Browsing by Author "Lassila, Marika Jenna Janina"

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.