Prognostic significance of SOX17 and WNT5a promoter methylation status in circulating cell-free DNA metastatic colorectal cancer patients

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RESEARCH ARTICLE

Hippokratia 2023, 27(1): 7-11

Vagionas A, Balgkouranidou I, Koukaki T, Biziota E, Amarantidis K, Kakolyris S, Xenidis N (†)
Department of Medical Oncology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece

Abstract

Aim: Carcinogenesis of colorectal cancer is a process involving genetic mutations and epigenetic alterations in its multiple phases. The most considerable epigenetic alteration occurring in colorectal cancer (CRC) tumorigenesis is the methylation-mediated silencing of tumor suppressor genes. The present study aimed to detect the methylation status of SOX17 and WNT5a promoters in cell-free DNA circulating in plasma of metastatic CRC patients and to investigate potential prognostic correlation.

Methods: A methylation-specific real-time polymerase chain reaction was utilized to investigate the methylation status of genes (SOX17 and WNT5a) promoter in the blood of 85 metastatic CRC patients.

Results: We found the SOX17 promoter methylated in 54/85 (63.5 %) while WNT5a was methylated in 39/85 (45.8 %) samples of the advanced CRC. All control samples were negative for SOX17 and WNT5a promoter methylation. Patients with metastatic CRC and methylated SOX17 and WNT5a promoter status had a significantly poorer outcome than patients with non-methylated ones.

Conclusions: Plasma SOX17 and WNT5a promoter methylation are frequent epigenetic events in advanced CRC. The reported correlations between the methylation status of genes (SOX17 and Wnt5a) promoter and poorer survival in patients with advanced CRC disease agree with the proposed role of SOX17 as a tumor suppressor gene. A more considerable CRC patient cohort is required to research these findings’ potential further and investigate whether SOX17 in plasma could serve as a useful prognostic biomarker in metastatic CRC. HIPPOKRATIA 2023, 27 (1):7-11.

Keywords: metastatic colorectal cancer, cell-free DNA, SOX17, WNT5a gene, DNA methylation, prognosis 

Corresponding author: Anastasios Vagionas, Medical Oncologist, MD, Director of Oncology of General Hospital of Kavala, 65500, Kavala, Greece, email: tvagionas@yahoo.com

Introduction

Colorectal cancer (CRC) is, in both genders, the third leading cause of cancer-related death, in part because of significant changes in risk factors, the introduction and dissemination of early detection tests, and improvements in treatment1,2. It is a multistep disease resulting from different alterations accumulation in the genome (mutations) and the epigenetic code (DNA methylation, histone modifications, etc.)3. DNA methylation plays a critical role as a modulator of main genomic functions such as tumor suppressor gene expression. These cumulative events initially induce adenoma emergence, which, in a proportion of patients, progresses into invasive adenocarcinoma with metastasis potential4. An essential and well-characterized event during this progression to adenocarcinoma is the activation of the Wnt-pathway5.

Wnt-pathway inactivation depicts an early event in CRC tumorigenesis predominantly caused by inactivating mutations in its main component, APC gene6. The methylation-mediated silencing of additional downstream or upstream Wnt signal-regulating components may propose an important alternative mechanism of constitutive Wnt-pathway activation in CRC, including AXIN27, CDH18, WNT5a 9, and SOX1710.

As already stated, methylation is fundamental in CRC progression, and many genes frequently modify the methylation levels of tumor cells compared to the normal colon mucosa. The SRY-related high mobility group box (SOX) family of transcription factors regulates stem/precursor cell development and function by repressing the canonical Wnt-signaling pathway. Analysis of CpG island methylation and gene expression in CRC cell lines showed that silencing of the SOX17 gene is associated with DNA methylation. By suppressing Wnt signaling, SOX17 plays a tumor suppressor role11. SOX17 has also shown significantly increased methylation in CRC cell lines in polypoid and non-polypoid adenomas and colon carcinomas compared to normal colon mucosa. It has also been shown that there is a functional relation between methylation and SOX17 silencing12. Silva et al, suggested that the methylation frequency of SOX17 increases during the neoplastic adenoma-carcinoma sequence concerning other Wnt antagonists. In screening studies involving DNA methylation in stool or plasma samples, authors underline the usefulness of SOX17 as a biomarker for the early detection of CRC.

The WNT5a gene, located at 3p14, is a tumor suppressor locus commonly deleted in multiple tumors. The WNT5a protein is classified as a non-canonical Wnt protein, and its role in tumorigenesis is still unclarified13. Studies have shown that increased WNT5a expression is essential for tumor progression and have nominated it as proto-oncogene14. On the other hand, WNT5a also inhibits tumor cell proliferation, whereas WNT5a expression is considered a good prognostic factor in patients with breast and colon cancer. Recent studies have confirmed that the most frequent mode of its inactivation in CRC is by tumor-specific methylation15.

Plasma cell-free DNA (cfDNA) is a principal component of Liquid biopsy that contains large amount of information that reflects the tumor’s molecular profile and can lead to its decoding in a less invasive way. Despite the preanalytical obstacles and the need for standardization, cfDNA circulating in plasma still remains a great source of novel molecular tumor biomarkers16,17.

Numerous studies have described in plasma samples and in the corresponding primary tumors, similar methylation patterns of tumor suppressor genes18. Methylation in peripheral blood usually reflects the methylation status of the primary tumor as numerous studies have already reported19. A blood-based test is noninvasive and versatile both for primary detection and postoperative follow-up for local or distant relapse20.

This study focuses on detecting the methylation status of the SOX17 and WNT5a genes in the cfDNA circulating in the plasma of metastatic CRC patients. In particular, we examined the dynamic of the methylation frequency between SOX17 and WNT5a in metastatic CRC (mCRC) patients. In plasma cfDNA samples from mCRC patients, we aimed to estimate the DNA methylation status prognostic significance regarding these two gene promoters.

Materials and Methods

Study design

Eighty-five samples collected at the beginning of the first-line therapy from mCRC patients comprised our study’s material. The methylation status of SOX17 and WNT5a genes in the cfDNA circulating in those plasma samples obtained were examined. All mCRC patients had a performance status of 0-1 and provided informed consent to participate in the study. Plasma circulating cfDNA was isolated from those CRC patients and 40 healthy individuals comprising the healthy volunteer group, totally independent regarding age but the gender-matched group who had not received any medical care and had no relation with the patients. The study was approved by the Scientific Committee of our Institution (Alexandroupolis University hospital, decision No ΕΣ6, date 28/09/2017). 

Collection of samples and plasma circulating cfDNA isolation

We collected peripheral blood in EDTA tubes, and samples were subsequently centrifuged for 10 min at 1500 x g, and the plasma was stored until further analysis at -80 oC. We extracted cfDNA from 1,200 mL of plasma using the MagCore automated nucleic acid Εxtractor (MagCore Plasma DNA Extraction kit) according to the manufacturer’s instructions. The DNA concentration was determined in the Qubit fluometer (Thermo Fisher Scientific).

Sodium bisulfite conversion

According to the manufacturer’s protocol, we performed sodium bisulfite conversion of up to 0.5 g cfDNA utilizing the EZ DNA Methylation Gold Kit (ZYMO Research Co., Orange, CA), using as the 100 % methylated control, the Universal Methylated Human DNA Standard (Zymo Research Corp., USA). We employed the dH2O and gDNA from the 100 % methylated control as negative and positive controls in each conversion reaction, respectively. We stored the converted DNA at -70 °C until it was used. 

Methylation Specific PCR (Real-time MSP)

We detected the methylation status of SOX17 and WΝΤ5a in cfDNA by real-time MSP using specific primer pairs for the unmethylated and methylated promoter sequences. In this study, both real-time MSP assays designed and used have been evaluated for their specificity and sensitivity. In the presence of 99.9 % of non-methylated sequences, they detected down to 0.1 % of the methylated. The assays are not quantitative, so a cut-off has not been established, but when an amplification signal is detected (0-40 cycles), the sample is reported positive. We performed the MSP reactions in the Qiagen Rotor-Gene instrument of a total of 20μl. We used as fully methylated (100 %) MSP positive control, human placental genomic DNA (gDNA; Sigma Aldrich) methylated in vitro with SssI methylase (NEB, Ipswich, MA), after sodium bisulfite conversion, while as a negative MSP control, we used the same unmethylated placental gDNA after sodium bisulfite conversion. To prepare serial dilutions of known concentrations, we mixed the unmethylated DNA control with 100 % methylated DNA to evaluate the analytical sensitivity. After optimizing for real-time MSP, the MSP primers from previous studies20 were used to detect SOX17 methylation, while the MSP primers described previously21 were used to detect the WNT5a gene.

Statistical analysis

We performed data statistical analysis utilizing IBM SPSS Statistics for Windows, Version 21.0 (IBM Corp., Armonk, NY, USA). We report the methylation status of SOX17, WΝΤ5a, and all other qualitative variables as frequencies and percentages (%). We calculated survival rates using the Kaplan-Meier method and determined the statistical difference between the survival curves with the log-rank test.

Results

Study’s population characteristics

Our study’s population consisted of 85 mCRC patients with a mean age 68.35 (±9.6) years, a median age of 70, and a range of 47-76 years. More than half (54.7 %) were males. 

Correlation between tumor parameters and SOX17 promoter methylation

We examined the methylation status of SOX17 in cfDNA obtained from 85 mCRC patients and observed a high SOX17 promoter methylation status in this group of patients (54/85, 63.5 %), while no promoter methylation was documented in the control group. A statistically significant correlation was seen between patients with methylated SOX17 status and poor differentiation (p =0.01). 

Correlation between tumor parameters and WΝΤ5a promoter methylation

We found the WΝΤ5a promoter to be unmethylated in the control group, while it was methylated in 39/85 (45.8 %) of the patients, and its methylation status was associated with poor differentiation and right-sided CRC (p =0.01 and p =0.021, respectively).

Survival analysis

After a 48-month median follow-up period, 81 of the 85 (95.2 %) patients presented disease progression, and 78 of the 81 (96.2 %) patients died. 

Correlation between survival and SOX17 promoter methylation

We evaluated the Kaplan-Meier estimates for the independent cohort of 85 advanced CRC patients concerning the methylation status of SOX17 promoter in cfDNA circulating in plasma. A significantly shorter progression-free survival period was observed in patients with methylated SOX17 gene in correlation with those with an unmethylated one (p <0.001). Additionally, patients with mCRC and a methylated SOX17 promoter status had statistically significantly poorer survival than those with an unmethylated SOX17 promoter status (p =0.001). Figure 1a and Figure 1b give Kaplan-Meier survival estimates according to the methylation status in mCRC. 

Figure 1: a) Overall survival and b) Progression-free survival of patients with metastatic colorectal cancer in relation to SOX17 methylation status.
OS: overall survival, PFS: progression-free survival.

Correlation between survival and Wnt5a promoter methylation

The Kaplan-Meier estimates for the entire cohort of patients have shown that patients with methylated Wnt5a gene presented a statistically shorter progression-free survival period in correlation with those with an unmethylated one (p <0.001). Additionally, as shown in Figure 2a and Figure 2b, patients with mCRC and a methylated WNT5a promoter status had statistically significantly poorer survival than those having an unmethylated WΝΤ5a promoter status (p =0.001).

Figure 2: a) Overall survival and b) Progression-free survival of patients with metastatic colorectal cancer in relation to WNT5a methylation status.
OS: overall survival, PFS: progression-free survival.

Correlation between survival and both genes’ promoter methylation

Thirty patients from the entire cohort presented simultaneous methylation of both genes. As demonstrated in shown in Figure 3a and Figure 3b, these patients presented a shorter progression-free survival and overall survival than those with both genes unmethylated and those with at least one gene unmethylated (p =0.001 and p =0.001, respectively).

Figure 3: a) Overall survival and b) Progression-free survival of patients with metastatic colorectal cancer in relation of both SOX17 and WNT5a methylation status.
OS: overall survival, PFS: progression-free survival.

Discussion

CRC remains a leading cancer-associated morbidity and mortality cause worldwide. The overall survival of CRC patients depends highly on the disease stage at diagnosis. The five-year survival estimates range from 85-90 % for patients with stage I to less than 10 % for patients with stage IV disease. Around half of all patients with CRC develop local recurrence or distant metastasis during their disease22.

In recent times, abnormal DNA methylation patterns have emerged as potential candidate cancer biomarkers23 to explore the molecular profile of tumorigenesis.  Most of these studies reveal promising aspects that could impact the treatment of CRC patients in the future.

A tumor suppressor role is attributed to the SOX17 gene through modulation of Wnt signaling. In the early stages of gastrointestinal tumorigenesis, SOX17 is induced by Wnt activation; however, progressively, during malignant progression, SOX17 is downregulated by methylation. Hence, SOX17 appears to prevent the malignant progression at the early stage of tumorigenesis. Its methylation-dependent silencing is additionally supported by the fact that SOX17 is commonly unmethylated in normal human tissues but highly methylated in various primary tumors, including CRC. Detection of aberrant promoter methylation of several Wnt inhibitors, including SOX17 and WNT5a, at different stages and time points along CRC progression, supplies a significant increment of methylation from adenoma to carcinoma. Therefore, Wnt signaling levels may alter during the evolution of cancer due to simultaneous or alternative methylation of the genes involved in the pathway. Literature on SOX17 promoter methylation status mainly includes studies performed at the primary tumor level. A previous study demonstrated that SOX17 promoter methylation in cfDNA obtained from operable gastric cancer patients is associated with the clinical outcome19. Similarly, in other types of cancer, including breast cancer, SOX17 has been found highly methylated in circulating tumor cells isolated from breast cancer patients and in corresponding cfDNA. Additionally, WNT5A, which is another canonical WNT inhibitor and noncanonical WNT member, was repeatedly methylated in the promoter region24,25. Consequently, expression of the WNT5A gene was downregulated or silenced, deteriorating or losing its antagonistic action against the anomalous activation of canonical WNT signaling.

The present study evaluated the frequency and potential prognostic significance of SOX17 and WNT5a promoter methylation in circulating cfDNA from mCRC patients. SOX17 and WNT5a methylation was detected in 63.5 % and 45.8 % of the mCRC patients, respectively. These observations indicate that SOX17 promoter methylation represents a frequent event in advanced disease. The observed high incidence of SOX17 methylation may also be relevant to the critical role of Wnt/β catenin signaling pathway in CRC progression. We suggest that SOX17 methylation and inactivation, along with a subsequent excessive Wnt/β catenin signaling, is a frequent but gradually occurring event that is noticeable at later disease stages, associating with a more aggressive tumor phenotype and a higher metastatic potential. This agrees with the observed significant association between unmethylated SOX17 promoter status and better survival in mCRC.  This survival difference was also seen in patients with unmethylated WNT5a gene. Patients with methylated WNT5a had a poorer prognosis than those with unmethylated one.

These data, in sum, demonstrate that SOX17 and WNT5a are critical in controlling the signaling of the Wnt/β catenin pathway, and SOX17 inactivation as a result of DNA methylation up-regulates this critical pathway, resulting in an accelerated disease progression. It is also demonstrated that the methylation of both genes gives prognostic solid results. Nevertheless, “liquid biopsy” could also be a potential surrogate for biomarker discovery through the epigenetic approach.

We conclude that SOX17 and WNT5a methylation is a frequent event in mCRC and, when present, is associated with bad prognosis, particularly in this group of mCRC patients. Further studies in a larger cohort of patients are required to explore and establish its prognostic significance further.

Conflict of interest

Authors declare no conflicts of interest. 

In memoriam

This paper is dedicated to the memory of Professor Nikolaos Xenidis (1966-2022). 

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394-424.
  2. Vogelaar I, van Ballegooijen M, Schrag D, Boer R, Winawer SJ, Habbema JD, et al. How much can current interventions reduce colorectal cancer mortality in the U.S.? Mortality projections for scenarios of risk-factor modification, screening, and treatment. Cancer. 2006; 107: 1624-1633.
  3. Aaltonen LA. The multistep process of colon carcinogenesis. Cytokines Mol Ther. 1996; 2: 111-114.
  4. Pan Y, Liu G, Zhou F, Su B, Li Y. DNA methylation profiles in cancer diagnosis and therapeutics. Clin Exp Med. 2018; 18: 1-14.
  5. Klaus A, Birchmeier W. Wnt signalling and its impact on development and cancer. Nat Rev Cancer. 2008; 8: 387-398.
  6. Schneikert J, Behrens J. The canonical Wnt signalling pathway and its APC partner in colon cancer development. Gut. 2007; 56: 417-425.
  7. Koinuma K, Yamashita Y, Liu W, Hatanaka. H, Kurashina, K, Wada T, et al. Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability. Oncogene. 2006; 25: 139-146.
  8. Kamiyama H, Noda H, Takata O, Suzuki K, Kawamura Y, Konishi F. Promoter hypermethylation of tumor-related genes in peritoneal lavage and the prognosis of patients with colorectal cancer. J Surg Oncol. 2009; 100: 69-74.
  9. Sun G, Wu L, Sun G, Shi X, Cao H, Tang W. WNT5a in Colorectal Cancer: Research Progress and Challenges. Cancer Manag Res. 2021; 13: 2483-2498.
  10. Zhang W, Glöckner SC, Guo M, Machida EO, Wang DH, Easwaran H, et al. Epigenetic inactivation of the canonical Wnt antagonist SRY-box containing gene 17 in colorectal cancer. Cancer Res. 2008; 68: 2764-2772.
  11. Voorham QJ, Janssen J, Tijssen M, Snellenberg S, Mongera S, van Grieken NC, et al. Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal carcinomas. BMC Cancer. 2013; 13: 603.
  12. Silva AL, Dawson SN, Arends MJ, Guttula K, Hall N, Cameron EA, et al. Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists. BMC Cancer. 2014; 14: 891.
  13. Zhu N, Qin L, Luo Z, Guo Q, Yang L, Liao D. Challenging role of Wnt5a and its signaling pathway in cancer metastasis (Review). Exp Ther Med. 2014; 8: 3-8.
  14. Griesmann H, Ripka S, Pralle M, Ellenrieder V, Baumgart S, Buchholz M, et al. WNT5A-NFAT signaling mediates resistance to apoptosis in pancreatic cancer. Neoplasia. 2013; 15: 11-22.
  15. Ray SK, Mukherjee S. Cell Free DNA as an Evolving Liquid Biopsy Biomarker for Initial Diagnosis and Therapeutic Nursing in Cancer- An Evolving Aspect in Medical Biotechnology. Curr Pharm Biotechnol. 2022; 23: 112-122.
  16. Lissa D, Robles AI. Methylation analyses in liquid biopsy. Transl Lung Cancer Res. 2016; 5: 492-504.
  17. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res.2001; 61: 1659-1665.
  18. Mansour H. Cell-free nucleic acids as noninvasive biomarkers for colorectal cancer detection. Front Genet. 2014; 5: 182.
  19. Balgkouranidou I, Karayiannakis A, Matthaios D, Bolanaki H, Tripsianis G, Tentes AA, et al. Assessment of SOX17 DNA methylation in cell free DNA from patients with operable gastric cancer. Association with prognostic variables and survival. Clin Chem Lab Med. 2013; 51: 1505-1510.
  20. Chimonidou M, Strati A, Malamos N, Georgoulias V, Lianidou ES. SOX17 promoter methylation in circulating tumor cells and matched cell-free DNA isolated from plasma of patients with breast cancer. Clin Chem. 2013; 59: 270-279.
  21. Panagopoulou M, Karaglani M, Balgkouranidou I, Biziota E, Koukaki T, Karamitrousis E, et al. Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers. Oncogene. 2019; 38: 3387-3401.
  22. Kim MS, Lee J, Sidransky D. DNA methylation markers in colorectal cancer. Cancer Metastasis Rev. 2010; 29: 181-206.
  23. Draht MX, Riedl RR, Niessen H, Carvalho B, Meijer GA, Herman JG, et al. Promoter CpG island methylation markers in colorectal cancer: the road ahead. Epigenomics. 2012; 4: 179-194.
  24. Jiang G, Lin J, Wang W, Sun M, Chen K, Wang F. WNT5A Promoter Methylation Is Associated with Better Responses and Longer Progression-Free Survival in Colorectal Cancer Patients Treated with 5-Fluorouracil-Based Chemotherapy. Genet Test Mol Biomarkers. 2017; 21: 74-79.
  25. Abdelmaksoud-Dammak R, Miladi-Abdennadher I, Saadallah-Kallel A, Khabir A, Sellami-Boudawara T, Frikha M, et al. Downregulation of WIF-1 and Wnt5a in patients with colorectal carcinoma: clinical significance. Tumour Biol. 2014; 35: 7975-7982.