The role of the stem cell marker OLFACTOMEDIN-4 and the microRNA generator DICER1 in colorectal carcinogenesis

The intestine is a pivotal organ which is divided into two anatomical parts: the small intestine and the large intestine (colon and rectum). Both parts are made up of single layered epithelium. This epithelium is composed of villi (protrusions) – found only in the small intestine - and crypts (invaginations) leading to an increase of the surface of the intestinal lumen whereby the uptake of nutrients and water is improved. Every five days, the intestinal epithelium is renewed whereby both, crypts and eventually villi, are filled up with new cells. The homeostasis of the crypts/villi relies on adult stem cells (SCs), especially crypt base columnar (CBC) cells, which are located at the base of the crypts. These are regulated by an active Wnt signaling pathway. A deregulation of the Wnt signaling pathway leads to cancer formation found in humans almost exclusively in the colon and rectum. Colorectal cancer (CRC) is worldwide the third most common cause for cancer related deaths. In the majority of CRC, origin and progress are caused by mutations in the adenomatous polyposis coli (APC) gene which encodes an essential component of the β-catenin destruction complex that is the central element of the Wnt signaling pathway. As a consequence of these mutations, the executor of the Wnt signaling pathway, β-catenin, which is in this context a transcription factor, cannot be downregulated any more. As a consequence target genes of β-catenin are expressed in an unregulated manner. These target genes regulate features of stem cell biology which confer cancer stemness, metastasis, EMT (epithelial-mesenchymal transition), chemoresistance and other characteristics to colorectal tumor cells. Interestingly, APC mutations have only an effect when they occur in the adult stem cells. Thus, the descendend tumor cells show characteristics of these cells and have been termed cancer stem cells (CSCs). Like adult stem cells in the normal crypt CSCs are the origin of cancer and are characterized by an activated - here deregulated - Wnt signaling pathway and thus, by the aforementioned features. Clinically, cancer death is caused in most cases by metastasis which is treated by chemotherapy from which most if not all CRCs escape by the development of chemoresistance which is an intrinsic feature of the CSCs. Therefore, CSC specific targeted therapies might be a promising therapeutic tool for a successful treatment of CRCs. One possibility is the interference of CSC sustaining molecules as these molecules are involved in the induction and maintenance of CSCs. Here, a promising molecule is olfactomedin-4 (OLFM4) which was discussed to be a CSC marker. But the role of OLFM4 as a CSC marker and important factor for tumorigenesis has been controversially described. Therefore, I investigated in the first part of my thesis the role of OLFM4 in CRC cells. I demonstrate that OLFM4 was expressed only in two out of 14 CRC cell lines. The assumption that OLFM4 was only expressed in cells with characteristics of CSCs and thus, was not detected in the cell lines as they possess only a small proportion of CSCs, was not confirmed. I found that CSCs showed a reduced OLFM4 expression and thus, OLFM4 was not coexpressed with other SC markers. These results indicate that OLFM4 is not a marker of CSCs in CRC. In order to analyze the functional role of OLFM4 in CRC cells, I overexpressed OLFM4 lentivirally. However, the overexpression of OLFM4 and thus, high OLFM4 protein levels did not influence the expression of CSC, EMT or differentiation marker. Likewise, OLFM4 did not play a functional role for proliferation, stemness and metastatic features. Therefore, this study demonstrates that OLFM4 is not a CSC marker and has no functional role for the driving activity in the process of colorectal carcinogenesis. Additionally, I evaluated in the second part of my thesis the role of the microRNAome (miRNAome) in colorectal carcinogenesis, the influence on CSC features and whether the miRNAome might be a tool for specific CSC targeted therapies. microRNAs (miRNAs) are generally downregulated in tumors whereby the miRNA loss promotes tumorigenesis. As the majority of the CRC cases are driven by an APC mutation in the SC compartment, I used for my investigations a mouse model with a conditional Apc knockout in CBC cells which develops efficiently intestinal adenomas. This mouse model was crossed with another mouse model harboring a conditional knockout of the essential miRNA generator Dicer1 to investigate the role of a loss of the miRNAome in murine Wnt driven intestinal tumors. In this part of my study I demonstrated that hetero- and homozygous deletion of Dicer1 in CBC cells, in combination with an Apc knockout, enhances significantly the number of adenomas. Moreover, deletion of Dicer1 resulted in smaller adenomas caused by reduced proliferation. Further analysis of DICER1 deletion in human CRC cell lines revealed that loss of DICER1 and thus, miRNAs led likewise to a decreased proliferation. Additionally, I showed that loss of miRNAs increased the expression/protein levels of CSC markers and CSC features indicating that loss of DICER1 promotes tumorigenesis. Moreover, I translated these mouse model/cell culture results into human colonic normal and tumor tissue as well as CRC. In a collection of different tissues (normal tissue, adenomas and cancers of stages I to IV), increased DICER1 levels were seen from normal tissue to adenomas followed by decreased levels during carcinoma progression. Increased levels of DICER1 were also found in the murine Wnt driven adenomas. In support with this I provided finally evidence that DICER1 expression is regulated by the Wnt signaling pathway thus already early in the beginning of the colorectal tumorigenesis. Thus, this data showed that DICER1 is a tumor suppressor in intestinal cancer and the loss of DICER1 and hence, of the miRNAome, influences CSC marker expression and marker protein levels as well as proliferation and CSC features. Therefore, the miRNAome might possibly become a therapeutic target for CSC targeted therapy.