Association of FoxP3/Scurfin Germline Polymorphism (C-2383T/rs3761549) with Colorectal Cancer
Annals of Colorectal Research: June 01, 2013, 1 (1); 12-6
June 30, 2013
Article Type: Research Article
March 2, 2013
April 4, 2013
M R, Hosseini
S V, Ghaderi
A. Association of FoxP3/Scurfin Germline Polymorphism (C-2383T/rs3761549) with Colorectal Cancer,
Ann Colorectal Res.
FoxP3 gene encodes a transcription factor with crucial roles in the development and function of regulatory T cells.
The present study was conducted to investigate whether the C-2383T (rs3761549) polymorphism in the promoter region of the FoxP3 gene is associated with colorectal cancer.
Patients and Methods:
The study groups consist of 108 patients (52 men and 56 women) with colorectal cancer and 187 (126 men and 61 women) healthy individuals. They were genotyped for the FoxP3 polymorphism at position -2383 C > T using polymerase chain reaction-restriction fragment length polymorphism method. Frequencies of the gene variants were analyzed separately in men and women since FoxP3 is an X-linked gene.
The numbers of female patients with C/C, C/T and T/T genotypes were 48 (85.7%), 7 (12.5%) and 1 (1.8%), while in female controls, they were 53 (86.9%), 8 (13.1%) and 0 (0%), respectively. In male patients and controls, frequencies of the C genotype were 49 (94.2%) and 118 (93.7%), and the T genotype were 3 (5.8%) and 8 (6.3%), respectively. Genotype frequencies were not significantly different between controls and colorectal cancer patients. Distance metastasis was significantly associated with the FoxP3 polymorphism as calculated using Pearson’s chi-squared test (P = 0.006 in men and P = 0.03 in women). While 50% of metastatic patients had T or C/T (or T/T) genotype, the percentage of this genotype in non-metastatic patients was 4% in men and 11.5% in women. However, P values did not remain significant if differences were calculated using two-sided Fisher׳s exact test (P = 0.11 in men, and P = 0.09 in women); this might be due to the low number of our metastatic patients. Other characteristics of patients including age, tumor grade and stage were not significantly associated with the polymorphism.
Our results supported an association between C-2383T FoxP3 polymorphism and metastatic colorectal cancer in southern Iran.
Copyright © 2013, Colorectal Research Center and Health Policy Research Center of Shiraz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
Colorectal carcinoma is one of the leading causes of cancer worldwide (
1). In Iran, this disease is the sixth most frequent cancer in men and the fifth in women ( 2). Colorectal carcinoma develops in a special microenvironment enriched in the enterobacterial antigens, and numerous myeloid and lymphoid cells, including infiltrating T cells into intestinal mucosa ( 3, 4). Enteric bacteria are known to induce pro-inflammatory cytokines, which provoke pro-angiogenic and tumor-enhancing factors such as NF-κB. The excessive inflammatory reaction and maintenance of intestinal homeostasis are essentially controlled by a subset of T-cells, called T regulatory cells (Tregs). Mice deficient in T cells were shown to be highly susceptible to inflammatory bowel diseases and to the development of carcinomas, which were prevented by adoptive transfer of Tregs, particularly those being positive for FoxP3 marker ( 3). FoxP3, also called scurfin, belongs to the forkhead/winged-helix family of transcriptional factors. It acts as a main regulator in the development and function of Tregs whose main functions are attenuation of inflammation and suppression of effector T cells. FoxP3 is known as a reliable marker for Tregs, previously identified by non-specific markers such as CD25 ( 5, 6). Alterations in the numbers of FoxP3+ Tregs are found in a variety of diseases. For example, patients with an autoimmune disease such as systemic lupus erythematosus have a significant decrease in the suppressive function of Tregs and reduced levels of FoxP3 molecule ( 7). In contrast, these cells accumulate in tumors and the peripheral blood of cancer patients and promote tumor growth through inhibition of anti-tumor immune responses. However, evidence suggests that in certain cancers, such as colorectal carcinoma, Tregs are advantageous to the host where they suppress bacteria provoked chronic inflammation related to carcinogenesis ( 3). In fact, in colorectal carcinoma, the relationship between the FoxP3+ Tregs abundance and favorable prognosis has been demonstrated. Tregs appear to suppress destructive inflammation in early stages of carcinogenesis, while they might attenuate the immune response against colorectal carcinoma at advanced stages ( 3). Immune related genes have been associated with colorectal cancer risk and progression ( 8). The human FoxP3 gene contains 11 coding exons, and maps to the p arm of the X chromosome. There are several studies in the case of FoxP3 polymorphisms and autoimmunity and allergy ( 9-13). However, a few publications have investigated FoxP3 polymorphisms in cancer ( 14, 15), and there is no published literature on Foxp3 polymorphism and colorectal cancer. In the present study, we aimed to study single nucleotide polymorphisms (SNP) in the promoter region of FoxP3 gene (C-2383T/rs3761549) in colorectal cancer patients. This SNP has been associated with endometriosis, a benign inflammatory lesion with malignant potential ( 11, 12, 16, 17). Moreover, the associations between this SNP and clinicopathological parameters of the patients were analyzed.
The present study was conducted to investigate whether the C-2383T (rs3761549) polymorphism in the promoter region of the
FoxP3 gene is associated with colorectal cancer.
3. Patients and Methods
The study was approved by the Ethics Committee of Shiraz University of Medical Sciences. All the participants were informed that blood samples would be used for genotyping, and their consent was obtained. One-hundred and eight patients with colorectal cancer were enrolled in this study. Histologically, adenocarcima was confirmed in all patients. Data on clinicopathological characteristics of patients were obtained from the patients' medical files (
Table 1). Fifty-two patients were men with a mean age of 62.5 ± 10.5 and age range of 39-82 years, and 56 were women with a mean age of 54.9 ± 12.7 and age range of 23-83 years. The control group consisted of 187 healthy individuals with no history of autoimmunity and malignancy. They comprised 126 men with a mean age of 62.0 ± 10.2 and age range of 39-82 years, and 61 women with a mean age of 54.9 ± 12.0 and age range of 23-83 years.
Table 1. Characteristics of 108 Colorectal Cancer Patients at Diagnosis
Characteristics Men (No. = 52) Women (No. = 56) Age at diagnosis, y, Mean ± SD 62.58 ± 10.5 54.9 ± 12.7 Median age, y 60.5 54.0 Tumor location, No. (%) Colon 44 (84.6) 51 (91.1) Rectum 8 (15.4) 5 (8.9) Tumor stage , No. (%) a I/II 19 (63.3) 15 (53.6) III/IV 11 (36.7) 13 (46.4) Histological grade, No. (%) Well differentiated 39 (75.0) 27 (48.2) Moderately or poorly differentiated 13 (25.0) 29 (51.8) Distant metastasis, No. (%) Yes 2 (3.8) 4 (7.1) No 50 (96.2) 52 (92.9)
aData on tumor stage were not available for all patients. 3.1. DNA analysis
Genomic DNA was extracted using conventional Proteinase K protocol. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for determination of
C/T FoxP3 polymorphism at position -2383 using the following primer pair: forward, 5'-CTGAGACTTTGGGACCGTAG-3' and reverse, 5'-TGCGCCGGGCTTCATCGACA-3'. PCR was performed in a volume of 15 μL, containing 0.6 μL (20 ng) of genomic DNA, 0.6 μL of each primer, 1.2 μL of Taq DNA polymerase, 0.45 μL dNTP, 1.5 μL buffer, 0.50 μL MgCl 2 and 9.6 μL of ddH 2O. Our PCR condition was initial denaturation at 94 ºC for 5 min, followed by 30 cycles of 94 ºC for 30 sec, 60 ºC for 30 sec, and 72 ºC for 30 sec, with the final extension of 72 ºC for 10 min. The PCR products then underwent RFLP reaction with restriction enzyme BseNI (Fermentas, Lithuania) and incubated at 65 ºC for 16 hours. Finally, the products were separated on 3% agarose gel and visualized by Ger-Red (Biotium-US) staining. The resulted bands were two fragments of 261 and 127-bp ( T/T in women and T in men), three fragments of 184, 127 and 77-bp ( C/C in women and C in men) or four fragments of 261,184, 127, and 77-bp ( C/T in women). 3.2. Statistical Analysis
Statistical analysis was performed using SPSS 11.5 software packages. The differences in the distribution of genotypes in different groups were calculated using Pearson’s chi-squared test except where stated. The difference between the ages of the patients according to the
FoxP3 polymorphism was calculated using one-way analysis of variance (ANOVA) in women and t-test in men.
FoxP3C-2383T SNP was studied in 108 colorectal cancer patients and 187 healthy controls. The frequencies of the gene variants were analyzed separately in men and women because FoxP3 is an X-linked gene. The frequency of the genotypes in the patient group did not differ from that in healthy controls in both male and female subgroups (P > 0.05) ( Table 2). Moreover, the mean age of colorectal cancer onset did not differ among patients by the FoxP3 polymorphism in men and women, as well. It was 62.2 ± 10.3 and 68.6 ± 14.5 years in carriers of the C and T genotypes in men, respectively. In women carrying the C/C and C/T (or T/T) genotype, it was 54.9 ± 13.5 and 55.1 ± 11.5 years, respectively.
Table 2. GenotypeFrequencies of FoxP3 Polymorphism in Colorectal Cancer Patients and Healthy Individuals
Genotypes a Case ( n= 108) Control ( n=187) Men, No. (%) C 49 (94.2) 118 (93.7) T 3 (5.8) 8 (6.3) Women, No. (%) CC 48 (85.7) 53 (86.9) CT 7 (12.5) 8 (13.1) TT 1 (1.8) 0 (0)
aComparing the patient groups with control subjects, P values (calculated using the chi-square test) were not significant.
Table 3 indicates the distributions of the genotypes in the studied groups by tumor grade, tumor stage and the presence of distant metastasis at diagnosis. The genotype frequencies were not significantly associated with tumor stage and grade, though a higher percentage of patients with T/T or C/T genotype was diagnosed as moderately/poorly adenocarcinoma than patients with the C/C genotype in female subgroup (5/8; 62.5% vs. 24/48; 50%). The only individual carrying the T/T genotype was a female patient whose disease, as poorly differentiated adenocarcinoma in the rectum, was diagnosed at stage IV with distant metastasis. Distance metastasis was significantly associated with the disease as calculated through Pearson’s chi-squared test with P = 0.006 in men, and P = 0.03 in women. While 50% of metastatic patients (1 out of 2 men and 2 out of 4 women) had T or C/T (or T/T) genotype, the percentage of this genotype in non-metastatic patients was 4% in men (2/50) and 11.5% in women (6/52). However, the P values did not remain significant when the differences were calculated using the two-sided Fisher׳s exact test (P = 0.11 in men, and P = 0.09 in women).
Table 3. Genotype Frequencies of FoXP3 Polymorphisms in Colorectal Cancer
Characteristics C in Men orCC in Women, No. T in Men or CT/TT in Women, No. P Value a Men Histological grade Well differentiated 37 2 0.73 Moderately or poorly differentiated 12 1 0.73 Tumor stage b I/II 19 0 Not determined III/IIV 11 0 Not determined Distant metastasis No 48 2 0.006 Yes 1 1 0.006 Women Histological grade Well differentiated 24 3 0.51 Moderately or poorly differentiated 24 5 0.51 Tumor stage I/II 13 2 0.87 III/IIV 11 2 0.87 Distant metastasis No 46 6 0.03 Yes 2 2 0.03
aP values calculated using the chi-squared test.
bThe sum is less than the total number of patients because some data were not available.
A master molecule in the development and function of Tregs is the transcriptional factor FoxP3. Tregs control both adaptive and innate immune responses through several pathways, notably the suppression of CD4
+ T-cells through competition for interleukin-2, inactivation of CD8 + T-cells via cell contact and transforming growth factor-β, and attenuation of inflammation through secretion of interleukin-10 ( 5, 6, 18). Colorectal cancer develops in the presence of huge numbers of organisms and foreign antigens that can provoke a destructive inflammatory reaction if they were not controlled by local regulatory mechanisms, particularly Tregs ( 3). The chronic inflammation can promote different aspects of tumorigenesis, and has contributed to colorectal cancer pathogenesis ( 3).
Several polymorphisms exist within the
FoxP3 gene, of which some have contributed to a number of diseases including allergy and autoimmune disease ( 4, 9-15). For example, Fodor et al. analyzed genotype distribution of the rs3761548 FoxP3 polymorphism in allergic rhinitis among the Hungarians. They found a protective effect of the rare FoxP3 rs3761548 genotype ( A/A) against allergic rhinitis in homozygous women, and susceptibility to the disease in women being either wild-type ( C/C) or heterozygous ( C/A) for the rare allele ( 10). Our group has recently shown the association of an immune response-related gene, named programmed death-1 (PD-1), with colon cancer ( 8). We, therefore, hypothesized that another immune-related gene FoxP3 is associated with colorectal cancer. We studied C-2383T (rs3761549) polymorphism in the promoter region of the FoxP3 gene, because this SNP ( C/T genotype) was associated with endometriosis ( 12, 13), a benign inflammatory lesion whose malignant transformation has been reported ( 16, 17). A few publications are available regarding cancer and FoxP3 polymorphisms. They essentially failed to demonstrate a significant association between the studied cancer and FoxP3 polymorphism ( 14, 15). We found that the genotype frequencies were not significantly different between controls and colorectal cancer patients. Moreover, the SNP was not significantly associated with clinicopathological characteristics of the patients including age, tumor grade and stage in both men and women. However, a significant association of metastatic colorectal cancer was observed with T genotype in men and C/T ( T/T) genotype in women. It has been shown that, in addition to lymphocytes, FoxP3 is aberrantly expressed in several tumor cells, including colorectal cancer cells. Although, the consequence of FoxP3 expression by tumor cells remains controversial, studies conducted on human samples have generally reported its association with metastasis ( 19). Whether -2383 C to T polymorphism of FoxP3 gene through direct effects on FoxP3 molecule and disturbance of a balanced immune response in T lymphocytes, and/or its aberrant expression by tumor cells, or indirectly through linkage disequilibrium with other genes contributes to colorectal metastasis needs more investigation. The limitation of our study was the low number of our metastatic patients. The difference between our metastatic and non-metastatic groups regarding the distribution of FoxP3 polymorphism did not remain significant when the differences were calculated using the two-sided Fisher's exact test, a likely more appropriate test when the data were unequally distributed among the cells of the table ( 20). In summary, for the first time, we investigated the association of FoxP3 polymorphism with colorectal carcinoma. Our results support a significant association between metastasis and FoxP3 polymorphism; this should be confirmed in a larger number of patients. Further research is also required to disclose the functional consequence of C/T (T/T) genotype of C-2383T polymorphism on FoxP3 molecule.