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Additional file 4. Wnt response is partly location specific. (A) Heatmap with average (n = 3) scaled counts, centred around the location mean. Right columns show summarized log2FC(Cβ/wt) and mean read count across locations. Using a likelihood ratio test, we identified genes, in which the reaction to Wnt activation depends significantly on the location. The location dependently upregulated genes were separated into 3 groups, where the upregulation was maximal in prSI, diSI or colon, respectively. (B) Genes differentially expressed between left and right TCGA colon cancer samples were tested for enrichment in Hallmark genes sets using EGSEA. (C) Using transcriptome data from TCGA [8] we tested, which Wnt target genes [32] were differentially expressed between left and right colon in the MSS APC mutated colon cancer samples. Wnt genes with a trend to differential expression between the colon cancer locations (adjusted p-value

Additional file 1. Validation of findings. (A) The expression of dsRed after Ctnnb1S transduction was examined by fluorescence microscopy including images merged with bright field, scale bar = 200 μm. (B) Heatmap depicts average (n = 3) scaled counts for both conditions of each location, right columns show summarized log2 fold changes, log2FC(Cβ/wt), and mean read count across locations. Genes represent our location signatures comprising each 200 most significantly (Wald-Test) upregulated genes comparing the wt samples of prSI, diSI or colon to all other locations’ wt samples, respectively. (C) Validation of location marker genes with quantitative PCR, n = 3, Student’s t-test, error bars represent S.E.M., mean fold change in vertical number. (D) Immunoblot of prSI location marker REG1, diSI location marker FABP6, colon location marker MUC1 and control GAPDH, protein lysates from organoids grown without (wt) or with transduced Ctnnb1S (Cβ). The loading control is exemplarily shown for the FABP6 membrane. (E) For genes which are significantly differentially expressed in the murine organoid locations in colon vs. small intestine and in TCGA normal samples from the left vs. right colon we find a significant correlation (Spearman) of the log2 fold changes (log2FC).

Background: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. Methods: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of β-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. Results: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. Conclusion: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

Additional file 3. Location signatures are downregulated upon Wnt activation in vivo. (A) To validate our findings in human in vivo samples we used TCGA data [8] with the summarized clinical characteristics of patients. *Transverse colon samples were depicted but not included in the differential expression analyses between the two sides, due to lack of more specific site information. The same holds for a single sample from normal transverse colon of a female patient (44 years). (B) Using transcriptome data from TCGA [8] we identified genes differentially expressed (adjusted p-value

Additional file 3. Location signatures are downregulated upon Wnt activation in vivo. (A) To validate our findings in human in vivo samples we used TCGA data [8] with the summarized clinical characteristics of patients. *Transverse colon samples were depicted but not included in the differential expression analyses between the two sides, due to lack of more specific site information. The same holds for a single sample from normal transverse colon of a female patient (44 years). (B) Using transcriptome data from TCGA [8] we identified genes differentially expressed (adjusted p-value

BACKGROUND: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. METHODS: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of β-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. RESULTS: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. CONCLUSION: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

Understanding the molecular pathogenesis of cancer is key to understand its Achilles’ heels to be able to improve treatment. The heterogeneity of cancer has been examined by major worldwide consortia efforts. For example, the Cancer Genome Atlas Project (TCGA) generated multiple levels of data such as whole-exome sequencing, array-based DNA methylation profiling, -mRNA sequencing, -microRNA sequencing and revolutionized how the heterogeneity of cancer can be classified on a molecular level. Next to important insights about the molecular basis of cancer, they provided a rich open source repository of data for more detailed studies. This thesis will describe and discuss results from characterizing pathomechanisms in colorectal cancer from the genetic, genomic and transcriptomic facets

The epithelial monolayer of intestine is one of the fastest renewing tissues of the human body, a process that is regulated by intestinal stem cells (ISCs) residing in crypt-like structures. These ISCs continuously divide to generate differentiated cells, while simultaneously engaging in ongoing neutral competition with each other for a position within the crypt. Whenever an ISCs acquires a mutation that confers a competitive advantage, the mutant ISC gradually replaces all normal ISCs which results in permanent fixation of a mutation within the crypt and the initiation of colorectal cancer (CRC). This thesis focuses on the competition between normal and mutant ISCs, with specific emphasis on mutations that activate the Wnt pathway such as loss of Apc or activation of Ctnnb1. We reveal that Apc-mutant ISCs act as ‘supercompetitors’ that actively force normal ISCs to differentiate and leave the crypt. Moreover, we demonstrate that boosting the fitness of normal ISCs can inhibit the competitive advantage of Apc-mutant and prevent adenoma formation. These findings are particularly relevant for familial adenomatous polyposis (FAP) patients that carry germline mutations in the APC gene and are predisposed to the development of CRC. Critically, understanding the molecular basis underlying the competition between normal and mutant cells can be used to develop novel chemoprevention strategies for FAP and other heritable cancer syndromes.