Identification of epigenetic silencing of the SFRP2 gene in colorectal cancer as a clinical biomarker and molecular significance

Abstract

Background: Several studies have suggested secreted frizzled-related protein 2 (SFRP2) gene as a potential clinical biomarker in colorectal cancer (CRC). However, its diagnostic role remains unclear. In this study, we aimed to investigate the significance of SFRP2 methylation levels in a large cohort of biological specimens (including blood, adipose and colonic tissues) from patients with CRC, thereby potentially identifying new biomarker utility.

Methods: We examined the expression (by qPCR) and methylation status (by 450 K DNA array and DNA pyrosequencing) of the SFRP2 gene in healthy participants (N = 110, aged as 53.7 (14.2), 48/62 males/females) and patients with CRC (N = 85, aged 67.7 (10.5), 61/24 males/females), across different biological tissues, and assessing its potential as a biomarker for CRC. Additionally, we investigated the effect of recombinant human SFRP2 (rhSFRP2) as a therapeutic target, on cell proliferation, migration, and the expression of key genes related to carcinogenesis and the Wnt pathway.

Results: Our findings revealed that SFRP2 promoter methylation in whole blood could predict cancer stage (I + II vs. III + IV) (AUC = 0.653), lymph node invasion (AUC = 0.692), and CRC recurrence (AUC = 0.699) in patients with CRC (all with p < 0.05). Furthermore, we observed a global hypomethylation of SFRP2 in tumors compared to the adjacent area (p < 0.001). This observation was validated in the TCGA-COAD and TCGA-READ cohorts, demonstrating overall hypermethylation (both with p < 0.001) and low expression (p < 0.001), as shown in publicly available scRNA-Seq data. Notably, neoadjuvant-treated CRC patients exhibited lower SFRP2 methylation levels compared to untreated patients (p < 0.05) and low promoter SFRP2 methylation in untreated patients was associated with poor overall survival (p < 0.05), when compared to high methylation. Finally, treatment with 5 µg of rhSFRP2 treatment in CRC cells (HCT116 cells) inhibited cell proliferation (p < 0.001) and migration (p < 0.05), and downregulated the expression of AXIN2 (p < 0.01), a gene involved in Wnt signaling pathway.

Conclusions: These findings establish promoter methylation of the SFRP2 gene as a prognostic candidate in CRC when assessed in blood, and as a therapeutic prognostic candidate in tumors, potentially valuable in clinical practice. SFRP2 also emerges as a therapeutic option, providing new clinical and therapeutical avenues.

Keywords: SRFP2; Colorectal cancer biomarkers; DNA methylation; Promoter methylation; Wnt signaling pathway.

Fig. 1

Promoter SFRP2 methylation in whole blood as a biomarker for colorectal cancer. A We measured the promoter methylation of SFRP2 gene from healthy participants (N = 31) and patients with CRC (N = 62) (p = 0.220). B Kaplan–Meier curve comparing the median of the SFRP2 promoter methylation as low and high methylation. The significance of differences is evaluated with the Log-rank test. We measured the promoter methylation of SFRP2 gene from patients with CRC in C early stage (I + II) (N = 42) and late stage (III + IV) (N = 39), (p = 0.044) D absence (N = 44) and presence (N = 35) of lymph node invasion (p = 0.014) and E absence (N = 58) and presence (N = 24) of recurrence (p = 0.013). Asterisks indicate significant differences between the groups according to the Mann Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001). F, G and H indicate ROC curve of stage (F), lymph node invasion (G) and Recurrence (H) in three models. Model 1 indicate SFRP2 methylation. Model 2 indicates clinical variables, such as age, sex, BMI, HDL, triglycerides, and tumor location. Model 3 includes Model 1 + Model 2. AUC Area under curve, CRC colorectal cancer, ROC receiver operating characteristic curve, Sens sensitivity, Spec specificity

Fig. 2

Diagnostic and prognostic value of SFRP2 methylation in tumor tissue from colorectal cancer. A Global β methylation was done with significant CpG sites between tumor and NAT. The CpG sites included in the global SFRP2 methylation in colon tissue in our participants were: cg14289246, cg05961809, cg23292160, cg25775322, cg23207990, cg22178613, cg10318528, cg11354906, cg00082664, cg03202804, cg05874561, cg04965141, cg25645268, cg14330641, cg23121156, cg06549216, cg10942078, cg20881942, cg23714408, cg05164933, cg14063488. B SFRP2 expression in colon samples from NAT (N = 11) and tumor area(N = 16). C Global β methylation was done with significant CpG sites between tumor and NAT in the TCGA-COAD. D SFRP2 expression in colon samples from NAT (N = 51) and tumor area (N = 308) in the TCGA-COAD cohort. E Global β methylation was done with significant CpG sites between tumor and NAT in the TCGA-READ. F SFRP2 expression in colon samples from NAT (N = 10) and tumor area(N = 94) in the TCGA-READ cohort. G Single cell RNA-seq analysis of SFRP2 gene in NAT, tumor core and tumor border. SFRP2 was mostly expressed in NAT and a small fraction of tumor core, in stromal cells and myofibroblasts. H We measured the promoter methylation of SFRP2 gene from patients with CRC no treated (N = 57) and treated (N = 24) with neoadjuvant therapy. I Kaplan–Meier curve comparing the median of the SFRP2 promoter methylation as low and high methylation in non-treated patients with CRC. The significance of differences is evaluated with the Log-rank test. Asterisks indicate significant differences between the groups according to the Mann Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001). COAD colon adenocarcinoma, NAT normal adjacent area, READ rectum adenocarcinoma, TCGA The cancer genome atlas

Fig. 3

SFRP2 inhibit cell proliferation and migration, through decreasing AXIN2 expression. A promoter methylation of SFRP2 in HCT116 cells. B Cells were seeded in 6-well plates at the concentration of 50.000 cell/well. Cells were treated with 1, 5 and 10 µM of AZA (5-Aza-2-deoxycytidine), during 72 h. SFRP2 gene expression was measured using qPCR and Probe qPCR kit (Takara, Spain). Gene normalization was used by PPIA. C Cells were treated at crescent concentration of rhSFRP2 at 0.1, 0.5, 1, 2.5 and 5 µg/mL of rhSFRP2 during 48 h. D wound healing Assay was conducted in the presence of 5 µg/mL of rhSFRP2. E–H gene expression in cell treated with vehicle or with 5 µg/mL of rhSFRP2 of AXIN2, CTNNB1, TIAM1 and VEGF. AZA azacytidine, CTNNB1 beta-catenin, ND No Detected, PPIA Peptidylprolyl Isomerase A, rhSFRP2 recombinant human secreted frizzled related Protein 2, TIAM1 T-cell lymphoma invasion and metastasis-inducing protein 1, VEGR Vascular endothelial growth factor