Functional Role of the COP9 Signalosome in Colorectal Cancer and Intestinal Homeostasis:Mechanistic Links to Cancerogenic Signaling Pathways in the Gut

Colorectal cancer is the third most prevalent cancer worldwide and a major cause of cancer-related death. In 2012, nearly 1.4 million new CRC cases were diagnosed. Most CRCs develop from benign polyps in the intestinal epithelium that transform into adenocarcinoma. This malignant transformation is accompanied by a series of genetic alterations named the “adenoma-carcinoma sequence”. In most cases, this sequence is initiated by mutations of APC or β-catenin, which affect the canonical Wnt signaling pathway and lead to β-catenin stabilization and to the activation of oncogenes. In addition, the COP9 signalosome subunit CSN5 was found to be overexpressed in the majority of colorectal adenocarcinomas. Investigating the possible link between β-catenin and CSN5 in CRC, our group recently discovered that CSN5 knockdown lowers β-catenin protein levels in the CRC cell line SW480. Further experiments showed that the knockdown of CSN5 decreased cell proliferation and apoptosis in CRC cells, likely as a result of inhibited Wnt signaling.The aim of the first part of this thesis was to identify the mechanism that underlies CSN5’s influence on β-catenin protein levels in CRC cells. Since central key proteins of the canonical Wnt signaling pathway are mutated and /or autonomous in CRC cells, an alternative β-catenin degradation pathway mediated by the ubiquitin E3 ligase SIAH-1 was investigated in HCT116 cells. CSN5 knockdown affected active β-catenin (ABC) levels and the expression of different β-catenin/Wnt target genes in HCT116 cells. Moreover, SIAH-1 knockdown also reduced β-catenin protein levels and β-catenin/Wnt target gene expression in this cell line, while SIAH-1 overexpression promoted β-catenin protein levels. A combined knockdown of SIAH-1 and CSN5 revealed that SIAH-1 might participate in CSN5’s effect on β-catenin/Wnt target gene expression, as the CSN5 knockdown effect was abolished in this case. Co-IP studies indicated that β-catenin, SIAH-1 and CSN5 proteins interact with each other in HCT116 cells and in vitro pulldown assays pointed to indirect interactions of these three proteins in a large protein complex. Furthermore, knockdown and overexpression approaches showed that CSN5 promotes SIAH-1 expression on mRNA and protein level. However, CSN5 also supported the degradation of SIAH-1 protein in short-term cycloheximide inhibition assays. Studies using the cullin RING ligase inhibitor MLN-4924, which reduces cullin NEDDylation, and a CUL-1/SIAH-1 Co-IP indicated that SIAH-1 might be a target of a CUL-1-based and thus CSN-regulated CRL. cDNA microarray analysis of SW480 cells following CSN5 knockdown and subsequent verification experiments using RT-qPCR revealed that CSN5 might regulate the expression of genes directly linked to the canonical Wnt signaling pathway. The mRNA levels of the Wnt receptor antagonist DKK1 were upregulated upon CSN5 knockdown, while the gene expression of DKK4 was downregulated when CSN5 levels were silenced. Furthermore, the expression of the Wnt ligand WNT6 was decreased upon CSN5 knockdown. Interestingly, the CSN5 knockdown effect on DKK1 was also observed in HCT116 cells and was inverted in CSN5-overexpressing SW480 cells. In addition, the CSN5 knockdown enhanced DKK1 protein levels and the secretion of DKK1 by Caco-2 and SW480 cells. Cell culture experiments using supernatant transfer and recombinant DKK1 treatment showed that the DKK1 concentrations secreted by cells subjected to a CSN5 knockdown are sufficient to inhibit canonical Wnt signaling in SW480 cells. These data clearly suggest that CSN5’s effect on β-catenin levels in CRC cells may be mediated by DKK1. Also, investigating the mechanism underlying CSN5’s impact on DKK1 mRNA expression, it became obvious that the YAP/TAZ transcription factors are not involved. Finally, MLN-4924 assays supported the hypothesis that DKK1 expression might be controlled by a repressor, which may be targeted by a CRL.Previous experiments of our group showed that the conditional and tissue-specific knockout of Csn5 in the mouse gut leads to the death of mice within two weeks. Within this time, a marked goblet cell hyperplasia occurred that was followed by the complete loss of the intestinal epithelium. As the differentiation of epithelial cells vitally depends on the Notch signaling pathway, the potential connection between CSN5, Notch signaling and epithelial differentiation was investigated in the second part of this thesis.CSN5 knockdown was found to decrease the levels of Notch intracellular domain (NICD), the transcriptional activator of Notch target gene expression, in CRC cells. This promoted the transcription of ATOH-1 and DLL-1, two genes that are repressed by Notch signaling and known as markers for goblet cells. In a next step, an ex vivo small intestine organoid approach was used to further investigate Csn’s functional role in mouse gut. Induction of a homozygous Csn5 or Csn8 knockout resulted in the death of organoids within four days, whereas a heterozygous knockout of these Csn subunits did not apparently affect the organoids. Interestingly, only the heterozygous knockout of Csn5 or Csn8 resulted in enhanced mRNA expression of Atoh-1 and Dll-1, supporting the hypothesis of a shift of differentiation towards secretory cell types. Of note, the enterocyte marker ‘alkaline phosphatase’ was reduced in Csn subunit knockout organoids and the homozygous Csn5 knockout seemed to increase the number of enteroendocrine cells as evidenced by IHC staining. In contrast to my prediction, no goblet cell hyperplasia became visible in the knockout organoids, pointing to the importance of other cell types such as immune cells for epithelial cell differentiation in vivo. Organoid death observed following homozygous knockout of Csn5 or Csn8 was accompanied by an increased degree of apoptosis and a reduced proliferation rate. In addition, the homozygous knockout of the Csn subunits resulted in nuclear accumulation of β-catenin and increased Atoh-1 protein levels as measured by IHC, although it was not possible to confirm these results by immunoblotting. These results indicate that the integrity of the Csn complex is crucial for intestinal epithelium viability.Taken together, this work revealed that the COP9 signalosome is a pivotal regulator of central signaling pathways mediating colorectal cancer development and intestinal homeostasis