SOX11 hypermethylation as a tumor biomarker in endometrial cancer

Abstract

We previously reported that SOX4 is overexpressed in endometrial cancer and that it partially contributes to hypermethylation of miR-129-2 and miR-203. The current study seeks to identify methylation and expression levels of the SOX gene family in endometrial carcinomas. Methylation levels of the 16 SOX gene family members were measured by combining bisulfite restriction analysis (COBRA), MassARRAY, and pyrosequencing assays of cell lines and endometrial cancer samples. Gene expression was determined by RT-qPCR. The methylation level of the SOX11 locus was correlated with clinicopathologic factors in primary endometrial tumors and in TCGA endometrial cohort. It was also examined in DNA of serum and endometrial specimens from a longitudinal cohort of early stage endometrial cancer patients. COBRA assays indicated that hypermethylation of SOX1, SOX2, SOX11, SOX14, SOX15, SOX17, and SOX18 was present in endometrial cancer cell lines and not in the normal control. SOX11 expression was reactivated only by a DNA methylation inhibitor. Moreover, aberrant DNA methylation of SOX11 was detected in the majority of endometrioid endometrial carcinomas (n=114) and none of the 22 adjacent normal endometrial samples (P<0.0001). The methylation status of SOX11 associated significantly with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.0001), and this finding was validated in TCGA endometrial cohort. Furthermore, SOX11 was not hypermethylated in serum DNA from early stage endometrial cancer patients. This study found that hypermethylation of SOX11 is common in endometrial carcinomas and strongly associates with microsatellite instability and MLH1 methylation.

Keywords: Biomarker; DNA methylation; Endometrial carcinoma; Epigenetics; SOX11.

Fig. 1. SOX11 is hypermethylated in endometrial cancer cells.

(a) Summary by COBRA of the methylation status of the SOX gene family in endometrial cancer cell lines (from left to right: AN3CA, HEC1A, Ishikawa, KLE, RL95–2, and SK-UT-1B) and one normal (N) pooled sample derived from two noncancerous endometria as a negative control. Black and white circles: 100% and 0% methylation. Gray circles: methylated. (b) Diagram of the SOX11 locus and relative locations of methylation assays. (c) Methylation levels of SOX11 in endometrial cancer cell lines, as revealed by COBRA analysis. SssI: methylated positive control; N: normal endometrium; +, AciI: restriction enzyme added; -: without AciI; u: unmethylated; and m: methylated. C, Methylated SOX11 in primary endometrial tumors as determined by COBRA assays. *: methylated.

Fig. 2.. Relative expression levels of SOX family genes in endometrial cancer cells after treatment with DAC and/or TSA.

Gene expression was determined by RT-qPCR and compared to untreated controls. GAPDH was used as an internal control gene. Error bar: SD; *: P<0.05 compared with untreated control of the same cell type.

Fig. 3.. Methylation of the SOX11 CpG island and clinicopathologic covariates in primary endometrioid endometrial carcinomas (EECs).

(a) Methylation profiles of SOX11 in 22 adjacent normal endometrial tissues and 114 primary tumors determined by MassARRAY analysis. (b) Dot plots demonstrating SOX11 hypermethylation in EEC tumors. (c–e) Dot plots indicating that methylation levels of SOX11 correlate with grade, MSI status, and MLH1 methylation. m: MLH1 methylated; u: MLH1 unmethylated.

Fig. 4.. Hypermethylation of SOX11 in TCGA endometrial cohort.

(a) Methylation analysis of 32 paired endometrioid endometrial (EEC) tissues. (b) Dot plots demonstrating SOX11 hypermethylation in primary EEC, serous, and mixed histological endometrial tumors. (c–e) Dot plots indicating that methylation levels of SOX11 associated with tumor grade, MLH1 methylation, and MSI status. m: MLH1 methylated; u: MLH1 unmethylated.

Fig. 5.. Hypermethylation of SOX11 in early stage endometrial tumors.

(a–b) DNA methylation of SOX11 CpG sites in early stage endometrial tumors and paired adjacent normal endometria as determined by pyrosequencing. (c–d) DNA methylation in serum DNA from early stage endometrial cancer patients before and after surgery as determined by pyrosequencing.