Cervical cancer is a common female genital tract malignancy with increasing incidence in recent years. Standard treatments of cervical cancer have been established. However, prognostic indicators for cervical cancer are still lacking. Enhancer of zeste homolog 2 (EZH2) is an important member of the polycomb group protein and directly or indirectly regulates expression of oncogenes and tumor suppressor genes.1 The EZH2 is highly expressed in malignant tumors, such as breast and prostate cancers, and is closely associated with the degree of malignancy of malignant tumors and prognosis in patients.2,3 The EZH2 mRNA expression in cervical carcinoma tissues is significantly higher than in normal cervical tissue, but it is not related to the diameter of primary cervical lesions.4
P53 is an acknowledged tumor suppressor gene, and its expressed protein (P53) is involved in cell cycle regulation and DNA repair. Studies5–7 have demonstrated that EZH2 can induce the inactivation of P53, indicating that EZH2 and P53 may play a certain role in the development of malignant tumors through their mutual adjustment and synergistic interaction.
Traditional immunohistochemical methods can detect the expression of EZH2 and P53 in a pathologic section and can be used for evaluating the relationship between the expression and such factors as the clinical stage, lymph node metastasis, and prognosis. However, the EZH2 and P53 expression has to be detected individually by immunohistochemical methods, which may lead to poor sensitivity and low accuracy of detection.
Few studies have focused on improvement in the detection of EZH2 and P53 expression. A new method for simultaneous detection of EZH2 and P53 expression is needed.
Quantum dots (QDs) are a new semiconductor crystal, a long-time multicomponent and multicolor dynamic real-time detection medium for various biomolecular markers in vivo or in living cells under physiologic conditions, which overcomes limitations of common labeling methods, such as poor stability, toxicity to cells, and limited observation.8 The QDs are primarily used in research on tumor-associated factors.
Therefore, the purposes of this study were to simultaneously detect the expression of EZH2 and P53 in cervical squamous cell carcinoma by QDs double immunofluorescent staining technique; to explore the relationship between EZH2 and P53 expression and clinical staging of cervical squamous cell carcinoma, lymph node metastasis, and prognosis; and to illustrate the clinical significance of the relationships.
MATERIALS AND METHODS
Information was collected about 168 patients with cervical squamous cell carcinoma undergoing surgical treatment from January 2005 to December 2006. The patients experienced abdominal or laparoscopic radical hysterectomy and pelvic lymphadenectomy performed by gynecologic oncology experts with more than 10 years of experience. All the patients had preoperative examination conducted by 2 experienced gynecologic oncology experts, and the clinical stage was determined according to the 2009 International Federation of Gynecology and Obstetrics staging system. All the excised tissues were sent for pathologic examination. After the surgery, 11 cases received only chemotherapy, 92 received only radiotherapy, and 37 received both therapies. The clinical information for all the participants is listed in Table 1.
The study was approved by the ethics committee of the First Affiliated Hospital of Sun Yat-sen University in Guangzhou, China, and consent was obtained from the patients and their families.
QDs Double Immunofluorescent Staining Method
The tissue sections were embedded with paraffin, dewaxed, hydrated, and immersed in 0.01-M citrate buffer (pH, 6.0) for antigen repair by microwave retrieval method. They then were incubated with primary antibody (rabbit antihuman monoclonal antibody for EZH2 and mouse antihuman monoclonal antibody for P53 [1C12]), stained using QDs Double Staining Kit (QDK001; Wuhan Jiayuan of Quantum Dot Technological Development Co, Ltd, Wuhan, China), and then followed up with incubation in QDs-IgG mixture.
After incubation, the sections were mounted and observed by ultraviolet excitation under a fluorescence microscope for locations where fluorescence occurred. Green color suggests EZH2 protein, whereas red indicates P53 protein. According to the qualitative fluorescent labeling method,9 3 to 4 fields of view were selected from each film under high magnification, and 100 cells per field were counted. Red color indicated P53 protein (Fig. 1A), green suggested EZH2 protein (Fig. 1B), and bright red/green meant positive cells. Positive expression is regarded if the positive cells account for greater than 30% of the total cells.
The expression of EZH2 and P53 simultaneously detected by QDs was classified into double positive (both were positively expressed in tissue sections) (Fig. 1C), single positive (one of them was positively expressed in tissue sections), and double negative (neither one was positively expressed in tissue sections).
The follow-up study was performed by specially assigned patients after 1, 3, and 6 months and 1 year postsurgery. After this, a visit was ensured to occur at least once a year. Follow-up study included gynecologic examination; gynecologic ultrasonography; gynecology tumor markers; magnetic resonance examination, if necessary; ultrasound of spleen and gallbladder; and urinary tract ultrasound.
Using SPSS 18.0 software, measurement data were analyzed with a t test and nonparametric tests, whereas count data were analyzed with χ2 and Fisher exact tests. Survival analysis with recurrence and death as the prognostic end points was conducted by the Kaplan-Meier method, and intergroup difference was evaluated by log-rank test. For all analysis, probability smaller than 0.05 (P < 0.05) was considered statistically significant.
The follow-up study lasted for 72.36 ± 9.83 months (60–84 months). It was conducted in 143 cases accounting for 89% of the total 168 patients, and the remaining 25 cases accounting for 11% of the patients were out of touch because of changes in telephone number or address and hence were classified as lost to follow-up. Among the 143 patients, 107 patients were tumor free, and 36 patients were found with tumor recurrence or metastasis (including 26 patients who became deceased).
Relationship Between EZH2/P53 Expression and Clinical Stages
Among the 168 patients, 84, 71, and 13 patients were in stages IIA, IB, and IA of cervical cancer, respectively. The 40 patients with stage IIA cervical cancer, 27 patients with stage IB cervical cancer, and 3 patients with stage IA cervical cancer showed double-positive EZH2/P53 expression, accounting for 47.62%, 32.39%, and 23.08% of the total patients with each stage of cancer, respectively. A total of 3 patients with stage IIA cancer, 4 patients with stage IB cancer, and 3 patients with stage IA cancer revealed double-negative expression, accounting for 3.57%, 5.63%, and 23.08% of the total patients with each stage of cancer, respectively.
A total of 41 patients with stage IIA cancer, 40 patients with stage IB cancer, and 7 patients with stage IA cancer were found with single-positive expression, accounting for 48.81%, 56.34%, and 53.85% of the total patients with each stage of cancer, respectively, which were statistically different (χ2 = 14.831, P = 0.005 < 0.01) (Table 2, Fig. 2A). The proportion of patients with double-positive expression was significantly (P < 0.05) higher, but the proportion with double-negative expression was significantly (P < 0.05) lower in the patients with stage IIA cancer than in those with stage IB and IA cancer and also lower in the patients with stage IB cancer than in those with stage IA cancer.
Relationship Between EZH2/P53 Expression and Lymph Node Metastasis
Pathologic examination revealed that 70 and 98 patients were with and without lymph node metastasis, respectively. The double-positive expression was found in 38 patients (54.29%) with lymph node metastasis and 32 patients (32.65%) without lymph node metastasis; the double-negative expression was detected in 1 patient (1.43%) and 9 patients (9.18%); and the single-positive expression was shown in 31 patients (44.29%) and 57 patients (58.16%), which were significantly different (χ2 = 6.076, P = 0.042 < 0.05) (Table 2, Fig. 2B).
The proportion of cases with double-positive expression was significantly (P = 0.003 < 0.05) higher, but the proportion with double-negative expression was significantly (P = 0.009 < 0.05) lower in patients with lymph node metastasis than in those without lymph node metastasis.
Survival Analysis of Patients With Different EZH2/P53 Expressions
The Kaplan-Meier survival analysis was conducted with the EZH2/P53 simultaneous expression as influencing factors and based on the results of QD detection.
Results showed that, in 143 patients at follow-up, the progression-free survival (PFS) of those with double-positive, single-positive, and double-negative expression was 70.60 ± 3.23, 66.51 ± 2.02, and 56.28 ± 3.14 months (mean [SD]), respectively, which were significantly different (χ2 = 9.161, P = 0.01 < 0.05). The overall survival (OS) of patients with double-positive, single-positive, and double-negative expression was also significantly different (χ2 = 9.767, P = 0.008 < 0.05), with 71.600 ± 2.277, 69.315 ± 1.742, and 60.828 ± 2.718 months, respectively. The PFS and OS were significantly higher (Ps = 0.001 and 0.003 < 0.001) in patients with double-negative expression than in those with double-positive expression but were not significantly different (Ps = 0.35 and 0.57 > 0.05) from those with single-positive expression, respectively (Fig. 3).
Polycomb group is a group of proteins regulating cell proliferation and differentiation. Among them, EZH2 protein can promote cell proliferation and inhibit cell differentiation and target gene expression and is closely associated with tumorigenesis. The EZH2 gene is a human gene discovered in 1996,10 located on chromosome 7q35. It is a homologous gene of Drosophila enhancer of zeste gene, and both have similar structure and function. The EZH2, the EZH2 gene product, is involved in the regulation of cell growth and can inhibit the target gene of chromatin and promote cell proliferation.
The EZH2 expression in tumors, such as urinary tract cancer, breast cancer, and lymphoma, has been reported.2,3,11 Its expression in advanced or early-stage tumors or metastatic tumors is higher than that in earlier-stage tumors and those without metastases, which may suggest the EZH2 gene is associated with tumor invasion and metastasis. Bachmann et al12 found that EZH2 can promote the occurrence, development, and invasion of endometrial cancer. A recent study demonstrated that EZH2 not only can inhibit the expression of the target gene in chromatin to promote cell proliferation and immortalization but also can act as a transcription inhibition factor to inhibit cell growth and differentiation and regulate gene activity at the gene level.8
Current studies showed that EZH2 cannot directly lead to tumorigenesis but can affect cell proliferation and differentiation by regulating expression of other genes or proteins, which may result in the occurrence of malignant cells. P53 is a recognized tumor suppressor gene. The expression levels of inactivated P53 in breast cancer tissue are positively correlated with EZH2.2 Tang et al7 found that the expression level of wild-type P53 is negatively correlated with that of EZH2 in human fibroblasts and confirmed that the wild-type P53 protein can bind with the EZH2 gene promoter to inhibit EZH2 expression, arrest human fibroblasts at the G2 phase, inhibit fibroblast proliferation, and induce apoptosis. In addition, Tang et al found that the reduction of normal P53 protein can demethylate the EZH2 promoter.
Wagener et al13 detected the expression of EZH2, P53, and P53 pathways-associated protein (including P63 and MDM2) in nasopharyngeal tissue sections and also revealed that EZH2 expression is positively correlated with all three of the proteins. In contrast, Bracken et al5 silenced EZH2 gene expression in osteosarcoma cells by RNA interference and found that EZH2 inhibition can suppress P53 gene expression. It is obvious that the interaction between these 2 genes is complicated, but the exact mechanism is unclear.
In recent years, biomarkers have been important for studies on various diseases, including cervical cancer. Fluorescent organic dye material is the most commonly used biomarker, but it cannot simultaneously detect multicomponents because its metabolic products may be toxic to cells and labeling time is short. In addition, it is easily quenched and cannot be used for long-term dynamic detection. The QDs are semiconductor nanocrystals smaller than 100 nm, and different QDs can emit different colors of light if stimulated by a single wavelength of excitation light. The QDs are characterized by high photochemical stability and long-lived fluorescence emission—which can facilitate detection—as well as little interference from background fluorescence in the detection signal and also low cytotoxicity.8
Since 1998, when QDs were first applied to the biomedical field, studies have been focused primarily on tumor-related factors: labeling biological molecules such as DNA and proteins inside and outside the cells14; multichannel and long-term visualizing of target proteins, molecular distribution, and signal transduction molecules15; and facilitating differentiation and identification of fluorescence spectroscopy of labeled biomolecule to dynamically trace cytokines.
The QDs’ immunohistochemical staining capability has been confirmed to be an alternative to traditional immunohistochemical staining in some cases.9,16,17
The present study demonstrated that the expression levels of EZH2 and P53 in tumor tissue and the proportion of cases with double-positive expression significantly increased with increasing clinical stages of cancer, also confirming that the expression of EZH2 and P53 was up-regulated with increasing stages of cervical cancer. These findings may suggest that the 2 proteins were involved in the development of cervical cancer. However, the nature of their interaction with each other remains undefined. The increase of EZH2 and P53 expression in patients with lymph node metastasis indicated that they may be involved in the metastasis of cervical cancer cells. Other studies have shown that they may participate in the metastasis of cervical cancer cells by affecting factors enhancing the ability of tumor cell migration, such as metastasis-related genes (MTA).18
High EZH2 expression could lead to histone H3-K27 methylation and up-regulated expression of MTA genes, thereby lowering the adhesion and enhancing the migration ability of cancer cells to penetrate the basement membrane, promote the occurrence and development of malignant tumors, and eventually develop into invasive cancer with distant metastasis.
The EZH2 and P53 expression here was found to be related with the clinical stage of cervical cancer and lymph node metastasis. The simultaneous detection of expressions of both proteins could partly predict the prognosis of cervical cancer, which was consistent with the results of previous studies indicating that EZH2/P53 expression can enhance tumor metastasis.5–7
We are aware of the limitations of this study, resulting from limited numbers of samples. Hence, the results need to be verified by more studies with more cases. The Kaplan-Meier analysis showed that the PFS and OS of patients with positive EZH2/P53 expression were significantly lower than those of patients with negative expression. These results agreed with those obtained from previous studies on breast cancer, prostate cancer, and endometrial cancer. Therefore, an in-depth study of EZH2 and P53 expression in cervical cancer and the related mechanisms is important for clarifying molecular mechanism of tumor development and for evaluating the progress of cervical cancer. Whether expression of EZH2 and P53 can be used as prognostic biomarkers deserves further study.
The QDs double fluorescent staining could evaluate EZH2 and P53 expression in tissue biopsy very well. The EZH2 and P53 may be involved in the occurrence and development of tumors through their synergistic interaction. The EZH2 and P53 expression was related with the clinical stage of cervical cancer, lymph node metastasis, and survival prognosis.
We thank every patient involved in this study.
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Keywords:© 2014 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.
EZH2; P53; Cervical cancer; Double quantum dot fluorescence staining; Prognosis