Gene expression analysis identifies theActivin/Inhibin signaling pathway as a target ofCSN5/JAB1 in colorectal epithelial cancer cells

CSN5 is a subunit of the constitutive photomorphogenesis (COP9) signalosome (CSN). This multifunctional 600 kDa protein complex functions as a deNEDDylase, regulating SCF E3 ubiquitin ligase mediated ubiquitin/26S proteasome (UPS) dependent protein degradation and signaling platform to control pivotal cellular processes such as protein degradation, signal transduction or the cell cycle. CSN5 harbors the deNEDDylase activity of the CSN, but also has several activities independent of its integration into the CSN complex. For example, it can act as a transcriptional coactivator for various transcription factors (e.g. AP-1, NF-κB, 53BP1 or HAND2). CSN5 also influences the nuclear export and degradation of proteins like p27, p53 or SMAD7. CSN5 also has been implicated in tumorigenesis in several cancer entities. At the same time CSN5 drives cell proliferation and is critical for cell homeostasis. Moreover, prior work in our laboratory demonstrated, that the conditional homozygous deletion of Csn5 in the intestinal epithelium is lethal for mice. It also causes severe changes in the cellular composition of the intestinal epithelium (Schütz, Bernhagen et al., unpublished data and Schütz A. PhD thesis, RTWH Aachen University, 2011). This finding hints towards the importance of CSN5 in the differentiation of cells in the intestinal epithelium. The differentiation of cells is a complex process which is regulated by the switching-on and -off of central genetic programs. The overall aim of my PhD thesis was to investigate the influence of CSN5 on the expression of genes in the intestinal epithelium. As a surrogate for the intestinal epithelium I used the human colorectal adenocarcinoma cell line SW480. Csn5 gene deletion was mimicked by siRNA-mediated gene silencing of CSN5.I showed that the knockdown of CSN5 led to a change in the gene expression profile of SW480 cells. Surprisingly the overall number of strongly regulated genes was unusually low. Only 44 genes showed a significant twofold change in expression while only 6 of them were differentially regulated by an at least three-fold range. These genes were ADP-ribosylation factor-like 14 effector protein-like (ARL14EPL), keratin associated protein 3-1 (KRTAP3-1), late cornified envelope protein 1F (LCE1F), β-actin-like protein 2 (ACTBL2), inhibin βA (INHBA) and glia maturation factor γ (GMFG). In further analysis I demonstrated by gene set enrichment analysis that the knockdown of CSN5 leads to the accumulation of differentially regulated genes in certain cellular processes e.g. KEGG_NOTCH_SIGNALING_PATHWAY, KEGG_TGF_BETA_SIGNALING_ PATHWAY, HALLMARK_WNT_BETA_CATENIN SIGNALING, CELL_CYCLE_PROCESS or REACTOME_CELL_CYCLE. Notably genes of different signaling pathways were negatively influenced by the CSN5 knockdown. These pathways (e.g. Notch, Wnt or TGF-β) have been described to be involved in the orchestration of the differentiation process in the intestinal epithelium and may constitue a link between CSN5 and the differentiation process of the intestinal epithelium. Furthermore, genes assigned to cell cycle processes were found to be influenced by the CSN5 knockdown. Gene onthology analysis revealed that CSN5 negatively influences genes in different processes regarding development or differentiation of a variety of tissues (e.g. epithelium, heart or skeletal system). This may further support the hypothesis that CSN5 plays a role in the regulation of differentiation.Based on these results and on the identification of inhibin βA in the knockdown experiment, I investigated the role of CSN5 in the activin/inhibin signaling pathway. This pathway belongs to the TGF-β superfamily of signaling pathways. There is also emerging evidence in the literature, supporting the hypothesis that this signaling pathway plays a role in the intestinal epithelium. I showed that the CSN5 knockdown influences the expression of different members of the activin/inhibin signaling pathway. Using RT qPCR I could validate that the knockdown of CSN5 led to an increase in inhibin βA expression and a decrease in inhibin α and inhibin βB expression. Despite this the overexpression of CSN5 did not influence the expression of any of the inhibin subunits. The knockdown of the CSN subunits 1 and 2 also led to a decreased inhibin α expression. The inhibin βA expression was also reduced by CSN1 knockdown in contrast to the CSN5 knockdown. Applying Western blot experiments, I could show that the knockdown of CSN5 led to a reduction of all studied CSN subunits (CSN1, 5 and 8). But the knockdown of CSN1 and 2 led to a significantly stronger reduction of CSN1 and 8 protein levels in comparison to CSN5. Thus, I hypothesized that CSN5 as part of the COP9-signalosome influences the inhibin α expression while inhibin βA expression may additionally be influenced by the CSN5 monomer.One of the best-described functions of the CSN is the regulation of cullin RING E3 ubiquitin ligases. These ligases mark proteins for proteasomal degradation. By knocking down cullin1 additionally to CSN5 I could restore inhibin βA expression almost back to normal. While the cullin1 knockdown alone did not influence the inhibin βA expression as measured by RT-qPCR. Inhibin α expression was also decreased by cullin1 knockdown, which even seemed to enhance the effects of CSN5 knockdown. The inhibition of cullin RING E3 ubiquitin ligases by MLN4924 treatment showed the same effect as the cullin1 or CSN5 knockdown for inhibin α. For inhibin βA inhibition by MLN4924 did not alter the effect of the CSN5 knockdown. On the other hand, MLN treatment alone led to a decrease in inhibin βA protein level.Another factor that may influence activin/inhibin subunit expression by CSN5 is the tumor suppressor protein p53. In the colon adenocarcinoma cell line HCT116 harboring a p53 knockout, I could show that CSN5 knockdown led to comparable results regarding inhibin βA expression, as in SW480 cells. It should be noted, that SW480 cells harbor a truncated p53. By contrast, wildtype HCT116 cells expressing normal levels of wildtype p53 showed contrary results. This indicates that p53 may be important for the influence of CSN5 on activin/inhibin subunit expression.On protein level I could not detect any changes in inhibin βA after CSN5 knockdown. Pilot experiments suggest that the possible enhanced protein synthesis may be compensated by enhanced secretion of inhibin βA. This may prevent an intracellular accumulation of inhibin βA.On functional level I could show that the supernatant transfer of CSN5 knockdown cells onto untreated cells results in a decrease of proliferation in the recipient cells. This inhibitory effect seems to be mediated by activins, as it was revertable by follistatin, an inhibitory protein of activins.This thesis provided evidence that CSN5 plays a role in the differentiation of the colon epithelium. This influence seems to be, at least in part, mediated by transcriptional effects of CSN5, as differential gene expression profiles were obtained in colorectal cancer cells upon CSN5 knockdown. In focusing on one of these regulated factors, my thesis showed that CSN5 influences activin/inhibin signaling pathway. This pathway may be a specific link between CSN5 and the homeostasis of the intestinal and especially the colonic epithelium. My results might be a start point, to take a closer look at the role of CSN5 or the COP9-signalosome in intestinal differentiation and homeostasis, as also the cancerogenesis of the intestine