RIP1 has emerged as a key mediator of survival and cell death in the context of cellular stress and inflammatory signaling. RIP1 has been reported to play an essential role in activation of NF-κB in response to TNF-alpha, TLR3 and DNA damage. Recent studies have elucidated a key role for RIP1 in both apoptotic and necrotic forms of cell death. In general, the prosurvival actions of RIP1 are likely to be mediated largely by activation of NF-κB. Post-translational modifications of RIP1, such as K63-linked polyubiquitination, may determine the nature of RIP1 signaling partners and the biological outcome. Receptor tyrosine kinase (RTK) signaling pathways are of central importance in cancer. Our study suggests that RIP1 play a role as a cell life/death switch in a classical oncogenic RTK signaling pathway in an interaction between EGFRwt and the oncogenic EGFR mutant EGFRvIII.
We propose a model for EGFRvIII-mediated activation of NF-κB that involves recruitment of components of the network that mediates TNFα induced NF-κB activation. Firstly, EGFRvIII recruits the ubiquitin ligases c-IAP1, c-IAP2 and TRAF2 to RIP1 and to EGFRvIII resulting in K63-linked ubiquitination of RIP1. EGFRvIII forms a signaling complex by recruiting RIP1, NEMO and TAK1 to EGFRvIII and also increases the association of RIP1 with NEMO and TAK1. Importantly, RIP1 is required for the recruitment of NEMO to EGFRvIII. An unusual feature of EGFRvIII mediated NF-κB activation is the requirement for the kinase activity of RIP1. Previous studies suggested that RIP1 kinase activity is dispensable for NF-κB activation. However, in a recent study it was shown that Etoposide-induced NF-κB activation occurs in two phases and that RIP1 kinase activity is required for the second phase (Biton and Ashkenazi, 2011
Our study demonstrates that RIP1 is required for EGFRvIII-mediated oncogenicity. Stable silencing of RIP1 in U87MG cells expressing EGFRvIII results in slowed proliferation of cells in culture and importantly, abrogates EGFRvIII mediated tumorigenicity in an in vivo orthotopic model. In our experiment, none of the eight animals with inoculated with RIP1-silenced EGFRvIII cells formed intracranial tumors over an observation period of eight months while all of the animals with control shRNA formed intracranial tumors in about 57 days. Presumably, the lack of EGFRvIII-mediated tumorigenicity results from a loss of the ability of EGFRvIII to activate NF-κB.
An intriguing observation is the unexpected finding that ligand mediated activation of EGFRwt abolishes EGFRvIII-mediated NF-κB activation using RIP1 as a switch. This is unexpected since both EGFRwt and EGFRvIII are known to activate NF-κB, and one would predict a synergistic effect. Synergistic interactions reported previously include EGFRvIII mediated induction of the EGFRwt ligand HB-EGF in an autocrine loop and paracrine effects of EGFRvIII on EGFRwt via an IL-6 mediated pathway (Inda et al., 2010
; Ramnarain et al., 2006
). The antagonistic effect of EGFRwt on EGFRvIII is highly novel and to our knowledge, antagonistic interactions between RTKs in the EGFR family have not been reported previously. EGFRvIII is expressed almost exclusively in tumors with EGFR gene amplification and EGFRwt overexpression. As discussed, immunohistochemical studies examining expression of EGFRwt and EGFRvIII and our data support the co-expression of EGFRwt and EGFRvIII in individual tumor cells. Our current studies show that ligand mediated activation of EGFRwt results in a dissociation of the EGFRvIII associated signaling complex composed of RIP1, NEMO and TAK1 as well as a separation of RIP1 from NEMO and TAK1. This is associated with loss of K63-linked RIP1 ubiquitination and a complete loss of NF-κB activation. We propose that activation of EGFRwt induces conformational changes in EGFRvIII leading to a loss of EGFRvIII signalosome, a hypothesis that will be examined in future studies.
Our study provides evidence that a RIP1 switch operates in EGFR signaling. Firstly, EGFRvIII forms a physical complex with RIP1 and associated signaling proteins, recruits ubiquitin ligases to RIP1 resulting in K63-linked ubiquitination of RIP1 leading to NF-κB activation via a RIP1 dependent pathway. Ligand-mediated activation of EGFRwt results in a dissolution of the EGFRvIII signalosome, leading to a loss of RIP1 association with key signaling proteins, such as NEMO, TAK1, EGFRvIII, deubiquitination of RIP1, and a complete loss of NF-κB activation. Addition of EGF now results in an association of RIP1 with FADD and Caspase-8. Thus, addition of EGF in the presence of EGFRvIII shifts RIP1 towards a cell death role. While exposure to EGF does not result in cell death when cells express a low level of EGFRwt, when EGFRwt is overexpressed to levels similar to those detected in actual GBM tumors, EGF becomes a death signal. This is remarkable since EGF is normally a trophic factor. This cell death has features of both apoptosis and necrosis and it requires the kinase activity of RIP1. Thus, the addition of EGF results in unleashing the RIP1 switch all the way from an EGFRvIII-RIP1-NF-κB oncogenic signal to a RIP1-FADD-Caspase 8 cell death signal. Although it is somewhat counter-intuitive that a growth factor receptor would induce cell death, there are precedents for life/death signaling switches. For example, Caspase-8, traditionally viewed as an apoptotic protein also has a role in survival (Dillon et al., 2012
). Similarly, increased expression of EGFRwt has previously been implicated in growth suppression and apoptosis (Armstrong et al., 1994
; Gill and Lazar, 1981
RIP1 is expressed in most GBMs and is co-expressed with EGFRvIII in tumors showing evidence of NF-κB activation, suggesting that the mechanisms detected in our experimental system may be operational in GBM. Thus, EGFRvIII mediated activation of NF-κB may be a major target for treatment. Since RIP1 mediates EGFRvIII mediated activation of NF-κB, inhibition of RIP1 using either chemical inhibition of silencing approaches could be a potentially important treatment in GBM. In addition, our data suggest that EGFRwt activation could be a mechanism for activating the cell death function of RIP1 in GBM. This is a very novel approach since so far all effort has been focused on inhibiting the EGFR in GBM with limited success (Karpel-Massler et al., 2009
). Furthermore, the availability of endogenous EGFRwt ligand(s) in the vicinity of EGFRvIII expressing GBM cells may influence the oncogenic effects of EGFRvIII by abrogating EGFRvIII mediated NF-κB activation and by diverting RIP1 into a cell death pathway that renders tumor cell populations vulnerable to targeted or conventional therapy.