the expression of Twist protein and that of E-cadherin and N-cadherin in endometrioid adenocarcinoma tissues
The Spearman rank correlation analysis showed that the expression of the Twist protein and that of the E-cadherin (r = −0.584, P < 0.05), N-cadherin protein (r = 0.460, P < 0.05) in endometrioid adenocarcinoma were significant correlated with statistical difference.
| > Discussion|| |
The development of human malignant tumors is a complex multigene, multistep, and multistage process. The expression imbalance and interaction of lots kinds of oncogenes and tumor suppressor genes leads to cell cycle disorders and uncontrolled proliferation that result in malignant changes. Most malignant tumor patients die of tumor recurrence and metastasis. Therefore, studies on the occurrence and development of malignant tumors, and the invasion and metastasis mechanism have increased over the past 20 years.
Recent studies suggest that Twist gene plays a key role in EMT and affects the apoptosis of tumor cells, inhibits tumor cell differentiation, promotes tumor angiogenesis, and participates in tumor drug resistance. The research performed by Yang et al. based on a series of experiments on mouse breast cancer cell line 4T1 in vitro and in vivo in 2005 showed that the upregulation of the Twist gene can significantly improve tumor cell invasion and intravascular infiltration. When RNA interference technology was used to knockdown the Twist gene expression in a high tumor metastatic cell line, the lung metastasis rate of the breast cancer cells decreases significantly. Ansieau et al. also found that Twist is a key gene in the promotion of cancer invasion and metastasis. This gene also helps cancer cells escape the immune system monitoring, thereby promoting tumor cell growth and spread to other organs. In a normal human body, the Twist gene in silent state can be activated when cancer occurs. Shibata et al. discovered through the follow-up of the cervical cancer patients that the prognoses of the patients in the Twist positive and negative expression groups have significant differences. Therefore, the expression of Twist in patients with cervical cancer is closely related with poor prognosis. Moreover, Twist can be considered an independent prognostic indicator. Kyo et al. used immunohistochemistry and Western blot analysis methods to detect the endometrial cancer tissues in 70 cases of invasion endometrial carcinoma patients. They found that 51% (high expression) of the Twist protein expressions were positively correlated with the tumor invasion depth and related with pelvic lymph nodes metastasis. Nevertheless, no significant correlation with age, FIGO staging, cell differentiation, body mass index (BMI), and menopause was observed.
The present study detected the expressions of the Twist protein in endometrioid adenocarcinoma and NETs. The positive expression rate of Twist in endometrioid adenocarcinoma was 86.8%, which was significantly higher than that in the NET (25.0%). In addition, the Twist expression exerted no influence on endometrioid adenocarcinoma patients' age, BMI, underlying disease, and vascular invasion but were related with the tumor FIGO clinical stage, tumor cell differentiation, and muscular layer infiltration.
EMT is an important process in the occurrence and development progression of cancer cells. Twist plays a key function in E-cadherin adjustment. Twist, as a basic helix-loop-helix transcription factor, can recognize and combine with the E-box sequence on the promoter of the target gene, consequently affecting the E-box promoter expression at the transcriptional level, inhibiting the expression of E-cadherin, and inducing the formation of EMT. Many growth factors and receptors and transcription factors can be induced in different cells to start EMT. These factors, either alone or in synergy, constitute the EMT signal conditioning network that acts on the migration, proliferation, invasion, and metastasis of tumor cells through the regulation and control of the Twist expression. However, the specific adjustment mechanism of the signal network is still confronted with many unsolved problems.
A positive E-cadherin expression inhibits tumor metastasis, which affects the invasion metastasis of tumor through many links. This study showed that the E-cadherin protein expression in endometrioid adenocarcinoma was significantly lower than that in NETs. The E-cadherin expression was low in endometrioid adenocarcinoma and associated with tumor FIGO staging, cell differentiation degree, and depth of muscular layer invasion. This result suggests that the downregulation of the E-cadherin expression may be an important indicator of clinical prognosis. Furthermore, a significant negative correlation existed between the Twist protein and E-cadherin expressions in endometrioid adenocarcinoma. Such correlation indicated that the Twist gene, as a transcription factor, may cause the loss of cell adhesion ability, thereby starting EMT and leading to the invasion and metastasis of endometrial cancer by inhibiting the E-cadherin transcription.
Both N-cadherin and E-cadherin are classical calcium adhesion proteins. In mature tissues, N-cadherin mainly mediates the adhesion among the brain, muscle, and vascular tissue cells. This protein is rarely expressed in epithelial tissues.
In the present study, N-cadherin was highly expressed in endometrioid adenocarcinoma and related with tumor FIGO staging, cell differentiation, and depth of muscular layer infiltration. This suggests that N-cadherin expression may become an important indicator for the clinical evaluation of prognosis. E-cadherin is commonly unexpressed in the tissue cells with positive N-cadherin expression. Therefore, a transition may exist between them. Twist was highly expressed in endometrioid adenocarcinoma. The tissues with high Twist expressions showed decreasing and no E-cadherin expression. Inhibition exists between the two mechanisms of expression, which is consistent with the relevant literature. In addition, we also found that Twist and N-cadherin upregulations are significantly positively correlated. This result indicates that Twist, as a transcription factor, can regulate and control E-cadherin and N-cadherin expressions to a certain extent. Twist may also play an important role in the occurrence, development, invasion, and metastasis of endometrioid adenocarcinoma. The relationship and interaction mechanism between twist, E-cadherin, and N-cadherin and their relationship with other genes need further verification. Further research will help reveal the molecular mechanism of the invasion and metastasis of endometrioid adenocarcinoma.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]