|COMMENTARY ON THE IZMIR STUDY
|Year : 2020 | Volume
| Issue : 2 | Page : 166-171
High potential of SOX21 gene promoter methylation as an epigenetic biomarker for early detection of colorectal cancer
Keivan Moradi1, Esmaeil Babaei1, Nayebali Rezvani2, Reza Safaralizadeh1, Homayoun Bashiri3, Mohammad Ali Hosseinpour Feizi1
1 Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
2 Department of Clinical Biochemistry, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Department of Gastroenterology, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Submission||14-Aug-2018|
|Date of Decision||13-Sep-2018|
|Date of Acceptance||22-Sep-2018|
|Date of Web Publication||17-May-2020|
Mohammad Ali Hosseinpour Feizi
Department of Animal Biology, Faculty of Natural Science, University of Tabriz, Tabriz
Source of Support: None, Conflict of Interest: None
Background: Despite the advances in screening during the past decades, colorectal cancer (CRC) still is a leading cause of cancer deaths worldwide. Therefore, the development of new diagnostic methods is necessary.
Aim: The aim of this study was to compare methylation changes of SRY-Box 21 (SOX21) gene promoter in tumor tissues and their normal adjacent mucosa in patients with CRC and to examine the relationship between the methylation levels and demographic/clinicopathological factors.
Materials and methods: A total of 41 CRC patients participated in the present study. After the extraction of DNA and bisulfite treatment of the samples, the methylation levels were determined by using the MethyLight method.
Statistical analysis: Two-sided Mann–Whitney U test was used to compare the median level of methylation in tumor tissues and their adjacent normal mucosa.
Results: The methylation rates in tumor tissue samples were significantly higher compared to their adjacent normal mucosa (P < 0.0001). No association between demographic/clinicopathological factors and methylation status observed in tumor tissues. A receiver operating characteristics curve was constructed and tissue samples exhibited a sensitivity of 80.5% and specificity of 97.6% for SOX21 promoter methylation.
Conclusion: The results of this study indicated the high potential of SOX21 gene promoter methylation as a candidate noninvasive diagnostic biomarker in stool and plasma of colorectal cancer patients. However, further studies with larger sample sizes are required to evaluate the specific role of SOX21 methylation as a biomarker for early detection of CRC.
Keywords: Biomarker, colorectal cancer, early detection, methylation, promoter
|How to cite this article:|
Moradi K, Babaei E, Rezvani N, Safaralizadeh R, Bashiri H, Feizi MA. High potential of SOX21 gene promoter methylation as an epigenetic biomarker for early detection of colorectal cancer. Indian J Cancer 2020;57:166-71
|How to cite this URL:|
Moradi K, Babaei E, Rezvani N, Safaralizadeh R, Bashiri H, Feizi MA. High potential of SOX21 gene promoter methylation as an epigenetic biomarker for early detection of colorectal cancer. Indian J Cancer [serial online] 2020 [cited 2021 Jan 20];57:166-71. Available from: https://www.indianjcancer.com/text.asp?2020/57/2/166/284477
| » Introduction|| |
More than one million new cases are annually diagnosed with colorectal cancer (CRC) worldwide. This type of cancer is the third most common cancer in males and the second in females. According to National Institutes of Health (NIH), the number of new cases and deaths of CRC during the years of 2011–2015 in the United States were 39.4 and 14.5 per 100,000 per year, respectively.
It has been shown that detection of CRC disease at an early stage can increase the 5-year survival rate over 90%. However, this survival rate decreases dramatically during the late stages of the disease. Besides, some other features like high mortality, a large number of incident cases, and long duration of disease manifestation make CRC an excellent disease to apply screening methods. Despite the advances in screening during the past decades, CRC still is a leading cause of cancer deaths worldwide. Although colonoscopy is considered as the gold standard for CRC detection, some limitations like invasiveness, the required bowel preparation, and the risk of bleeding reduces the general acceptance of this screening method.
The occurrence of genetic mutations and epigenetic alterations in a multistep process leads to colorectal cancer. DNA methylation at CpG islands in the promoter part of genes is an epigenetic mechanism in which a methyl (CH3–) group is added to the cytosine bases to form 5-methylcytosine (5-MC). Once a gene is methylated at CpG islands, it usually stops performing the normal function and becomes silent.
Since hypermethylation of CpG islands is common in the neoplastic tissue of patients with CRC and is not commonly found in their adjacent normal tissue, DNA hypermethylation is a promising class of CRC screening biomarkers. After the approval of gene promoter hypermethylation in neoplastic tissues with high sensitivity and specificity, it can be considered as a potential optimal biomarker for early detection of CRC in the stool or serum of patients.
SOX21 (SRY-Box 21) gene is located on chromosome 13q31-q32 and encodes a transcription factor.,SOX21 gene promoter methylation was first reported by Mitchell et al. in 2014 as a candidate biomarker for stool-based assays in CRC patients. By study on a panel of genes, Mitchell et al. already reported that DNA methylation controls the expression of SOX21 gene and introduced it as a suitable biomarker for CRC diagnosis. Although these authors showed that the level and frequency of SOX21 gene promoter methylation in non-neoplastic colorectal tissues is very low in comparison to CRC tumor tissues, the number of normal samples studied in their study was very limited (only six samples).
Our goal was to investigate the status of methylation of this gene in the stool and plasma of CRC patients and to evaluate its capability as a clinical test; here, we compared methylation changes of SOX21 gene promoter in tumor tissues and their normal adjacent mucosa using quantitative MethyLight assays from 41 CRC patients who underwent surgical resection. Our study is unique in the analysis of more normal tissues on paired samples than the Mitchell et al. study. Also, the relation between the SOX21 gene methylation and demographic/clinicopathological factors has not been studied by Mitchell et al. and we examined this issue for the first time.
| » Materials and Methods|| |
After the approval of the study by the Ethics Committee of Kermanshah University of Medical Sciences, a total of 41 CRC patients participated in this study and the informed consent was obtained from all of them. Tumor and adjacent normal tissue samples (>10 cm away from the tumor) were collected during surgery of CRC patients from Bistoon Hospital (Kermanshah, Iran) in a 3-year period (2016–2018). None of the patients were under chemotherapy or radiotherapy prior to the surgical procedure. All of the samples were stored at −80°C until DNA was extracted.
QIAamp DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany; cat. no. 51104) was used for the extraction of tissue samples (20 mg of each sample) according to the manufacturer's recommendations. The extracted DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
Genomic DNA treatment with sodium bisulfite yields uracil from unmethylated cytosines, while leaves methylated cytosines unchanged. Here, 500 ng of genomic DNA was modified by EpiTectH Bisulfite Kit (Qiagen, Germany; cat no. 59826) according to the manufacturer's protocol. After the elution in 15 mL of TE buffer, the bisulfite-treated DNA was stored at −20°C until processing.
In order to detect methylated DNA sequences from bisulfite-treated templates, a pair of primers and a TaqMan probe specified to anneal the fully DNA methylated at SOX21 gene promoter were designed using Beacon Designer™ (version 8.13; www.premierbiosoft.com/molecular_beacons; Premier Biosoft International, Palo Alto, CA, USA). Here, we used MethyLight technique as a fluorescence-based quantitative real-time PCR (Polymerase Chain Reaction), highly specific, sensitive, and reproducible assay. The ALU-C4 element was used as a reference control using the primer and probe sequences as previously described., Primer and probe sequences for SOX21 gene and ALU-C4 element are shown in [Table 1].
|Table 1: SOX21 gene and ALU-C4 element primer and probe sequences for the MethyLight assays|
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The MethyLight reactions were performed on a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc.) using the EpiTect® MethyLight PCR + ROX Vial kit (Qiagen GmbH; cat. no. 59496) according to the manufacturer's protocol. The final volume of each reaction was 20 μl containing 2 μl 10X primer-probe mix containing 200 nM probe and 400 nM of each forward and reverse primers, 10 μl of 2X EpiTect MethyLight Master mix (without ROX), 0.4 μl 50X ROX Dye Solution, 2 μl (20 ng) bisulfite-treated DNA template, and 5.6 μl RNase-free water. The PCR conditions were 95°C for 3 minutes, followed by 50 cycles at 95°C for 5 seconds, 64°C for 10 seconds and 73°C for 20 seconds.
In order to establish the standard curve, a fully methylated control (EpiTect PCR Control DNA; cat. no. 59655; Qiagen GmbH) was used to calculate the percentage of methylated reference (PMR) of the samples. PMR is the degree of methylation of each sample relative to the fully methylated control and was calculated using the following formula: [(SOX21/ALU-C4) sample/(SOX21/ALU-C4) positive control] × 100.
To assess the sensitivity and specificity, a receiver operating characteristics (ROC) curve was constructed. In order to differentiate patients based on their methylation status, the point on the ROC curve at which (sensitivity + specificity) was maximal was considered as the optimal cut-off PMR value. Finally, the patients were classified as positive (PMR>cut-off value) or negative (PMR≤cut-off value) subjects.
Statistical data analysis was carried out using SPSS software (version 25.0; SPSS, Inc., Chicago, IL, USA). Two-sided Mann–Whitney U test was used to compare the median level of methylation in tumor tissues and their adjacent normal mucosa. In order to measure the association between classified demographic/clinicopathological characteristics with the median level of methylation, two-sided Mann–Whitney U test was used. P values ≤0.05 were considered significant for all described tests.
| » Results|| |
The median age of the 41 CRC patients was 59 years (range, 31–80). The ratio of the number of men to the number of women was 0.86 (19/22), and all patients had a Kurdish ethnic background. By histology, 73.2% of tumors were adenocarcinoma and 26.8% were mucinous carcinomas.
According to the PMR results [Figure 1], all 41 CRC tumor tissues had a low or high degree of methylation in their SOX21 gene promoter; 8 (19.51%), 12 (29.27%), and 21 (51.22%) of the tumor tissues showed a PMR ≤2.83, 2.83 <PMR≤10, and PMR>10, respectively. In comparison, 11 (26.83%) and 30 (73.17%) of the normal adjacent tissues had a PMR = 0 or PMR ≤2.83, respectively [Figure 1]. Samples positive for ALU-C4 and negative for SOX21 methylation were considered as PMR = 0.
|Figure 1: Methylation levels of SOX21 gene promoter in tumor tissues and their normal adjacent mucosa according to PMR results (PMR = 0; white cells, 0<PMR≤2.83; gray-25% cells, 2.83< PMR≤10; gray-50% cells, PMR>10; black cells)|
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The median level of PMR was 10.26 (range 0.66–66.62) in tumor tissues and 0.13 (0–2.83) in adjacent normal tissues. The methylation rates in tumor tissue samples were significantly higher compared to their adjacent normal mucosa (P < 0.0001) [Table 2]. The relationship between the demographic/clinicopathological features and the methylation level of SOX21 gene promoter in the group of tumor tissues is summarized in [Table 3]. No association was observed between demographic/clinicopathological factors and methylation level in tumor tissues (P > 0.05).
|Table 2: SOX21 gene PMR value of tissue samples and their normal adjacent mucosa in patients with colorectal cancer|
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|Table 3: Methylation level of SOX21 gene promoter in tumor tissues of CRC patients and its relationship with classified demographic/clinicopathological characteristics|
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In order to predict tissue diagnosis (cancer vs. normal tissue), a ROC curve was constructed and tissue samples exhibited a sensitivity of 80.5% and specificity of 97.6% for SOX21 methylation marker [the area under curve (AUC) = 0.953] [Figure 2]. The point on the ROC curve at which (sensitivity + specificity) was maximal was considered as the optimal cut-off PMR value (PMR = 2.75). According to this PMR cut-off, 33 (80.49%) and 8 (19.51%) patients were considered as positive and negative in methylation status, respectively.
|Figure 2: Receiver operating characteristics curve for methylation of SOX21 gene promoter in CRC tumor tissues versus normal adjacent mucosa|
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| » Discussion|| |
In recent years, there have been many studies on the identification and description of epigenetic biomarkers as promising cancer biomarkers in the scientific literature. At the moment, a number of methylation markers of colorectal cancer have been approved by FDA (Food and Drug Administration) and are available in commercial tests. EpiproColon® (for SEPTIN9 gene methylation detection) and Cologuard (for NDRG4 and BMP3 gene methylation detection) are two examples of these commercial tests that are able to detect colorectal cancer at the early stages. However, it should be noted that many studies are required to transform a potential marker into a diagnostic commercial test.
In the last 2 decades, many studies have been conducted on the use of a single or combination of DNA methylation biomarkers for colorectal cancer diagnostic purposes.,,,,,,,,, In the latest review by Lam et al.,VIM, SEPT9, ITGA4, OSM4, GATA4, and NDRG4 were the most validated genes to study further their promoter methylation in CRC stool or plasma samples for diagnostic purposes. An important result of these studies is the need to carry out several studies to transform a potential biomarker into a diagnostic commercial test.
SOX21, a candidate tumor-suppressor gene, is involved in brain tumorigenesis. SOX21 was seen to be downregulated in high-grade glioma compared to low-grade glioma. The expression of SOX21 and SOX2 overlap in glioma tissues and cell lines. Unlike the oncogenic activity of SOX2, SOX21 overexpression decreases proliferation and induces apoptosis. It has been suggested that this function is due to the binding to and downregulation of SOX2 protein.,
Although limited studies have been conducted on SOX21 gene, these studies have focused on its expression, not its methylation. In a study conducted by Yang et al.,SOX21 gene expression decreased in patients with oral squamous cell carcinoma. The authors of this article have suggested that this reduction in expression is probably due to hypermethylation of the promoter of SOX21 gene. Moreover, according to Kandimalla et al. study,SOX21 gene promoter is hypermethylated in bladder tumors. However, methylation of this gene has not been observed in control samples.
To our knowledge, the studies by Mitchell et al. are the only two studies performed on SOX21 gene promoter methylation in colorectal cancer so far., According to the first study, these authors showed that 82% of colon tissue samples (67 samples) and 0% of matched normal tissues (six samples) were hypermethylated. Subsequently, they evaluated SOX21 gene promoter in plasma samples from colorectal cancer cases and control subjects. These authors reported a sensitivity of 85% and a false positive rate of 50% in CRC and normal plasma samples, respectively.
In the present study, real-time methylation-specific PCR (MethyLight) was used to determine the methylation status of the SOX21 gene promoter. MethyLight is an ideal tool to quantitatively measure methylation level even in small volumes of DNA in clinical samples. The results of the present study showed that CRC tumor tissues exhibit a higher degree of methylation than their normal adjacent mucosa (P < 0.0001) [Table 2].
Here, we obtained a sensitivity of 80.5% and a specificity of 97.6% based on an optimal cut-off PMR (PMR = 2.75) for SOX21 gene promoter methylation. Although the number of normal samples examined by us (41 samples) is much more than numbers examined by Mitchell et al., our results are consistent with their results.
To our knowledge, there is no report on the relationship between SOX21 gene promoter methylation status and demographic/clinicopathological factors in the literature. The median PMR value of tissue-based SOX21 methylation in CRC tended to increase across the transition from moderately/well-differentiated to poorly differentiated status and from tumor-node-metastasis (TNM) stage I/II to TNM stage III/IV, but the difference was not significant (P = 0.796 and P = 0.605, respectively). Overall, the results of this study showed that there is no significant difference between the demographic/clinicopathological features and the methylation status of SOX21 gene promoter in CRC patients.
Despite the fact that colonoscopy is the gold standard for the screening of colorectal cancer patients, its invasiveness is a limitation to this method. Therefore, the development of new diagnostic methods with a noninvasive nature is necessary. The limitations of this study include the following. First, patients participated in this study all belong to a local area (Kermanshah province, Iran), and similar studies should be conducted in other populations. Second, the sample size is relatively small and this will reduce the test power of this study.
| » Conclusion|| |
SOX21 gene promoter methylation showed a sensitivity of 80.5% and a specificity 97.6%. These results are comparable to the sensitivity and specificity of most robust DNA methylation-based biomarkers reviewed by Lam et al. In conclusion, the results of this study along with two studies conducted by Michell et al. indicated the high potential of SOX21 gene promoter methylation as a candidate noninvasive diagnostic biomarker in stool and plasma of colorectal cancer patients. However, further studies with larger sample sizes are required to evaluate the specific role of SOX21 methylation as a biomarker for early detection of CRC.
The authors would like to thank the staff of the Medical Genetics Laboratory, Reference Laboratory and Kermanshah University of Medical Sciences for their assistance during the present study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| » References|| |
Coppedè F, Migheli F, Lopomo A, Failli A, Legitimo A, Consolini R, et al
. Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism. Epigenetics 2014;9:621-33.
Howlader N, Noone A, Krapcho M, Miller D, Bishop K, Altekruse S. SEER Cancer Statistics Review, 1975-2015, National Cancer Institute. Bethesda, MD: National Cancer Institute; 2015. 2015.
Moghimi-Dehkordi B, Safaee A. An overview of colorectal cancer survival rates and prognosis in Asia. World J Gastrointest Oncol 2012;4:71-5.
Geiger TM, Ricciardi R. Screening options and recommendations for colorectal cancer. Clin Colon Rectal Surg 2009;22:209-17.
Simon K. Colorectal cancer development and advances in screening. Clin Interv Aging2016;11:967-76.
Deaton AM, Bird A. CpG islands and the regulation of transcription. Genes Dev 2011;25:1010-22.
Mori Y, Olaru AV, Cheng Y, Agarwal R, Yang J, Luvsanjav D, et al
. Novel candidate colorectal cancer biomarkers identified by methylation microarray-based scanning. Endoc-Relat Cancer 2011:ERC-11-0083.
Li WH, Zhang H, Guo Q, Wu XD, Xu ZS, Dang CX, et al
. Detection of SNCA and FBN1 methylation in the stool as a biomarker for colorectal cancer. Dis Markers 2015;2015.
Yang CM, Wang TH, Chen HC, Li SC, Lee MC, Liou HH, et al
. Aberrant DNA hypermethylation-silenced SOX21-AS1 gene expression and its clinical importance in oral cancer. Clin Epigenetics 2016;8(1):129.
Caglayan D, Lundin E, Kastemar M, Westermark B, Ferletta M. Sox 21 inhibits glioma progression in vivo
by forming complexes with Sox 2 and stimulating aberrant differentiation. Int J Cancer 2013;133:1345-56.
Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, et al
. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer 2014;14:54.
Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, et al
. Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 2005;33:6823-36.
Rezvani N, Alibakhshi R, Vaisi-Raygani A, Bashiri H, Saidijam M. Detection of SPG20 gene promoter-methylated DNA, as a novel epigenetic biomarker, in plasma for colorectal cancer diagnosis using the MethyLight method. Oncol Lett 2017;13:3277-84.
Draht MX, Goudkade D, Koch A, Grabsch HI, Weijenberg MP, van Engeland M, et al
. Prognostic DNA methylation markers for sporadic colorectal cancer: A systematic review. Clin Epigenetics 2018;10:35.
Ahmed D, Danielsen SA, Aagesen TH, Bretthauer M, Thiis-Evensen E, Hoff G, et al
. A tissue-based comparative effectiveness analysis of biomarkers for early detection of colorectal tumors. Clin Translational Gastroenterol 2012;3:e27.
Leong K, Wei W, Tannahill L, Caldwell G, Jones C, Morton D, et al
. Methylation profiling of rectal cancer identifies novel markers of early-stage disease. Br J Surg 2011;98:724-34.
Cassinotti E, Melson J, Liggett T, Melnikov A, Yi Q, Replogle C, et al
. DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps. Int J Cancer 2012;131:1153-7.
Müller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, et al
. Methylation changes in faecal DNA: A marker for colorectal cancer screening? Lancet 2004;363:1283-5.
Tetzner R, Model F, Weiss G, Schuster M, Distler J, Steiger KV, et al
. Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer. Clin Chem 2009;55:1337-46.
Cesaroni M, Powell J, Sapienza C. Validation of methylation biomarkers that distinguish normal colon mucosa from cancer patients from normal colon mucosa of patients without cancer. Cancer Prev Res 2014:7;717-26.
Holdenrieder S, Dharuman Y, Standop J, Trimpop N, Herzog M, Hettwer K, et al
. Novel serum nucleosomics biomarkers for the detection of colorectal cancer. Anticancer Res 2014;34:2357-62.
Xiao W, Zhao H, Dong W, Li Q, Zhu J, Li G, et al
. Quantitative detection of methylated NDRG4 gene as a candidate biomarker for diagnosis of colorectal cancer. Oncol Letters 2015;9:1383-7.
Lam K, Pan K, Linnekamp JF, Medema JP, Kandimalla R. DNA methylation based biomarkers in colorectal cancer: A systematic review. Biochim Biophys Acta 2016;1866:106-20.
Ferletta M, Caglayan D, Mokvist L, Jiang Y, Kastemar M, Uhrbom L, et al
. Forced expression of Sox 21 inhibits Sox 2 and induces apoptosis in human glioma cells. Int J Cancer 2011;129:45-60.
Kandimalla R, van Tilborg AA, Kompier LC, Stumpel DJ, Stam RW, Bangma CH, et al
. Genome-wide analysis of CpG island methylation in bladder cancer identified TBX2, TBX3, GATA2, and ZIC4 as pTa-specific prognostic markers. Eur Urol 2012;61:1245-56.
Mitchell SM, Ho T, Brown GS, Baker RT, Thomas ML, McEvoy A, et al
. Evaluation of methylation biomarkers for detection of circulating tumor DNA and application to colorectal cancer. Genes 2016;7(12):125.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]