Insulin-like growth factor receptor activity in cancer biology & therapy responses

The Insulin-like Growth Factor 1 Receptor (IGF-1R) has an essential function in normal cell growth and in cancer progression. However, anti-IGF-1R therapies have mostly been withdrawn from clinical trials owing to a lack of efficacy and predictive biomarkers. IGF-1R activity and signalling in cancer cells is regulated by its C-terminal tail, and in particular, by a motif that encompasses tyrosines 1250 and 1251 flanked by serines 1248 and 1252 (1248- SFYYS-1252). Mutation of Y1250/1251 greatly reduces IGF-1-promoted cell migration, interaction with the scaffolding protein RACK1 in the context Integrin signalling, and IGF- 1R kinase activity. Here we investigated the phosphorylation of the SFYYS motif and characterise the conditions under which this motif may be phosphorylated under. As phosphorylated residues, the SFYYS motif may also serve to recruit interacting proteins to the IGF-1R. To this end we identified a novel IGF-1R interacting partner which requires phosphorylated residues in the SFYYS motif to interact with the IGF-1R. This interaction was found to be IGF-1-dependent, and required the scaffold protein RACK1. The interaction of this binding protein with the IGF-1R likely functions to promote maximal phosphorylation of Shc and ERK in IGF-1-stimulated cell migration, and may be important for IGF-1 signalling in cancer cells. Lastly, we have investigated possible kinases that may confer resistance or sensitivity to the IGF-1R kinase inhibitor BMS-754807. In this screen we identified ATR as a mediator of resistance and showed that suppression or chemical inhibition of ATR synergised with BMS-754807 to reduce colony formation. This work has contributes to our understanding of IGF-1R kinase regulation and signalling and suggests that administration of anti-IGF-1R drugs with ATR inhibitors may have therapeutic benefit.

The role of presenilin 1 and presenilin 2 in IL-1β-, LPS- and TNFα- mediated signalling events

The importance of γ-secretase protease activities in development, neurogenesis and the immune system are highlighted by the diversity of its substrates and phenotypic characterization of Presenilin (PS)-deficient transgenic animals. Since the discovery of Amyloid precursor protein (APP) and it’s cleavage by γ-secretase complexes, over 90 other type I membrane proteins have been identified as γ-secretase substrates. We have identified interleukin-1 (IL-1) receptor type I (IL-1R1), toll-like receptor 4 (TLR4) and tumour necrosis factor-α (TNFα) receptor-1 (TNFR1) as novel substrates for - secretase cleavage, which play an important role in innate immunity. In this study, using PS-deficient cells and PS-knockout animal models we examined the role of PS proteins, PS1 and PS2, in IL-1R1-, TLR4- and TNFR1- mediated inflammatory responses. Data presented show that in response to IL- 1β, lipopolysaccharide (LPS) or TNFα, immortalised fibroblasts from PS2- deficient animals have diminished production of specific cytokines and chemokine, with differential reduction in nuclear factor-κB (NF-κB) and (mitogen activated protein kinase) MAPK activities. In contrast, no defect in the response to IL-1β, LPS or TNFα was observed in PS1-deficient immortalised fibroblasts. These observations were confirmed using bone marrow-derived macrophages from PS2-null mice, which also display impaired responsiveness to IL-1β- and LPS, with decreased production of inflammatory cytokines. Furthermore, in whole animal in vivo responses, we show that PS2-deficient animals display ligand (IL-1β, LPS and TNFα)-dependent alterations in the production of specific pro-inflammatory cytokines or chemokines. Importantly, this reduced responsiveness to IL-1β, LPS or TNFα is independent of γ- secretase protease activity and γ-secretase cleavage of TNFR1, IL-1R1 or TLR4. These observations suggest a novel γ-secretase-independent role of PS2 in the regulation of innate immune responsiveness and challenge current concepts regarding the regulation of IL-1β-, LPS- and TNFα-mediated immune signalling.

The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons

Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.

Investigation of p75 neurotrophin receptor and the role of its post-translational modifications in tumour cell motility and invasiveness

The p75 neurotrophin receptor (p75NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners - including the TNF receptor associated factor 6 (TRAF6) E3 ubiquitin ligase - to produce cellular responses such as apoptosis, survival, and inhibition of neurite outgrowth. Recently,p75NTR was also shown to undergo γ-secretase-mediated regulated intramembrane proteolysis, and the receptor ICD was found to migrate to the nucleus where it regulates gene transcription. Moreover, γ-secretase-mediated proteolysis was shown to be involved in glioblastoma cell migration and invasion. In this study we report that TRAF6-mediated K63-linked polyubiquitination at multiple or alternative lysine residues influences p75NTR-ICD stability in vitro. In addition, we found that TRAF6-mediated ubiquitination of p75NTR is not influenced by inhibition of dynamin. Moreover, we report beta-transducin repeats-containing protein (β-TrCP) as a novel E3- ligase that ubiquitinates p75NTR, which is independent of serine phosphorylation of the p75NTR destruction motif. In contrast to its influence on other substrates, co-expression of β-TrCP did not reduce p75NTR stability. We created U87-MG glioblastoma cell lines stably expressing wild type, γ-secretaseresistant and constitutively cleaved receptor, as well as the ICD-stabilized mutant K301R. Interestingly, only wild-type p75NTR induces increased glioblastoma cell migration, which could be reversed by application of γ-secretase inhibitor. Microarray and qRT-PCR analysis of mRNA transcripts in these cell lines yielded several promising genes that might be involved in glioblastoma cell migration and invasion, such as cadherin 11 and matrix metalloproteinase 12. Analysis of potential transcription factor binding sites revealed that transcription of these genes might be regulated by well known p75NTR signalling cascades such as NF-κB or JNK signalling, which are independent of γ-secretase-mediated cleavage of the receptor. In contrast, while p75NTR overexpression was confirmed in melanoma cell lines and a patient sample of melanoma metastasis to the brain, inhibition of γ-secretase did not influence melanoma cell migration. Collectively, this study provides several avenues to better understand the physiological importance of posttranslational modifications of p75NTR and the significance of the receptor in glioblastoma cell migration and invasion.