The main goal of our research activity is the elucidation of the molecular and genetic basis of esophageal and colorectal tumor initiation and progression, and the translation of this knowledge into more accurate diagnosis, prevention and therapeutic intervention. This broad approach reflects the different interests and research and clinical activities of the faculties involved in theme 3.1 and ranges from the functional analysis of specific tumor suppressor- and onco-genes, to more clinically-related aspects such as molecular grading, the development of pre-clinical mouse models, the application of genomic and expression profiling to GI-tract tumors, and the development of molecular markers as prognostic predictors and potential therapeutic targets.

Notably, much of the research focus within this sub-theme has been aimed at the isolation and characterization of cancer stem cells (CSCs) from the above mentioned GI tract malignancies. CSCs represent not only an ideal model study for the mechanisms underlying tumor initiation, malignant progression, and metastasis to distant sites, but are also unique targets for the development of diagnostic, prognostic and therapeutic applications. 

The molecular basis of Barrett’s metaplasia and esophageal adenocarcinoma

The metaplastic changes observed in Barrett’s esophagus (BE) and the increased esophageal adenocarcinoma (EA) risk characteristic of BE patients are of great interest as they represent an ideal model to study how environmental changes can reprogram resident stem cells and thus trigger tumorigenesis. In collaboration with the Dept. of Surgery (prof. dr. J. van Lanschot), we recently initiated a novel research line towards the isolation and characterization of CSCs from EAs obtained from Barrett’s patients. The initial results show that a minority of EA cells (1*10-5) have tumor-initiating properties. Moreover, the analysis of EA cell lines have revealed a role of Wnt/b-catenin signalling in modulating cancer stemness in esophageal malignancies.

With regard to esophageal malignancies the departments of Surgery, Gastroenterology, Medical Oncology and Pathology of the Erasmus university Medical Center Rotterdam have a longstanding interest in research and clinical management of BE including EA. More than 550 BE patients are currently under surveillance at the Erasmus MC and yearly about 50 EA patients undergo resection. In collaboration with the Dept. of Surgery (Prof. H. Tilanus) research is performed on the molecular aberrations present in BE and EA. For this research more than 200 frozen EA and 18 BE samples are collected. In addition, 33 EA xenografts were established in nude mice and two EA cell lines were generated. For the investigations also all known EA cell lines were obtained and in an international effort the origins of these cell lines were verified. It appeared that three EA cell lines, amongst which the two common most used ones (SEG-1 and BIC-1) are not EA cell lines, but a known lung and colon carcinoma cell line, respectively. In addition, research was initiated on EA and esophageal squamous cell carcinoma patients with regard to a possible correlation with oncogene and tumor suppressor gene polymorphisms.

A mouse model for metaplasia and the role of Wnt signalling in cancer stemness

In our attempt to develop mouse models for epithelial tumorigenesis by targeting mutations in members of the Wnt pathway, we have generated a mouse model for mammary metaplasia, adenocarcinoma and lung metastasis (Apc1572T). Although the primary tumor does not affect the GI tract, this model is of great interest to study how metaplastic changes underlie malignancy through reprogramming of the resident stem cell. We have successfully isolated CSCs from these mammary tumors, established a gene signature that distinguish them from their normal counterparts and more differentiated tumor cells, and shown that they are capable of forming distant metastases in a broad spectrum of organs when injected into the tail vein. Notably, bulk tumor cells fail to do so upon tail vein injection. 

Molecular genetic analysis of hereditary colorectal cancer

This approach has led to the characterization of a large number of mutations in the APC gene (responsible for FAP) and in the MSH2, MLH1, and MSH6 genes (responsible for HNPCC) allowing the characterization of their mutation spectra, the presymptomatic and prenatal diagnosis, the development of novel mechanistic insights in genotype-phenotype correlations, and the identification of a pre-symptomatic target population of gene carriers to carry out new molecular-target-based trials.

In collaboration between the departments of Pathology (Dr. K. Biermann and Dr. W.N.M.Dinjens), Gastroenterology and Hepatology (Prof. Dr. E.J. Kuipers and Dr. M.E. van Leerdam), Clinical Genetics (Dr. A. Wagner) and Public Health (Prof. Dr. E.W. Steyerberg) a study (called LIMO and coordinated by the Erasmus MC, Rotterdam, The Netherlands) is performed to determine whether improvement of Lynch syndrome diagnostics can be obtained by the performance of microsatellite instability (MSI) analysis in colorectal cancer (CRC) patients up to the age of 70 years. MSI-analysis is performed in a prospective consecutive series of 1,000 newly diagnosed CRC patients ≤ 70 years, and the results are expected in 2010.

Isolation and characterization of CSCs from pre-clinical in vivo models for intestinal carcinogenesis

The generation of transgenic pre-clinical in vivo models for intestinal carcinogenesis has assisted the study of the signal transduction pathways underlying epithelial tumor formation and progression, and in the development of pre-clinical in vivo models for drug testing. Many of these mouse models are currently being employed worldwide for their close resemblance of the human clinical course of the disease. In particular, we are now concentrating on our compound Apc/KRAS preclinical mouse model for intestinal cancer that recapitulates the genetic and histo-pathologic changes observed in the majority of colon cancers in man. In short, we were able to isolate by FACS a minority population of CSCs from the Apc/KRAS intestinal adenocarcinomas (CD24hi/CD29+) characterized by tumor initiating capacity as shown by transplantation in NOD-SCID recipient mice. Expression profiling of these CSCs have revealed a unique signature which is now being applied to analyse human CRCs and its capacity to predict disease outcome.

Quiescent and cycling stem cells in intestinal homeostasis and cancer

Recently, a cycling stem cell type was isolated from the mouse intestinal epithelium that is capable of differentiating into all four intestinal types. To date however, the quiescent, label-retaining, intestinal stem cell of the GI tract has not been characterized yet. During the last 2 years, we have succeeded in doing so, by developing and applying a sophisticated transgenic model to label quiescent stem cells with a hystone 2B reporter gene fused to GFP. The FACS-isolated quiescent stem cells are able to regenerate the differentiation types characteristic of the intestinal epithelium. Also, they enter the cell cycle and start dividing as a response to tissue injury. Notably, these cells appear to be closely related to Paneth cells, a differentiated type normally only found in the upper GI tract but not in the colon, which is known to arise in the large intestine in the presence of inflammatory diseases.

We also applied the H2B-GFP transgenic model to isolate quiescent tumor cells from the compound Apc/KRAS mouse model. The initial results point to the presence of such quiescent tumor cells within the previously isolated fraction of CSCs (CD24hi/CD29+). The identification of a population of dormant CSCs is of great clinical relevance as they may well underlie metastases appearing in colon cancer patients, even decades after the surgical removal of the primary tumor. 

Development and assessment of chemopreventive and therapeutic approaches for colorectal cancer

Experimental pharmacological interventions are first being tested in the pre-clinical animal models that we developed. In a second phase, selected drugs are tested in trials with individuals with an hereditary predisposition to intestinal cancer. We have successfully applied this general scheme to test Aspirin and resistant starch (Na-butyrate) as chemopreventive agents. In close collaboration the University of Newcastle, the CAPP trial in HNPCC patients has shown that a 600 mg daily dose of Aspirin reduces the tumor burden of HNPCC patients by at least 50%. We are now expanding these studies to the role of Aspirin in sensitizing CSCs for DNA damage-induced apoptosis.

Esophageal adenocarcinoma and Barrett’s epithelium

The development of better prognostic parameters (e.g. molecular markers and ploidy status) which can predict malignant development. Isolation and further characterization of CSCs from BE and EA patients. Establishment of a mouse model for BE/EA.

Intestinal adenomas, adenocarcinomas and metastases

Given the exciting results obtained during the last 5 years, the study of stem cell differentiation and its role in development and tumorigenesis is expected to become a central one in the coming years. We plan to concentrate on the role of the Wnt signalling pathway both in differentiation and tissue-specific fate determination in homeostasis, and in cancer initiation and progression.

Also, the tools developed until now to study CSCs in mouse and man will form the basis for the isolation and analysis of the so-called migrating cancer stem cells, i.e. the CSC fraction that detach from the primary tumor and eventually metastasize distant organ sites. CSCs have been shown to be intrinsically resistant to chemo- and radio-therapy. Part of this phenotype is due to the expression of interleukin-4. As Aspirin is known to inhibit IL-4 gene expression, we plan to analyze its capacity to sensitize CSC to chemo- and radio-therapy in both mouse models and as a co-adjuvant therapy in colon cancer patients.

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4.      Gaspar C, Cardoso J, Franken P, Molenaar L, Morreau H, Möslein G, Sampson J, Boer JM, de Menezes RX, Fodde R. Cross-species comparison of human and mouse intestinal polyps reveals conserved mechanisms in adenomatous polyposis coli (APC)-driven tumorigenesis. Am J Pathol. 2008 May;172(5):1363-80.

5.      Fodde R, Brabletz T. Wnt/beta-catenin signaling in cancer stemness and malignant behavior. Curr Opin Cell Biol. 2007 Apr;19(2):150-8.

6.      Janssen KP, Alberici P, Fsihi H, Gaspar C, Breukel C, Franken P, Rosty C, Abal M, El Marjou F, Smits R, Louvard D, Fodde R, Robine S. APC and oncogenic KRAS are synergistic in enhancing Wnt signaling in intestinal tumor formation and progression. Gastroenterology. 2006 Oct;131(4):1096-109.

7.      Cardoso J, Molenaar L, de Menezes RX, van Leerdam M, Rosenberg C, Möslein G, Sampson J, Morreau H, Boer JM, Fodde R. Chromosomal instability in MYH- and APC-mutant adenomatous polyps. Cancer Res. 2006 Mar 1;66(5):2514-9.

8.      Boonstra JJ, van der Velden AW, Beerens EC, van Marion R, Morita-Fujimura Y, Matsui Y, Nishihira T, Tselepis C, Hainaut        P, Lowe AW, Beverloo BH, van Dekken H, Tilanus HW, Dinjens WNM. Mistaken identity of widely used esophageal adenocarcinoma cell line TE-7.  Cancer Res. 2007 Sep 1;67(17):7996-8001.

9.      Koppert LB, van der Velden AW, van de Wetering M, Abbou M, van den Ouweland AM, Tilanus HW, Wijnhoven BP, Dinjens WNM.Frequent loss of the AXIN1 locus but absence of AXIN1 gene mutations in adenocarcinomas of the gastro-oesophageal junction with nuclear beta-catenin expression.  Br J Cancer. 2004 Feb 23;90(4):892-9.

10.  Boonstra JJ, van Marion R, Beer DG, Lin L, Chaves P, Ribeiro C, Dias Pereira A, Roque L, Darnton SJ, Altorki NK, Schrump DS, Klimstra DS, Tang LH, Eshleman JR, Alvarez H, Shimada Y, van Dekken H, Tilanus HW, Dinjens WNM. Contaminated Cell Lines Threaten the Development of Treatment Strategies for Esophageal Adenocarcinoma. J Natl. Cancer Inst. Accepted for publication.