The role of LUBAC in tissue homeostasis and cancer

• Main Author: Taraborrelli, L.
• Published: University College London (University of London) 2017
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.747097

Linear ubiquitination is a recently discovered type of ubiquitination crucial for the regulation of different immune signalling pathways and cell death. Linear ubiquitin linkages are generated by the linear ubiquitin chain assembly complex (LUBAC), the only E3 ligase identified so far capable of catalysing these linkages de novo under physiological conditions. LUBAC is composed of three proteins: HOIL-1, SHARPIN and HOIP, with HOIP being the catalytic component. SHARPIN-deficient mice (cpdm) develop multi-organ inflammation due to excessive tumour necrosis factor (TNF)-mediated cell death. To better understand the mechanisms of how LUBAC regulates cell death and inflammation in vivo, the physiological role of different LUBAC components in tissue homeostasis was investigated. Here, I report that HOIP deficiency in mice results in embryonic lethality at mid-gestation due to excessive endothelial cell death and disruption of the yolk sac vasculature, implying that linear ubiquitination is crucial for developmental processes. Secondly, I present that the skin inflammation characteristic of cpdm mice is attenuated by TLR3 deficiency, indicating that TLR3 promotes the cpdm morbidity. Thirdly, in this thesis I illustrate the in-vivo effects of HOIP and HOIL-1 deficiency in murine keratinocytes (HoipE-KO and Hoil-1E-KO mice). HoipE-KO and Hoil-1E-KO mice display severe dermatitis and die around four to six days after birth. The skin of HoipE-KO and Hoil-1E-KO mice is characterised by increased cell death followed by inflammation, hyperproliferation and keratinocyte differentiation defects. Keratinocyte-specific deletion of HOIP in adult mice also results in excessive cell death, inflammation and skin structure disruption, indicating that LUBAC is essential to regulate skin homeostasis in young and adult mice. TNFR1 co-deletion rescues HoipE-KO and Hoil-1E-KO mice from postnatal lethality. However, in adulthood, Tnfr1KO;HoipE-KO and Tnfr1KO;Hoil-1E-KO mice develop inflammatory skin disease which is also characterised by increased cell death. To genetically dissect the modalities of cell death implicated in these models RIPK3/MLKL-mediated necroptosis and Caspase-8-mediated apoptosis were abrogated in these mice. Whereas genetic deletion of RIPK3 or MLKL does not affect the phenotype of HoipE-KO and Hoil-1E-KO mice, co-deletion of RIPK3 or MLKL with Caspase-8 does not only rescue these mice from early lethality and skin inflammation, but also from the late inflammation and lethality that occurs in Tnfr1KO;HoipE-KO and Tnfr1KO;Hoil-1E-KO mice. Hence, whilst RIPK3/MLKL-mediated necroptosis does not contribute to the phenotype of HoipE-KO and Hoil-1E-KO mice, Caspase-8-mediated apoptosis is the main driver of the lethal inflammation. The in-depth genetic analysis is supported by in-vitro experiments showing that HOIP-deficient primary murine keratinocytes (PMKs) display decreased cell viability which is rescued by inhibition of caspases and, importantly, also by inhibition of the kinase activity of RIPK1, but not that of RIPK3. Collectively, these results show that absence of HOIP or HOIL-1 in keratinocytes leads to excessive TNFR1-dependent and -independent apoptosis which causes lethal inflammation in mice. Finally, I also report the implication of LUBAC in lung cancer. HOIP or HOIL-1 deficiency in A549 cells drastically decreases TRAIL- and TNF-induced gene-activatory signalling and cytokine production. In a KRAS- and p53-mutated mouse model of NSCLC, absence of HOIP significantly improves the survival of these tumour-bearing mice, suggesting that LUBAC has pro-tumorigenic functions. Collectively, the work presented in this thesis identifies a crucial role of LUBAC in embryonic development, skin homeostasis and lung cancer progression. Whilst in physiological conditions linear ubiquitination is essential to prevent cell death thereby allowing embryogenesis and maintaining tissue homeostasis, in a cancer scenario it is likely to promote tumour progression. Therefore, this work determines a vital role for linear ubiquitination in controlling cell death and inflammation in embryogenesis, tissue homeostasis and cancer.