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Analyzing and modulating the epigenetic landscape of CDX2 as well as studying its function in colorectal cancer pathology

English title Analyzing and modulating the epigenetic landscape of CDX2 as well as studying its function in colorectal cancer pathology
Applicant Zlobec Inti
Number 166578
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pathologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Clinical Cancer Research
Start/End 01.06.2016 - 31.10.2019
Approved amount 305'040.00
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Keywords (5)

colorectal cancer; CDX2; epigenetics; methylation; prognosis

Lay Summary (German)

Lead
CDX2 is an important gene regulating intestinal differentiation. Interestingly, the loss of CDX2 protein in colorectal cancer patients is associated with unfavorable prognosis. In this study, we investigate the regulation of CDX2 and determine whether the epigenetic landscape in colorectal cancer can be therapeutically targeted.
Lay summary

Das Homeobox Caudal-Typ 2 Gen CDX2 ist erforderlich für die Differenzierung, Proliferation und den Unterhalt des intestinalen Zell-Phänotyps. Unsere Gruppe hat in früheren Studien gezeigt, dass die Expression des CDX2-Proteins in bis zu 20% aller kolorektalen Karzinome reduziert ist. Diese Resultate korrelieren mit De-differenzierung, der Bildung von Metastasen und verschlechterten Aussichten in der Behandlung dieser Patienten. Vorläufige Daten aus einem Panel für Zell-Linien aus kolorektalen Karzinomen bestätigen, dass eine Hypermethylation des Promoters die wahrscheinlichste Ursache für den Verlust der CDX2-Expression ist, weil die CDX2-Expression durch die Behandlung mit DNA Methyltransferase-Inhibitoren (DNMTi) wieder hergestellt werden kann. Wir versuchen hier zu zeigen, dass epigenetische Modifikationen, die einen Verlust der CDX2 Expression zur Folge haben, klinisch relevant sind und mit epigenetischen Therapien rückgängig gemacht werden können. Da solche Therapien einen „globalen“ Effekt auf die Demethylierung haben ist es unser Ziel, die Methylierung und damit die Sperre des CDX2-Promoters mit Hilfe der neusten Genomeditierungs-Technologie aufzuheben und dadurch den CDX2-Promoter zu reaktivieren. Im Erfolgsfall könnte dieser gezielte Ansatz auch auf andere Tumor-Suppressor Gene oder Krebsarten angewandt werden und hätte somit beachtliches pharmazeutisches Potential.

 

Direct link to Lay Summary Last update: 20.05.2016

Responsible applicant and co-applicants

Employees

Publications

Publication
CDX2 in colorectal cancer is an independent prognostic factor and regulated by promoter methylation and histone deacetylation in tumors of the serrated pathway
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.
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
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.

Collaboration

Group / person Country
Types of collaboration
UC Davis Genome Center United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Swiss Pathology Days Talk given at a conference CDX2 loss in colorectal cancer is an independent adverse prognostic factor and can be restored by de-methylation and inhibition of histone deacetylation 10.11.2018 Thun, Switzerland Zlobec Inti; Uth Kristin;
European Congress of Pathology Poster Regulation of CDX2 in Colorectal Cancer via Methylation and Histone Deacetylation 09.09.2017 Amsterdam, Netherlands Uth Kristin;
European Congress of Pathology Poster CDX2 promoter methylation is a mechanism of protein loss in the serrated pathway to colorectal cancer 09.12.2016 Köln, Germany Zlobec Inti;


Associated projects

Number Title Start Funding scheme
173219 Identification and analysis of PU.1 cell death pathways to improve leukemia therapy 01.05.2017 Project funding (Div. I-III)
183501 Installing a Hyperion CyTOF mass cytometry platform for high-dimensional single cell analysis at the University of Bern 01.10.2019 R'EQUIP

Abstract

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.
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