Abstract
The loss of the caudal homeobox 2 protein (Cdx2) is associated with poor prognosis in colorectal cancer (CRC) patients. However, the mechanisms that generate the loss of expression and the interplay with other genetic and transcriptomic aberrations remain unknown. Understanding the regulatory mechanism and interaction processes of CDX2, through a multi-omic approach, would help to change the genomic/transcriptomic tumor pattern, favoring sensitivity to standard treatment. CDX2 promoter methylation and mutations were studied by Miseq in both 7 CRC cell lines and 50 CRC patients. Azaytidine was used as demethylation treatment. A parallel study using CRISPR-dCAS9 fused with the catalytic TET and DNMT domains was required for the expression reversal studies. Determination of CDX2 status has been performed using a new approach developed in-house. A custom gene panel was used to study other genomic aberrations. Transcriptomic analysis by RNAseq was assessed through differential expression by DESeq2. Among the analyzed CG positions, a promoter island with an increased methylation rate tumor/normal and inverse correlation with expression was identified. Azacytidine treatment showed a dose-dependent correlation between methylation and expression (p<0.001). We also demostrated a specific reversion of CDX2 expression using CRISPR-dCAS9, subsequently changes in FOLFOX sensitivity was observed (p<0.05). Patients with CDX2- had higher levels of methylation in the studied CpG island than those with CDX2+. NGS showed no functional mutations or copy-number changes on CDX2-. Upstream mutations displayed an interaction between CDX2- and mutations in FLNA. Transcriptomic analysis revealed changes in INFa response pathway and MAPK proliferation genes. CDX2 promoter hypermethylation is the main regulatory mechanism of CDX2 expression. Loss of CDX2 interacts with immune and proliferation pathways at genomic and transcriptomic levels, conferring an agresive phenotype. Epigenetic engineering modifications of CDX2 could favor increase the sensitivity to standar therapy in this setting.
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