Clin Transl Oncol DOI 10.1007/s12094-013-1037-x
RESEARCH ARTICLE
Clinical significance of FOXP3 expression in human gliomas L. Wang • B. Zhang • X. Xu • S. Zhang X. Yan • F. Kong • X. Feng • J. Wang
•
Received: 17 September 2012 / Accepted: 27 March 2013 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2013
Abstract Objective Studies have demonstrated that the transcription factor forkhead box P3 (FOXP3) is expressed not only in regulatory T cells, but also in some cancer cells. This study aims to clarify whether or not FOXP3 expression occurs in human gliomas and investigate the clinical significance of this expression in gliomas. Methods We detected FOXP3 protein expression in 40 glioma samples, 3 normal brain tissue samples, and 4 normal tonsil tissue samples using immunohistochemical L. Wang and B. Zhang contributed equally to this work and should be considered as co-first authors. L. Wang S. Zhang Graduate School, Tianjin Medical University, No. 22 Qixiangtai Road, Tianjin 300070, China L. Wang S. Zhang J. Wang (&) Tianjin Neurosurgical Institute, Tianjin Huanhu Hospital, 122# Qixiangtai Road, Tianjin 300060, China e-mail:
[email protected] B. Zhang Clinical Laboratory, Tianjin Huanhu Hospital, 122# Qixiangtai Road, Tianjin 300060, China e-mail:
[email protected] X. Xu X. Feng Key Laboratory for Critical Care Medicine of the Ministry of Health, Tianjin First Center Hospital, No. 24 Fukang Road, Tianjin 300192, China X. Yan F. Kong Department of Pathology, Tianjin Huanhu Hospital, 122# Qixiangtai Road, Tianjin 300060, China J. Wang Department of Neurosurgery, Tianjin Huanhu Hospital, 122# Qixiangtai Road, Tianjin 300060, China
staining and western blot. The expression of FOXP3 protein was also detected in five glioma cell lines by western blot. We also evaluated the association of FOXP3 expression with clinical pathological grades, prognosis, and recurrence. Results Western blot analysis showed that the expression of FOXP3 protein was upregulated in high-grade glioma (HGGS) samples compared with low-grade samples. The cell line U87 showed the highest FOXP3 expression, while U373 had the lowest expression. Immunohistochemical analysis detected FOXP3 protein in 35 out of the 40 (87.5 %) glioma samples and high levels of FOXP3 were observed in 26 out of the 27 (96.3 %) high-grade gliomas samples. Statistical analysis suggested that the upregulation of FOXP3 is significantly correlated with the histologic grade of gliomas (P \ 0.05) and that patients with high expression of FOXP3 protein exhibit a poorer prognosis than those with low FOXP3 expression. Conclusions Our findings suggest that FOXP3 expression in glioma cells has a crucial function in the development of HGGS and is associated with the malignant biological behavior of HGGS. Keywords cells
Glioma FOXP3 Prognosis Regulatory T
Introduction Forkhead box P3 (FOXP3), a member of the forkhead/ winged helix family of transcription factors, was initially identified as a specific molecular marker and a crucial regulator of the development and function of CD4?CD25? immunosuppressive regulatory T cells (Tregs) [1]. The induction of FOXP3 expression converts
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normal T cells into suppressor T cells [2]. However, recent studies have indicated that FOXP3 is expressed not only in Treg cells, but also in tumor cells, such as melanoma cells, pancreatic carcinoma cells, and breast carcinoma cells [3– 5]. Furthermore, FOXP3 shows promote and repressive functions in different types of tumor cells [3, 4]. The correlation between FOXP3 expression in tumor cells and the pathological characteristics of certain tumors has been elucidated [6]. However, whether or not FOXP3 is expressed in glioma cells remains unclear. As one of the most aggressive tumors, the prognosis of malignant gliomas remains very poor. Although some improvements in survival have been achieved over the last 10 years, patients with malignant glioma ultimately die of tumor progression. The cumulative 1-year survival rate of glioma is only 30 % and the median overall survival time is 53 weeks [7]. Nearly 70 % of all patients with glioblastoma survive \1 year and approximately 90 % die within 18 months [8]. The low survival rate of glioma is attributed to its high degree of invasiveness, which is associated with the suboptimal therapeutic efficacies of surgery, radiotherapy, or chemotherapy [8–10]. Thus, understanding the molecular mechanism underlying the progression of glioma and development of new markers with significant effectiveness for early diagnosis, as well as potential targets for astrocytoma-directed therapies are of great clinical value. Recent studies suggest that malignant glioma patients are profoundly immunosuppressed. A fundamental understanding of which types of glioma have immune resistance mediated by Tregs is required to develop and initiate specific immunotherapeutic approaches to target these cells [11]. In this study, we investigated the expression of FOXP3 by glioma cells and explored the correlations between the World Health Organization (WHO) grades of glioma and FOXP3 expression levels to clarify whether or not FOXP3 expression in glioma cells is associated with the development and malignant biological behavior of gliomas.
Materials and methods Patients and samples Forty glioma tissue samples were collected from 17 females and 23 males with ages ranging from 12 years to 72 years in Tianjin Huanhu Hospital from February 2010 to August 2010. The samples included 13 glioma cases of grades I–II, 10 cases of grade III, and 17 cases of grade IV. All cases were newly diagnosed gliomas as defined by the WHO criteria. Clinical annotation consisted of patient age, survival time, and confirmation of no prior therapy. Three samples of normal brain tissues from non-functioning
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cerebral cortex around the epileptic foci of patients with epilepsy were collected from Tianjin Huanhu Hospital and four samples of tonsil tissues collected from Tianjin Children’s Hospital. Normal tonsil tissues with no obvious invasion and edema from postoperative pediatric patients with tonsillitis, who underwent tonsillectomy were also obtained. This study was approved by the ethics committee of Tianjin Huanhu Hospital and informed consent was obtained according to the Declaration of Helsinki. Cell culture Glioma cell lines LN229, U87, U251, U373, A172, and human renal epithelial cell line 293 were cultured in Dulbecco’s modified essential medium supplemented with 10 % fetal bovine serum, 100 units/mL penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B in a humidified atmosphere containing 5 % CO2 at 37 °C. All reagents were obtained from Invitrogen (Cergy Pontoise, France). Immunohistochemistry Tissue sections (4 lm) were deparaffinized with xylenes and rehydrated with decreasing concentrations of alcohol solutions. Endogenous peroxide activity was quenched with 3 % hydrogen peroxide for 15 min. Heat-induced antigen retrieval was performed using EDTA antigenic retrieval buffer in a microwave oven. The sections were incubated with 1 % bovine serum albumin to block nonspecific binding and then incubated again overnight at 4 °C with mouse anti-human FOXP3 antibody (1:100; clone 236A/E7, eBioscience). For negative controls, the primary antibody was replaced with normal mouse serum. After washing, the tissue sections were treated with biotinylated goat anti-mouse secondary antibody (EarthOx) and further incubated with streptavidin–horseradish peroxidase complex (Zymed). Tissue sections were immersed in 3-amino9-ethyl carbazole, counterstained with 10 % Mayer’s hematoxylin, dehydrated, and then mounted in Crystal Mount. To confirm that cells with FOXP3 expression were indeed gliomas, we performed double staining with antiglial fibrillary acidic protein (anti-GFAP) and anti-FOXP3. The degree of immunostaining of formalin-fixed, paraffin-embedded sections was viewed and scored separately by two independent investigators who were blinded to the histopathologic features and patient data of the samples. Scores were determined by combining the proportion of positively stained tumor cells with the intensity of staining. Scores given by the two independent investigators were averaged for further comparative evaluation of FOXP3 expression. The proportion of positively stained tumor cells was graded as follows: 0 = no positive tumor cells, 1 B 10 % positive tumor cells, 2 = 10–50 % positive
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tumor cells, and 3 C 50 % positive tumor cells. Cells at each intensity of staining were graded as 0 = no staining, 1 = weak staining, light yellow, 2 = moderate staining, yellowish brown, and 3 = strong staining, brown. The staining index was calculated as follows: staining index = staining intensity 9 proportion of positively stained tumor cells. Through this assessment method, we evaluate the expression of FOXP3 in gliomas by staining index (scored as 0, 1, 2, 3, 4, 6, or 9). Immunohistochemical staining for protein expression in tumor and normal tissues was quantitatively analyzed using an AxioVision Rel.4.6 computerized image analysis system assisted by an automatic measurement program (Nikon). In brief, the stained sections were evaluated at 100 9 magnification and ten representative staining fields of each section were analyzed to verify the mean absorbance, which represents the strength of staining signals as measured per positive pixel. The mean absorbance data were statistically analyzed using t test to compare the average mean absorbance difference between different groups of tissues. P \ 0.05 was considered statistically significant. Western blot Western blot analysis was performed as previously described [12, 13]. Glioma tissues and cell extracts were obtained by direct dissolution using a protein extract reagent according to the manufacturer’s directions (Bioteke Corporation). Protein (40 lg/lane) that was quantified by a Pierce BCA protein assay kit (Cat. #23225, Thermo Scientific) was separated by 12 % SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5 % non-fat dry milk in TBST for 1 h and then incubated with primary antibodies overnight at 4 °C. After washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (1:5,000) for 1 h at room temperature and detected by an ECL kit (Thermo Co., Ltd.). Follow-up To analyze the association between FOXP3 expression and patients survival, we followed up the patients for 2 years. The survival was recorded for all samples as follows: the time in days elapsed from resection to the day of death or the time in days elapsed from resection to the current day. Recurrence refers to tumors enlarged by more than 10 % in volume post-operation.
characteristics. Bivariate correlations between the studied variables were calculated by Spearman’s rank correlation coefficients. Survival curves were plotted by the Kaplan– Meier method using GraphPad Prism 5.0 and compared by the logrank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. P \ 0.05 was considered statistically significant.
Results FOXP3 expression in different grades of glioma tissues The results showed the wide expression of FOXP3 in the nucleus of tonsil specimens (Fig. 1a). Almost, no positive FOXP3 expression was observed in the cells of normal brain tissue samples (Fig. 1b). Among the 40 glioma specimens, FOXP3 expression was nearly undetected in samples of grade I (Fig. 1c), but found in the interstitial lymphocytes of samples of grade II, featuring unctate distribution, location in the nucleus, and common appearance in the stromal perivascular area (Fig. 1d). The number of positively expressing cells in high-grade gliomas (HGGS) III–IV (Fig.1e, f) was far higher than that in lowgrade gliomas (LGGS) I–II (Fig.1c, d). FOXP3 expression can be observed in the parenchyma of minority high-level astrocytomas, which showed irregularly shaped colored nucleus and sizes 1–2 times larger than those of lymphocyte nuclei. The number of colored cells was about 5.01 % in high-grade astrocytomas. Moreover, after performing double staining with anti-GFAP and anti-FOXP3, we found cells simultaneously show FOXP3 expression in the nuclei and GFAP expression in the cytoplasm (Fig. 1g). Figure 1g indicate that not all glioma cells have positive FOXP3 expression only poorly differentiated glioma cells show positive FOXP3 expression. Smaller and round cells that only show FOXP3 expression without positive GFAP expression may be infiltrated lymphocytes. Western blot analysis was conducted to determine the levels of FOXP3 protein expression in glioma, tonsil, and normal brain tissues. Strong positive FOXP3 expression was observed in the four tonsil specimens (Fig. 1h) and negative FOXP3 expression was observed in two normal brain tissue samples. As shown in Fig. 1h, FOXP3 expression was upregulated in HGGS samples compared with LGGS. Furthermore, relative band density analysis showed that the HGGS/LGGS ratio of FOXP3 expression could be as high as 7.2-fold.
Statistical analysis FOXP3 expression in glioma cell lines All statistical analyses were carried out using the SPSS19.0 software package. The v2 test was used to analyze the relationship between FOXP3 expression and pathological
FOXP3 protein expression was detected in glioma cell lines, including U87, U251, A172, LN229, and U373,
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Clin Transl Oncol Fig. 1 Immunohistochemical and Western blot results of FOXP3 expression in glioma tissues and the representative staining pattern of FOXP3. a Tonsil specimens. b Normal brain tissue. c, d Low-grade gliomas. e, f High-grade gliomas. g Double staining with anti-FOXP3 and anti-GFAP in high-grade gliomas (1 indicating double positive expression of GFAP and FOXP3 and 2 indicating single positive expression of GFAP). h FOXP3 expression in tonsil, gliomas, and normal brain tissues. The upper panel shows representative FOXP3 expression in different grades of gliomas and normal brain and tonsil tissues, while lower panels indicate the relative band density values of FOXP3 protein compared with b-actin (mean ± SD, n = 3)
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using Western blot analysis (Fig. 2). All cell lines showed positive FOXP3 expression. Among them, U87, the most malignant line, showed the strongest expression whereas U373 showed the weakest expression. No FOXP3 expression was detected in the 293 cell line (Fig. 2).
results imply that the progression of astrocytoma may be associated with increased FOXP3 expression.
Correlation between the clinical information of patients and FOXP3 expression level
After 2 years follow-up, we estimated the association between FOXP3 expression and patient survival time. The results showed that the patients survived \15 months, 82.6 % (19/23) patients were high FOXP3 expression. Although the patients survived more than 15 months, only 41.2 % (7/17) patients were high FOXP3 expression. A statistically significant difference was observed between two groups, which survived less or more than 15 month (P \ 0.05; Table 1). Spearman’s analysis also showed a correlation between FOXP3 expression and patient survival time (r = 0.2419, P = 0.0002; Table 2). Kaplan–Meier analysis was performed to calculate the effect of FOXP3 expression on survival time. The median survival time of patients whose tumors showed high levels of FOXP3 expression was only 11 months, whereas those with low levels of FOXP3 expression was longer than 20 months (Fig. 3). However, Cox regression multivariate survival analysis suggested that FOXP3 expression level is not an independent prognostic factor for assessing patient outcomes (P = 0.7280; Table 3).
To determine whether or not the expression level of FOXP3 is associated with the clinical characteristics of gliomas, we analyzed the percentage of high FOXP3 expression based on the gender, tumor location, age, tumor diameter, WHO differential grade, and time before recurrence. FOXP3 was detected in 35 of the 40 glioma cases (87.5 %) and not detected in the five cases of LGGS (I–II). High levels of FOXP3 expression were detected in 26 of 27 high-grade gliomas cases (96.3 %), and only one case of grade III glioma showed a low level of FOXP3 expression. Positive FOXP3 expression was found to be strongly correlated with the pathologic grades of the patients (P = 0.0000) and term to recurrence (P = 0.0001), but showed no difference among ages (P = 0.6661) or genders (P = 0.1692) (Table 1). Spearman’s correlation analysis further revealed a correlation between FOXP3 expression level and term to recurrence (P \ 0.005) (Table 2). These
Association between FOXP3 expression and patient survival time
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Clin Transl Oncol Table 1 Correlations of FOXP3 expression with clinic pathologic characteristics of glioma Num of patients
Ratio of high FOXP3 expression, num (%)
v2
P value
Male
23
17 (73.9)
1.8898
0.1692
Female
17
9 (52.9)
B60
19
13 (68.4)
0.1862
0.6661
[60 Location
21
13 (61.9) 0.1465
0.7019
0.1648
0.6847
Characteristic
Gender
Age (years)
Parenchyma
30
20 (66.7)
Ventricular
10
6 (60.0)
B3
24
15 (62.5)
[3
16
11 (68.7)
Diameters (cm)
WHO grade I–II
13
0
IIIb
10
9 (90.0)
b
0.0000
b
17
17 (100)
b
0.0000, 0.1840
20 (90.9)
15.4684
0.0001
7.3758
0.0066
IV
Fig. 2 Expression of FOXP3 in different glioma cell lines detected by Western blot. The upper panel shows representative Western blot results, while the lower panel provides the relative band density values of FOXP3 protein compared with b-actin (mean ± SD, n = 3). All of the glioma cell lines positively expressed FOXP3. U87 showed the highest level of FOXP3 expression
Time to recurrencea B3 months 22 [3 months
18
6 (33.3)
B15 months
23
19 (82.6)
[15 months
17
7 (41.2)
19.2214 30.0000,c1.7654
Survival
a
Discussion
Recurrence refers to the tumor volume increased by 10 % compared with that postoperation
b c
FOXP3 expression was initially believed to be restricted to hematopoietic cells and tissues; however, recent reports have demonstrated that other tissues and cell lines, including some tumor cells, are able to express FOXP3 [14]. The roles of FOXP3 gene or protein in different tumor cells remain controversial. Several studies suggest that the FOXP3 plays a critical role in suppressing the development of several kinds of tumors, such as prostate and breast [4, 15]. Zuo [4, 16] showed that FOXP3 is expressed in normal breast epithelial cells and downregulated in breast cancer tissues. FOXP3 acts as a transcriptional repressor of oncogene Skp2 and HER-2/ErbB2, which play key roles in breast cancer development [4, 16]. Jung [17] recently reported that FOXP3 upregulation requires p53 functions in human breast and colon carcinoma cells. Treatment with the genotoxic agents doxorubicin or etoposide could induce FOXP3 expression in p53-positive carcinoma cells but not in cells without p53 functions [17]. Moreover, knockdown of endogenous wild-type p53 using RNA interference
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Comparative to grade I–II Comparative to grade III
Table 2 Spearman correlation analysis between FOXP3 and clinic pathologic factors Variables
FOXP3 spearman correlation
P
Gender
0.0723
0.2381
Age
0.0583
0.4219
WHO grade
0.2918
0.0000
Survive
0.2419
0.0002
Recurrence
0.2401
0.0003
inhibits FOXP3 induction by genotoxic agents and exogenous expression of p53 in cells without p53 restores FOXP3 induction by DNA-damaging stresses. FOXP3 knockdown also reduces the p53-mediated growth inhibitory response to DNA-damaging agents [17].
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and demonstrated FOXP3? Tregs most frequently in GBM, but very rarely in low-grade astrocytomas; however, the presence of FOXP3? Tregs within GBMs did not associate with the median survival time, however, the authors did not mention the FOXP3 expression in glioma cells. Consistent with the reports [3, 18], we found that FOXP3 was not expressed in normal brain tissues and low expressed in LGGS (I–II), mainly existing in the tumor stroma, especially around the blood vessels. Sporadic expression can be seen in the tumor cells in grade III glioma and higher expression may be observed in glioblastoma (IV), particularly in the nuclei of tumor cells. Our result was disparity with the pattern of FOXP3 expression observed in differentiated thyroid carcinoma, in which the high expression was observed in the cytoplasm, but not in nuclei [18]. Our results imply that the levels of FOXP3 expression are positively correlated with glioma grade. However, the mechanism of FOXP3 expression in HGGS requires further investigation. During our manuscript’s revision, it has been reported that FOXP3 could be as a pro-apoptotic factor in glioma, overexpression of FOXP3 in glioma cells result in the appearance of apoptotic nuclei, DNA fragments, activation of effector caspases, and increased PARP-cleavage [20]. The results in this report are discrepancy with our findings. Our results derived from glioma samples suggest that FOXP3 probably play pro-metastatic action in vivo. Thus, the function of FOXP3 in glioma needs to be further investigated. We also found that all glioma cell lines expressed FOXP3; the positive rate was higher in glioma cell lines detected by western blot than by IHC in glioma tissues. The possible reasons for this discrepancy were that western blot is more sensitive than histochemical staining and it was easier for FOXP3? glioma cells to proliferate in vitro; FOXP3 can be induced in glioma cell lines in vitro culture. We followed up our patients 2 years after operation and found that the recurrence of gliomas also is significantly
Fig. 3 Kaplan–Meier curves with univariate analyses of patients with low FOXP3-expressing (bold line) versus high FOXP3-expressing tumors. Patients with higher levels of FOXP3 expression had shorter survival times than those with lower ones
A previous study demonstrated that FOXP3 expression is extensively downregulated in prostate cancer tissue at early stages and that the growth of prostate cancer cell lines can been strongly inhibited by transfection with FOXP3 gene. The same researchers also demonstrated that FOXP3 is necessary and sufficient to repress the transcription of the c-myc gene, which is one of the most commonly overexpressed oncogenes in prostate cancer [15]. Thus, FOXP3 may act as a critical tumor suppressor gene for prostate cancer. Conversely, several studies have revealed that FOXP3 is significantly overexpressed in carcinomas derived from pancreatic and thyroid tissues compared with non-malignant tissues [3, 18]. FOXP3 expression is related to the aggressiveness and immune evasion in cancers [3, 18]. These results indicate the FOXP3 may act as a tumor promoter in some kinds of tumors. Thus, the FOXP3 gene or protein exerts different functions in different types of tumors. Heimberger [19] reported significant differences in the prevalence of FOXP3? Tregs between tumors of different grades, between different pathologic types of tumors Table 3 Univariate and multivariate analysis of different prognostic parameters in patients with astrocytomas by cox regression analysis
No. patients
Univariate analysis
Multivariate analysis
P
Regression coefficient (SE)
P
Relative risk
0.1491
0.4721 (0.2291)
0.2617
1.2972
0.0001
1.7211 (0.2718)
0.0000
4.4727
0.0000
1.2287 (0.2391)
0.7280
0.9281
Age (years) B60
19
[60 21 Astrocytoma histology (WHO grade) I–II
13
III
10
IV
17
FOXP3 Low
14
High
26
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associated with the level of FOXP3 expression even in different glioblastomas. We suppose that the FOXP3 itself or genes regulated by FOXP3 may be associated with glioma recurrence. The expression of FOXP3 was significantly correlated with patient survival. However, FOXP3 expression is not an independent risk factor for prognosis because not all glioma cells showed FOXP3 expression. In conclusion, our findings suggest that FOXP3 expression in glioma cells has crucial functions in the development of HGGS and may be associated with the malignant biological behavior of HGGS. The results also suggest the application prospects of FOXP3 as a marker for the recurrence of astrocytic tumors. FOXP3 may become a new molecular marker of gliomas and a new target for glioma immunotherapy. However, its clear biological function in glioma cells requires further investigation. Acknowledgments This work was supported by the grant from the Tianjin Science and Technology Committee (11JCYBJC12100, 12ZCDZSY17700), the National Natural Science Foundation of China (81101911), the Tianjin Health Bureau Science and Technique Foundation (11KG115), and State Key Clinical Department of Neurosurgery. Conflict of interest
None.
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