GRAIL1 stabilizes misfolded mutant p53 through a ubiquitin ligase-independent, chaperone regulatory function.

Frequent (>70%) TP53 mutations often promote its protein stabilization, driving esophageal adenocarcinoma (EAC) development linked to poor survival and therapy resistance. We previously reported that during Barrett's (BE) progression to EAC, an isoform switch occurs in the E3 ubiquitin ligase RNF128 (aka GRAIL - gene related to anergy in lymphocytes), enriching isoform 1 (hereby GRAIL1) and, stabilizing the mutant p53 protein. Consequently, GRAIL1 knockdown degrades mutant p53. But how GRAIL1 stabilizes the mutant p53 protein remains unclear. In search for a mechanism, here we performed biochemical and cell biology studies to identify that GRAIL has a binding domain (315-PMCKCDILKA-325) for Hsp40/DNAJ. This interaction can influence DNAJ chaperone activity to modulate misfolded mutant p53 stability. As predicted, either the overexpression of a GRAIL fragment (Frag-J) encompassing the DNAJ binding domain, or a cell permeable peptide (Pep-J) encoding the above 10 amino acids, can bind and inhibit DNAJ-Hsp70 co-chaperone activity thus degrading misfolded mutant p53. Consequently, either Frag-J or Pep-J can reduce the survival of mutant p53 containing dysplastic BE and EAC cells and inhibit growth of patient-derived dysplastic BE organoids (PDOs) in 3D cultures. The misfolded mutant p53 targeting and growth inhibitory effects of Pep-J is comparable to simvastatin, a cholesterol lowering drug, that can degrade misfolded mutant p53 also via inhibiting DNAJA1, although by a distinct mechanism. Implications: We identified a novel ubiquitin ligase independent, chaperone regulating domain in GRAIL and further synthesized a first-in-class novel misfolded mutant p53 degrading peptide having future translational potential.

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Isoform alterations in the ubiquitination machinery impacting gastrointestinal malignancies.

The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.

LATS1 Regulates Mixed-Lineage Kinase 3 (MLK3) Subcellular Localization and MLK3-Mediated Invasion in Ovarian Epithelial Cells.

Mixed-lineage kinase 3 (MLK3) activates mammalian mitogen-activated protein kinase (MAPK) signaling pathways in response to cytokines and stress stimuli. MLK3 is important for proliferation, migration, and invasion of different types of human tumor cells. We observed that endogenous MLK3 was localized to both the cytoplasm and the nucleus in immortalized ovarian epithelial (T80) and ovarian cancer cells, and mutation of arginines 474 and 475 within a putative MLK3 nuclear localization sequence (NLS) resulted in exclusion of MLK3 from the nucleus. The large tumor suppressor (LATS) Ser/Thr kinase regulates cell proliferation, morphology, apoptosis, and mitotic exit in response to cell-cell contact. RNA interference (RNAi)-mediated knockdown of LATS1 increased nuclear, endogenous MLK3 in T80 cells. LATS1 phosphorylated MLK3 on Thr477, which is within the putative NLS, and LATS1 expression enhanced the association between MLK3 and the adapter protein 14-3-3ζ. Thr477 is essential for MLK3-14-3-3ζ association and MLK3 retention in the cytoplasm, and a T477A MLK3 mutant had predominantly nuclear localization and significantly increased invasiveness of SKOV3 ovarian cancer cells. This study identified a novel link between the MAPK and Hippo/LATS1 signaling pathways. Our results reveal LATS1 as a novel regulator of MLK3 that controls MLK3 nuclear/cytoplasmic localization and MLK3-dependent ovarian cancer cell invasion.

Osmotic and heat stress-dependent regulation of MLK4ß and MLK3 by the CHIP E3 ligase in ovarian cancer cells.

Mixed Lineage Kinase 3 (MLK3), a member of the MLK subfamily of protein kinases, is a mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) that activates MAPK signalling pathways and regulates cellular responses such as proliferation, invasion and apoptosis. MLK4ß, another member of the MLK subfamily, is less extensively studied, and the regulation of MLK4ß by stress stimuli is not known. In this study, the regulation of MLK4ß and MLK3 by osmotic stress, thermostress and heat shock protein 90 (Hsp90) inhibition was investigated in ovarian cancer cells. MLK3 and MLK4ß protein levels declined under conditions of prolonged osmotic stress, heat stress or exposure to the Hsp90 inhibitor geldanamycin (GA); and MLK3 protein declined faster than MLK4ß. Similar to MLK3, the reduction in MLK4ß protein in cells exposed to heat or osmotic stresses occurred via a mechanism that involves the E3 ligase, carboxy-terminus of Hsc70-interacting protein (CHIP). Both heat shock protein 70 (Hsp70) and CHIP overexpression led to polyubiquitination and a decrease in endogenous MLK4ß protein, and MLK4ß was ubiquitinated by CHIP in vitro. In untreated cells and cells exposed to osmotic and heat stresses for short time periods, small interfering RNA (siRNA) knockdown of MLK4ß elevated the levels of activated MLK3, c-Jun N-terminal kinase (JNK) and p38 MAPKs. Furthermore, MLK3 binds to MLK4ß, and this association is regulated by osmotic stress. These results suggest that in the early response to stressful stimuli, MLK4ß-MLK3 binding is important for regulating MLK3 activity and MAPK signalling, and after prolonged periods of stress exposure, MLK4ß and MLK3 proteins decline via CHIP-dependent degradation. These findings provide insight into how heat and osmotic stresses regulate MLK4ß and MLK3, and reveal an important function for MLK4ß in modulating MLK3 activity in stress responses.