Introduction

Colorectal cancer is one of the most frequent malignant tumors worldwide. With increasing use of prognostic biomarkers, molecularly stratified treatment of colorectal cancer is improving in clinical practice. However, identifying stable and easily detectable biomarkers for clinical use remains a challenge. Among these biomarkers, epigenetic changes, especially DNA methylation, are the most promising candidates for clinical practice. Prominent examples of such methylation markers are SEPTIN9, the Secretin Receptor (SCTR), and Engrailed Homeobox 1 (EN1). The methylation preferentially affects CpG islands within promoter regions of these genes. Aberrant methylation of CpG islands in the promoter region can be associated with the silencing of tumor suppressor genes and promotes tumorigenesis and tumor growth.

Aims & Methods

Our goal is to develop an innovative diagnostic tool for the reliable detection of malignant colon tissue based on the recognition of specific methylation patterns. This reporter system will detect hypermethylated genetic regions in vivo using luminescence. Suitable biomarkers for this approach will be identified by methylation profiling of colorectal carcinoma cell lines as well as organoids from patients with colorectal cancer.
Our reporter system consists of two constructs. The first construct, the key element of which is the methyl-binding domain of Methyl-CpG-binding domain protein 1 (MBD1), recognizes hypermethylated CpG islands that are frequently located within the promoter region. The second construct can bind with high specificity to the genomic region of interest via the sgRNA of a deadCas9. When the two components combine in close proximity, a SplitTEV protease induces the newly developed flipNluc luciferase. The emitted luminescence signal then detects the tumor in a highly specific manner and can be quantified. In addition, whole-genome bisulfite sequencing on colon carcinoma cell lines HCT116 and CaCo2 and organoids from colon biopsies were used to identify additional specific hypermethylated regions. The raw data was analyzed bioinformatically using Bismark and the R package DSS.

Results

We verified the functionality of our new assay by assessing the induction of the newly developed flipNLuc luciferase by the TEV protease. We detected a significant increase in luminescence, confirming the functionality of the luciferase. Thereafter, we validated the reporter system by analyzing the methylation marker genes EN1 and SCTR, which are typically hypermethylated in colorectal carcinoma. In particular, significantly increased luminescence was measured compared to the unmethylated housekeeping genes GAPDH and GPI. To optimize the sensitivity and specificity of our new detection system, additional genes with differentially methylated regions were identified during bioinformatic analysis of the sequencing data. Currently, we are adapting our reporter system to these newly identified genes to validate their potential role as new specific biomarkers.

Conclusion

Reliable DNA methylation biomarkers for clinical practice in the diagnosis of colorectal cancer are still limited. The developed method allows the highly specific identification of malignant tissue based on the detection of hypermethylated CpG islands. Furthermore, our method can be used for targeted screening for new methylation markers. By replacing the sgRNAs of deadCas9, our newly developed method is not limited to the detection of colon tumors and can be adapted to the diagnosis of other tumor entities.