colorectal cancer; CDX2; epigenetics; methylation; prognosis
Graule Janina, Uth Kristin, Fischer Elia, Centeno Irene, Galván José A., Eichmann Micha, Rau Tilman T., Langer Rupert, Dawson Heather, Nitsche Ulrich, Traeger Peter, Berger Martin D., Schnüriger Beat, Hädrich Marion, Studer Peter, Inderbitzin Daniel, Lugli Alessandro, Tschan Mario P., Zlobec Inti (2018), CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway, in Clinical Epigenetics
, 10(1), 120-120.
Nolte Sarah, Zlobec Inti, Lugli Alessandro, Hohenberger Werner, Croner Roland, Merkel Susanne, Hartmann Arndt, Geppert Carol I, Rau Tilman T (2017), Construction and analysis of tissue microarrays in the era of digital pathology: a pilot study targeting CDX1 and CDX2 in a colon cancer cohort of 612 patientsCDX1 and CDX2 analysis using ngTMA and DIA, in The Journal of Pathology: Clinical Research
, 3(1), 58-70.
The homeobox caudal-type 2 gene CDX2 is required for differentiation, proliferation and maintenance of an intestinal cell phenotype. Our group has shown that CDX2 protein is absent or markedly reduced in up to 20% of colorectal cancers, which correlates with de-differentiation, metastatic spread and unfavorable patient outcome. Our preliminary data using a panel of colorectal cancer cell lines confirms that promoter hypermethylation is a likely cause of CDX2 expression loss, since CDX2 expression can be recovered upon treatment with DNA methyltransferase inhibitors (DNMTi). Here, we aim to show that epigenetic modifications leading to CDX2 loss are clinically and functionally relevant and that these effects can be reversed by epigenetic therapies. Since these therapies have ‘global’ effects on demethylation, we aim to overcome the methylation blockade of CDX2, by establishing a state-of the-art method for specific targeting of the CDX2 promoter for re-activation. To achieve these aims, we will first determine whether CDX2 promoter hypermethylation causes CDX2 protein loss in human colorectal cancers and we will assess the relationship of CDX2 with the general methylator phenotype. Second, we take advantage of a fully-characterized cohort of colorectal cancer patients (n=750) to determine the potential prognostic and predictive relevance of CDX2 hypermethylation and protein loss. Third, we will identify novel transcriptional regulators of CDX2 based on transcription binding site prediction (e.g. GATA family members and p300) by Chromatin Immunoprecipitation (ChIP) and validate results using other promoter assays. The function of these transcription factors in colorectal cancer cell lines after knock-down will be assessed. Our findings will be validated using fresh frozen material from our high-quality biobank. Fourth, we will determine the effects of DNMTis and histone deacetylase inhibitors (HDACi) on methylated and unmethylated cell lines in vitro and CDX2 expression. Chromatin structure will be investigated by analysing open and repressed histone marks and their effect on CDX2 restoration. To re-activate silenced CDX2 promoters we propose to use CRISPR technology to target either a transcriptional activation domain or the catalytic domain of the demethylating TET2 protein to the human CDX2 promoter. Small guide (sg) RNAs will be designed and tested to target nuclease inactive Cas9 fused to either VPS64, a transcriptional activator, or to the catalytic domain of TET2, a demethylating enzyme to the CDX2 promoter region. To further increase the chance of successful re-activation of the CDX2 promoter, we take advantage of an amplifier system based on a polypeptide scaffold (Sun Tag) fused to the inactive Cas9 protein. The Sun Tag will be able to recruit multiple VPS64 or TET2 proteins, fused to single chain variable fragment specific for the Sun polypeptide, to the CDX2 promoter region. We will establish CDX2 knockdown colon cancer cell lines and compare expression of CDX2 in methylated, unmethylated and genomically modified cell lines, then perform functional assays to determine effects on proliferation, migration and invasion. This will allow us to determine whether CDX2 expression and its function can be reversed upon modification. This proposal uses the latest genome editing technologies to uncover new aspects of CDX2 in colorectal cancers. Not only will the clinical and biological relevance of this gene be detailed, but new regulatory aspects of CDX2 will be highlighted. Additionally, successful targeted re-activation of an already-methylated promoter, as we are proposing to do in the project, may be further generalized to other important tumor suppressor genes and could have significant therapeutic potential.