Colorectal Cancer Early Detection in Stool Samples Tracing CpG Islands Methylation Alterations Affecting Gene Expression.

Ana Florencia Vega-Benedetti,Andrea Casadei-Gardini,Mario Scartozzi,Loredana Moi,Angelo Restivo,Sandra Orrù,Francesco Cabras,Pina Ziranu,Luigi Zorcolo,Andrea Pretta,Eleonora Lai,Federica Fortunato,Eleonora Loi,Patrizia Zavattari,Letizia Ciccone,Marco Puzzoni

doi:10.3390/ijms21124494

Ana Florencia Vega-Benedetti, Andrea Casadei-Gardini + Show 14 more

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https://doi.org/10.3390/ijms21124494

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Abstract

Colorectal cancer (CRC) is a major cause of cancer mortality. Early diagnosis is relevant for its prevention and treatment. Since DNA methylation alterations are early events in tumourigenesis and can be detected in cell-free DNA, they represent promising biomarkers for early CRC diagnosis through non-invasive methods. In our previous work, we identified 74 early altered CpG islands (CGIs) associated with genes involved in cell cross-talking and cell signalling pathways. The aim of this work was to test whether methylation-based biomarkers could be detected in non-invasive matrices. Our results confirmed methylation alterations of GRIA4 and VIPR2 in CRC tissues, using MethyLight, as well as in stool samples, using a much more sensitive technique as droplet digital PCR. Furthermore, we analysed expression levels of selected genes whose promoter CGIs were hypermethylated in CRC, detecting downregulation at mRNA and protein levels in CRC tissue for GRIA4, VIPR2, SPOCK1 and SLC6A3. Most of these genes were already lowly expressed in colon normal tissues supporting the idea that cancer DNA methylation targets genes already barely expressed in the matched normal tissues. Our study suggests GRIA4 and VIPR2 as biomarkers for early CRC diagnosis using stool samples and confirms downregulation of genes hypermethylated in CRC.

Highlights

Colorectal cancer (CRC) is the third most common cancer worldwide and one of the most frequent cause of cancer death
We focused on four genes, Glutamate Ionotropic Receptor AMPA Type Subunit 4 (GRIA4), vasoactive intestinal peptide receptor 2 (VIPR2), SPOCK1 and SLC6A3, whose CpG islands (CGIs) were hypermethylated in CRC in our previous study
Further methylation analyses were performed in 10 paired samples for GRIA4 and VIPR2, confirming their alteration in CRC

Summary

Introduction

Colorectal cancer (CRC) is the third most common cancer worldwide and one of the most frequent cause of cancer death. Diagnosis has a relevant importance for its prevention and treatment [1]. To improve diagnosis in the early stage of colorectal cancer, researchers focused on the identification of biomarkers at genetic and epigenetic levels [2,3,4,5,6]. Colonoscopy is a costly and invasive procedure, screening tests on stool samples have been developed. These tests include faecal occult blood test (FOBT) and faecal immunochemical test (FIT) but, they can offer indications for possible CRC, their specificity and sensitivity are still limited [7]

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