Pasparakis, Manolis - A 6

The IKK/NF-κB signalling cascade regulates immune and inflammatory responses and cell survival and plays an important role in tumour development. In this project we aim to study the mechanisms of NF-κB function in carcinogenesis focusing particularly on the role of NF-κB signalling in stem cells.

NF-κB is widely recognised as a critical player in cancer. However, studies in mice revealed important but contradictory functions of IKK/NF-κB signaling in different tissues and tumour models. For example, inhibition of NF-κB in the intestinal epithelium protected mice from colon cancer, but NF-κB inhibition in the epidermis was suggested to enhance skin carcinogenesis. Moreover, NF-κB inhibition in hepatocytes inhibited hepatocellular carcinoma (HCC) formation in the Mdr2-/- mouse model of inflammation-driven liver carcinogenesis, but enhanced the development of liver tumours induced by the chemical carcinogen DEN. In addition, mice with liver parenchymal cell (LPC)-specific ablation of NEMO (NEMOLPC-KO) spontaneously developed chronic steatohepatitis and HCC, further supporting a tumor suppressing function of IKK/NF-κB signaling in hepatocytes. Here we studied the mechanisms of NF-κB function in different mouse models of cancer.

To dissect the mechanisms driving hepatitis and HCC development in NEMOLPC-KO mice we focused on hepatocyte death that was identified early on as a key component of the disease. We could show that LPC-specific knockout of FADD protected the NEMO-deficient hepatocytes from death, inhibited liver damage and prevented the development of HCC (Fig. 1) (Ehlken et al, 2014). Interestingly, combined ablation of TNFR1, Fas and TRAILR in liver parenchymal cells did not prevent hepatocyte death, hepatitis and HCC in NEMOLPC-KO mice, suggesting that death receptor independent signaling drives liver cell death, inflammation and cancer in this model. In addition, we found that RIPK3-deficiency did not prevent the development of hepatitis and HCC, showing that necroptosis does not play a critical role in the NEMOLPC-KO mice. In contrast, inhibition of RIPK1 kinase activity, by crossing to knock-in mice expressing kinase inactive RIPK1D138N, inhibited hepatocyte apoptosis and hepatitis and prevented HCC in NEMOLPC-KO mice (Fig. 1) (Kondylis et al, 2015). Therefore, NEMO deficiency sensitizes hepatocytes to death receptor independent, RIPK1/FADD-mediated apoptosis, which causes chronic liver damage culminating in the development of liver tumours.

Earlier studies using IκBα super-repressor mediated NF-κB inhibition in in human keratinocytes promoted RAS-mediated transformation in a xenograft model, suggesting that NF-κB acts as a tumour suppressor in the skin. To address the role of NF-κB in skin cancer we generated and utilized mice with epidermal keratinocyte-specific knockout of RelA/p65 (RelAE-KO mice). RelA deficiency prevented skin cancer induced by a single application of the chemical carcinogen DMBA followed by repeated application of the tumour promoting agent TPA, showing that NF-κB acts as a tumour promoter in keratinocytes. Mechanistic studies showed that RelA deficiency sensitized keratinocytes to DMBA-induced death and inhibited TPA-induced cytokine production (Kim & Pasparakis, 2014). Taken together, our results showed that NF-κB activation in epidermal keratinocytes promotes inflammation-associated tumour development by regulating the survival of cells exposed to DNA damage and the establishment of a tumour-promoting inflammatory microenvironment.

CYLD is a tumour suppressor initially identified as the gene mutated in familial cylindromatosis, a benign form or skin cancer. CYLD is a deubiquitinating enzyme previously suggested to regulate NF-κB and Wnt signalling but also TNFR1-induced cell death. We studied the mechanisms of CYLD function in intestinal and skin carcinogenesis using genetic mouse models. We found that loss of CYLD catalytic activity enhanced chemical carcinogenesis in both the skin and the gut, suggesting that CYLD protects epithelial cells from genotoxic stress induced tumourigenesis. Mechanistically, we found that CYLD catalytic activity is required for optimal stabilisation and activation of p53 by catalysing the removal of ubiquitin chains from p53. Our results showed that CYLD, which efficiently cleaves K63- and M1-linked ubiquitin chains but exhibits little activity against K48-linked chains, removes both K63 and K48 chains from p53. These results suggest that p53 is decorated with mixed or/and branched K63/K48 Ub chains and that CYLD removes K48 chains indirectly by cleaving K63 linkages, resulting in enhanced stabilisation and activation of p53. These studies identified CYLD as a deubiquitinase facilitating DNA damage-induced p53 activation, suggesting that regulation of p53 responses to genotoxic stress contributes to the tumour suppressor function of CYLD (Fernandez-Majada et al, 2016).

Based on our previous findings, we hypothesize that NF-κB may have an important stem cell-specific function in cancer. However, currently existing conditional mouse models are based on DNA recombinases introducing permanent genomic alterations, which are transmitted to the daughter cells. Thus, any mutation introduced in stem cells will be inherited to all differentiated cells derived from the targeted stem cells resulting in the entire tissue carrying the respective mutation. Because of this limitation, DNA recombinase assisted gene targeting methodologies do not allow the analysis of the stem cell-specific role of NF-κB. In this project we aim to study the role of NF-κB in stem cells in vitro using intestinal organoid cultures and in vivo by generating new mouse models allowing the temporally controlled, stem cell-specific manipulation of NF-κB. For this purpose, we have generated mice expressing the tetracycline transactivator (rtTA) by the endogenous Lgr5 gene. In order to manipulate IKK/NF-κB signalling specifically in stem cells, the Lgr5-rtTA mice will be crossed with mice expressing constitutively active IKK2 (IKK2ca), dominant negative IKK2 (IKK2DN) and IκBα super-repressor (IκBαSR) transgenes under the tetracycline-inducible minimal promoter. In these animals we will study the role of NF-κB in stem cells in tissue homeostasis and in tumour development.

Our work has provided new mechanistic insights into the complex function of NF-κB in cancer development. Despite considerable progress, our understanding of the molecular and cellular pathways controlled by NF-κB that are critical for cancer development remain incompletely understood. In the future, we will focus on studying the role of NF-κB signalling in stem cells in cancer development using the new mouse models generated within this project.