The role of E3 ubiquitin ligases in the development and progression of glioblastoma

Abstract

Despite recent advances in our understanding of the disease, glioblastoma (GB) continues to have limited treatment options and carries a dismal prognosis for patients. Efforts to stratify this heterogeneous malignancy using molecular classifiers identified frequent alterations in targetable proteins belonging to several pathways including the receptor tyrosine kinase (RTK) and mitogen-activated protein kinase (MAPK) signalling pathways. However, these findings have failed to improve clinical outcomes for patients. In almost all cases, GB becomes refractory to standard-of-care therapy, and recent evidence suggests that disease recurrence may be associated with a subpopulation of cells known as glioma stem cells (GSCs). Therefore, there remains a significant unmet need for novel therapeutic strategies. E3 ubiquitin ligases are a family of >700 proteins that conjugate ubiquitin to target proteins, resulting in an array of cellular responses, including DNA repair, pro-survival signalling and protein degradation. Ubiquitin modifications on target proteins are diverse, ranging from mono-ubiquitination through to the formation of polyubiquitin chains and mixed chains. The specificity in substrate tagging and chain elongation is dictated by E3 ubiquitin ligases, which have essential regulatory roles in multiple aspects of brain cancer pathogenesis. In this review, we begin by briefly summarising the histological and molecular classification of GB. We comprehensively describe the roles of E3 ubiquitin ligases in RTK and MAPK, as well as other, commonly altered, oncogenic and tumour suppressive signalling pathways in GB. We also describe the role of E3 ligases in maintaining glioma stem cell populations and their function in promoting resistance to ionizing radiation (IR) and chemotherapy. Finally, we consider how our knowledge of E3 ligase biology may be used for future therapeutic interventions in GB, including the use of blood-brain barrier permeable proteolysis targeting chimeras (PROTACs).

Fig. 1. The ubiquitin system and families of E3 ubiquitin ligases.

a Ubiquitin is ligated to proteins via a cascade involving E1, E2 and E3 ubiquitin ligases. E3s attach ubiquitin via specific lysine residues, which determine protein fate following ubiquitin conjugation. Ligation is carried out by a family of four distinct E3 ligases (b).

Fig. 2. The role of E3 ubiquitin ligases in RTK signalling in GB.

RTKs are transmembrane receptors containing extracellular, transmembrane and intracellular portions. The extracellular domain interfaces with the extracellular milieu allowing expressing cells to react and adapt to extracellular signals. Upon binding their cognate ligands, RTKs undergo autophosphorylation leading to downstream signalling [156]. In this way, RTKs transduce extracellular cues as signals into a cell. EGF signalling is a prototypical example of altered RTK signalling in GB. Activation of the receptor leads to phosphorylation of c-terminal residue tyrosines, facilitating the docking of Src homology (SH) domain-containing proteins and activation of several pathways, including the MAPK pathway, signal transducers and activators of transcription (STAT) signalling and Src-dependent phosphoinositide 3-kinase (PI3K)/Akt signalling [157]. At the membrane, E3s such as c-CBL can decrease oncogenic signalling by promoting EGFR ubiquitylation and turnover. RTK signalling converges on several pathways including the phosphoinositide 3-kinase (PI3K)/Akt pathway. Activation-dependent phosphorylation of RTKs (such as EGFR) facilitates the binding of class 1a PI3K via SH2 domains. This leads to the activation of PI3K and subsequent phosphorylation of phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 4,5-triphosphate (PIP₃) [158]. Via pleckstrin homology (PH) domains, Akt binds PIP₃ where it is phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK-1). An important negative regulator of this pathway is the phosphatase and tensin homologue (PTEN), a protein whose gene is mutated/deleted in 41% of GB cases. Akt signalling can also be modulated via negative feedback loops with phosphatases including PH domain leucine-rich repeat protein phosphatase (PHLPP) [159, 160]. The MAPK pathway is an important downstream signalling pathway activated by RTKs. As  one of the most commonly mutated pathways in human cancer, it translates  extracellular signals into cellular phenotypes such as proliferation, differentiation, migration and invasion [161]. Following RTK activation, son of sevenless (Sos) is recruited to the plasma membrane via interaction with Grb2. Sos is a guanine nucleotide exchange factor (GEF) which promotes the activation of membrane-bound RAS via binding of guanosine triphosphate (GTP) with Ras. Active Ras can then promote multiple oncogenic cellular responses. In contrast, GTPase-activating proteins (GAPs) accelerate Ras-mediated GTP hydrolysis and act as negative regulators of Ras signalling and are therefore tumour suppressors [162]. Red icons represent E3 ligases and their functions as observed in GB models. Where specific substrates are yet to be identified, E3 ligases appear as pink icons.

Fig. 3. The role of E3 ubiquitin ligases in maintaining GSCs.

GSCs are a population of de-deferentiated cells responsible for treatment failure and GB propagation. E3 ligases directly affect transcriptional programmes that mediate the differentiation of GSCs through direct interaction with or alteration of the stability of transcription factors involved in the stem cell transcriptional programme. Red icons represent E3 ligase functions shown directly in GSC/differentiated cell models. Pink icons represent reduced E3 ligase expression/activity. Orange coloured icons represent E3 ligase functions identified in neural stem cell or GB models.

Fig. 4. The role of E3 ubiquitin ligases in GB cell death.

The goal of therapeutic intervention in cancer is to kill or remove tumour cell populations. At the receptor level, E3 ligases form multi-component complexes to moderate cell death signalling from death receptors such as TRAIL-R2. In addition, similar complexes can moderate dichotomous outcomes (death or survival) from TNFR1 and EGFRvIII receptor activation. Downstream of this, E3 ligases also moderate DNA damage responses and intrinsic apoptosis activation. Red icons represent E3 ligases and their functions as described in GB models.