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.

Chaperones and Ubiquitin Ligases Balance Mutant p53 Protein Stability in Esophageal and Other Digestive Cancers.

The incidence of esophageal adenocarcinoma (EAC) and other gastrointestinal (GI) cancers have risen dramatically, thus defining the oncogenic drivers to develop effective therapies are necessary. Patients with Barrett's Esophagus (BE), have an elevated risk of developing EAC. Around 70%-80% of BE cases that progress to dysplasia and cancer have detectable TP53 mutations. Similarly, in other GI cancers higher rates of TP53 mutation are reported, which provide a significant survival advantage to dysplastic/cancer cells. Targeting molecular chaperones that mediate mutant p53 stability may effectively induce mutant p53 degradation and improve cancer outcomes. Statins can achieve this via disrupting the interaction between mutant p53 and the chaperone DNAJA1, promoting CHIP-mediated degradation of mutant p53, and statins are reported to significantly reduce the risk of BE progression to EAC. However, statins demonstrated sub-optimal efficacy depending on cancer types and TP53 mutation specificity. Besides the well-established role of MDM2 in p53 stability, we reported that individual isoforms of the E3 ubiquitin ligase GRAIL (RNF128) are critical, tissue-specific regulators of mutant p53 stability in BE progression to EAC, and targeting the interaction of mutant p53 with these isoforms may help mitigate EAC development. In this review, we discuss the critical ubiquitin-proteasome and chaperone regulation of mutant p53 stability in EAC and other GI cancers with future insights as to how to affect mutant p53 stability, further noting how the precise p53 mutation may influence the efficacy of treatment strategies and identifying necessary directions for further research in this field.