UBCH5 Family Members Differentially Impact Stabilization of Mutant p53 via RNF128 Iso1 During Barrett's Progression to Esophageal Adenocarcinoma

Abstract

Background & aims: TP53 mutations underlie Barrett's esophagus (BE) progression to dysplasia and cancer. During BE progression, the ubiquitin ligase (E3) RNF128/GRAIL switches expression from isoform 2 (Iso2) to Iso1, stabilizing mutant p53. However, the ubiquitin-conjugating enzyme (E2) that partners with Iso1 to stabilize mutant p53 is unknown.

Methods: Single-cell RNA sequencing of paired normal esophagus and BE tissues identified candidate E2s, further investigated in expression data from BE to esophageal adenocarcinoma (EAC) progression samples. Biochemical and cellular studies helped clarify the role of RNF128-E2 on mutant p53 stability.

Results: The UBE2D family member 2D3 (UBCH5C) is the most abundant E2 in normal esophagus. However, during BE to EAC progression, loss of UBE2D3 copy number and reduced expression of RNF128 Iso2 were noted, 2 known p53 degraders. In contrast, expression of UBE2D1 (UBCH5A) and RNF128 Iso1 in dysplastic BE and EAC forms an inactive E2-E3 complex, stabilizing mutant p53. To destabilize mutant p53, we targeted RNF128 Iso1 either by mutating asparagine (N48, 59, and 101) residues to block glycosylation to facilitate β-TrCP1-mediated degradation or by mutating proline (P54 and 105) residues to restore p53 polyubiquitinating ability. In addition, either loss of UBCH5A catalytic activity, or disruption of the Iso1-UBCH5A interaction promoted Iso1 loss. Consequently, overexpression of either catalytically dead or Iso1-binding-deficient UBCH5A mutants destabilized Iso1 to degrade mutant p53, thus compromising the clonogenic survival of mutant p53-dependent BE cells.

Conclusions: Loss of RNF128 Iso2-UBCH5C and persistence of the Iso1-UBCH5A complex favors mutant p53 stability to promote BE cell survival. Therefore, targeting of Iso1-UBCH5A may provide a novel therapeutic strategy to prevent BE progression.

Keywords: Barrett’s Esophagus; Esophageal Adenocarcinoma; RNF128-UBCH5 Complex; p53 Protein Stability.

Graphical abstract

Figure 1

Single-cell RNAseq analysis of patient-matched tissue biopsy specimens to examine co-expression patterns for RNF128 and key E2 gene mRNAs across esophageal cell type clusters. (A) Schematic (assistance from BioRender.com) of human sample biopsy specimens of matched normal SE and BE, with (B) tissue size details, (C) representative inverted microscope images, and (D) representative sections of frozen biopsy specimens from matched patient biopsy specimens of BE and SE. (E) A total of 13,332 cells were analyzed using UMAP cluster plots from SE (n = 2) and BE (n = 2) biopsy specimens. A total of 14 distinct molecular clusters were identified, with the top 200 genes per cluster listed in Supplementary Table 2. Using the top gene expression signatures composed of 5–10 characteristic genes expressed in each cluster, we define the cell type identity for each cluster. (F–H) Representative expression of known established markers in the literature for epithelial (CDH1) vs lamina propria (VIM) clusters, TP63 identifies the cell types specific to SE epithelium, and EPCAM and TFF3 are validated markers to identify metaplastic BE cells (cells with zero expression are shown in gray). We observed that cluster 0 is enriched for BE-specific cells vs clusters 1, 2, 3, 4, 5, 7, and 8, which represent SE-specific cell types. (I) RNF128 expression is higher in BE-specific clusters when compared with SE or lamina propria clusters. (J) Extraction and remapping (UMAP) of BE (cluster 0) cell types. (K) Cluster 0 vs cluster 1 identifies SE vs BE cell types. (L) Louvain analysis showing expression levels (color) vs percentage of cells expressing the gene (size of circle) shows that RNF128 is expressed specifically in the BE cluster (cluster 0; with expression of BE markers EPCAM, TFF1, TFF2, and TFF3 vs SE cluster 1; expressing stratification markers KRT4 and KRT15). We observed that E2 family member UBE2D3 is highly expressed vs UBE2D1, which has low expression in BE cluster 0.

Figure 2

Single-cell RNA sequencing of nondysplastic BE to characterize cell types and expression patterns. (A) Patient biopsy specimens (n = 2) characterized at the single-cell level. A total of 7684 cells were analyzed for a final cell count of cell expression of 4293 for UMAP analysis. Clustering analysis showed 12 predictive distinct molecular cell clusters within the BE biopsy specimens. Cell-type classification clusters are characterized using known genes, and genes per cluster can be found in Supplementary Table 3. (B) Using known markers for lamina propria vs epithelial cells, we classified clusters based on epithelial (C0, C2, C3, C5, and C9) vs lamina propria (C4, C6, C7, C8, C10, and C11) (cells with zero expression are shown in gray). (C) Characterization of esophageal SE vs BE cell types using known markers TP63 vs EPCAM, clusters 0 and 2 are SE cells vs clusters 1, 3, 5, and 9, which express columnar-epithelial marker EPCAM (squamous cells do not express EPCAM but are positive for the basal-squamous-esophagus marker TP63). (D and E) Characterization of intestinal-type (MUC2; VIL) (clusters 3, 5, and 9) vs cardia-stomach (CLDN18; TFF2) cluster 1. (F) Quantification of cell distribution from each patient biopsy specimen into each cluster type. (G) Cell-type–specific expression levels of RNF128 (E3-ligase) vs E2-conjugating enzyme genes UBE2D1, UBE2D2, UBE2D3, UBE2E1, and UBE2I. (H) Dot-plot analysis by cluster showing normalized z-score gene expression levels (depicted by the color) vs percentage of cells expressing the gene (dot size represents the proportion of cells in each cluster expressing the marker). BE-CARD, BE cardia-like; BE-INT, intestinal-like; NE, normal (esophageal) epithelium.

Figure 3

RNAseq-based expression analysis of top candidate E2-conjugating enzymes for RNF128. Panels show gene-based RNAseq data extracted from a 65-sample BE progression-related cohort with mRNA from NDBE (n = 6), LGD admixture (10%–100%) without HGD (n = 20), HGD admixture (10%–100%; n = 28), and EAC (n = 11) tissue samples resected from HGD or EAC patients. For (A) UBE2D1, (B) UBE2D3, (C) UBE2E1, and (D) UBE2I horizontal subpanels are as follows. (Ai–Di) Violin-style boxplots, with ANOVA P values for NDBE plus LGD vs HGD or EAC group comparisons. (Aii–Dii) Expression changes in a patient subset (n = 8) with matched LGD and EAC (n = 7) or HGD (n = 1), with paired t test P values for each E2 shown. (Aiii–Diii) RNAseq-based comparison of expression and expression-derived copy number estimates (using recursive median smoothing of genes mapping north and south of target loci chromosomal locations in genome version hg38) for UBE2D1 (10q21.1), UBE2D3 (4q24), UBE2E1 (3p24.2), and UBE2I (16p13.3). Pearson correlations using all 65 samples are shown. (Aiv–Div) shows expression correlations (Pearson, using all 65 samples) to the RNF128 Iso2/Iso1 log₂ ratio. RPKM, Reads Per Kilobase of transcript per Million mapped reads.

Figure 4

Confirmation of UBE2D3 changes in other BE cohorts, and RNF128 to specific E2 copy number correlations in the ESCA TCGA cohort for esophageal cancer. (A and B) Normalized deposited data from 2 GEO EAC cohorts from Kimchi et al (GEO series GSE1420) and Wang et al (GEO series GSE26886), which support the loss of UBE2D3 expression between BE and EAC, as well as the observed high UBE2D3 expression in both SE and BE. Both studies applied histopathology-driven tissue dissection and U133 Affymetrix expression array platforms, but different analytic methodologies. P values represent unequal variances t tests applied between BE and EAC groups in each cohort. (C) Lack of correlated expression between UBE2D1 and UBE2D3 across the 65 RNAseq samples in the BE progression cohort. (D–G) RNAseq by expectation maximization (RSEM) expression vs capped relative linear copy number plots in TCGA EAC samples (n = 87) from ESCA cohort for (D) UBE2D1, (E) UBE2D3, (F) UBE2E1, and (G) UBE2I.

Figure 5

RNF128 Iso1 binds tighter with UBCH5 family members. (A) V5-tagged RNF128 (either Iso1 or Iso2) were co-transfected either with UBCH5A or UBCH5C as indicated in CpA cells. Twenty hours after transfection, cells were treated with proteasomal inhibitor MG132 (2 μmol/L for 4 hours) before harvest and immunoprecipitation followed by immunoblotting using indicated antibodies. (B) To test the interaction ability between RNF128 Iso1 and Iso2 with either WT or catalytic-dead (C85A) mutant UBCH5A, we performed co-transfection, immunoprecipitation, and immunoblot analysis as described earlier using the indicated antibodies. (C) Quantitative reverse-transcription polymerase chain reaction showing exogenous Iso1 or Iso2 transcript expression in CpA and CpD cells along with ACTB (housekeeping) compared with cell lines transfected with empty vector. Cycle threshold (Ct) expression values, rather than ΔΔCt ratios are presented, given the lack of RNF128 construct in the vector controls. Error bars represent the triplicate Ct range for each condition. (D) DDK-tagged UBCH5A (either WT or C85A mutant) and UBCH5C proteins were overexpressed in HEK293 cells, and 24 hours after transfection cell lysates were subjected to immunoprecipitation using FLAG-M2 beads. Thoroughly washed beads then were incubated with equal volumes of in vitro transcribed and translated V5-tagged either RNF128 Iso1 or Iso2. Unlike cells, Iso2 synthesis was approximately 1.9-fold higher compared with Iso1. Compared with input, 100-fold more amounts of Iso1 and Iso2 were used for interaction studies and the percentage binding was calculated as shown after normalization of input band intensity using ImageJ software (National Institutes of Health, Bethesda, MD). Lower panel: Expression of different UBCH5 family members in HEK293 cells. DDK, DYKDDDDK tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HC, Heavy Chain; IP, Immunoprecipitation; LC, Light Chain.

Figure 6

Asparagine (N) glycosylations of Iso1 are important for protein stability, whereas loops at proline residues impact UBCH5 interaction to modulate functionality. (A) Crystal structure of RNF128 Iso1 (modified from Protein Data Bank 3ICU) showing asparagine (N) and proline (P) residues. (B) Steady-state levels of wild-type and glycosylation-deficient mutants of RNF128 (N48A, N59A, N101A, or triple mutant 3NA) in CpA cells (short exposure [SE] and long exposure [LE]). (C) Similar studies as described earlier showing steady-state levels of P54A and P105A mutants compared with WT and 3NA mutants of Iso1. (D) CpA cells were transfected with either WT, 3NA, or P105A Iso1 mutants. Twenty-four hours after transfection, cells were treated with cycloheximide (50 μg/mL) for the indicated times followed by immunoblotting. (E) To calculate protein half-life, relative band intensities were determined from 3 independent experiments using ImageJ (National Institutes of Health), normalized to the amount of protein at the initial time point and plotted (means ± SD). (F) UBCH5A (WT) protein showed weaker interaction with RNF128 Iso1 P54A and P105A mutants compared with WT Iso1. (G) RNF128 Iso1 (WT, 3NA, P54A, and P105A) proteins were synthesized using Transcription and Translation coupled in vitro to transcription and translation systems. In vitro ubiquitination assay of His-tagged p53 (WT) was performed using the earlier translated E3s along with the recombinant UBCH5A. Subsequently, p53 protein was immunoprecipitated using a specific antibody followed by immunoblotting using indicated antibodies. Fold-change of p53 polyubiquitinated species generated were quantified relative to WT Iso1-V5. CHX, Cycloheximide; DDK, DYKDDDDK tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, Immunoblotting; IP, Immunoprecipitation.

Figure 7

UBCH5A catalytic activity is important in maintaining RNF128 Iso1 stability by protecting it from β-TrCP1–mediated degradation. (A) RNF128 Iso1 and Iso2 steady-state levels were determined in CpA after co-overexpression of either UBCH5A WT or C85A mutants. (B) To determine the effect of UBCH5A catalytic activity on the stability of Iso1, CpA cells overexpressing the indicated proteins were either left untreated or treated with MG132 followed by immunoblotting using the indicated antibodies. (C) Iso1 and Iso2 either WT or β-TrCP1 recognizing phosphodegron-deficient (SA) mutants were overexpressed in multiple cell lines (CpA, CpD, HepG2, and HEK293). Twenty-four hours after transfection, cell lysates were subjected to immunoblotting using the indicated antibodies. (D) Different Iso1 mutants (WT or SA mutant) were co-overexpressed in CpA cells either with WT, C85A, or K144R mutant UBCH5A. Cell lysates were prepared 24 hours after transfection followed by immunoblotting. (E) Interaction of RNF128 Iso1 (WT and SA mutant) with β-TrCP1 in the presence of UBCH5A (WT and C85A mutants). Twenty hours after transfection cells were treated with MG132 for 4 hours and cell lysates were subjected to immunoprecipitation followed by immunoprecipitation as indicated. DDK, DYKDDDDK tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsc70, Heat shock cognate 71 kDa protein; IP, Immunoprecipitation.

Figure 8

Targeting of RNF128 Iso1 via alteration of UBCH5A catalytic activity to destabilize mutant p53. (A) Upper left panel: Table showing UBE2D family member genes (UBE2D1–4), protein (UBCH5A–D) and their respective chromosomal locations. Upper right panel: Percentage sequence homology between UBCH5 family members. Lower panel: Pairwise amino acid sequence alignments between UBCH5A–D isoforms performed using the CLASTALW multiple sequence alignment tool. Redboxes identify 61PF62, C85. and 95PA96 residues as discussed in the article. (B) The RNF128 Iso1-V5 constructs were co-expressed with the indicated UBCH5A mutants (WT, P61A/G, and P95A/G). Twenty hours after transfection, cells were treated with MG132 as described earlier, followed by immunoprecipitation and immunoblotting as indicated. (C) Similar co-transfection was conducted in CpA cells as described earlier and cell lysates were subjected to immunoblotting with the indicated antibodies. (D) CpA (left panel) and HEK293 (right panel) cells overexpressing P53^R175H-DDK mutant were co-transfected with different UBCH5A mutants as indicated and 48 hours after transfection mutant p53 levels were determined using immunoblotting as indicated. (E) As described earlier, p53R248Q and p53R273H mutants showed down-regulation upon overexpression of P61G and P95A mutant UBCH5A in CpD (upper panel) and HEK293 cells (lower panel). (F) UBCH5A (WT, P61G, P95A, K144R, and C85A mutants) were overexpressed in CpD, and 48 hours after transfection cells were plated at clonal density to perform a clonogenic assay. Survival fractions upon UBCH5A overexpression are shown in the top panel and the corresponding cell lysates were subjected to immunoblot analysis using the indicated antibodies. DDK, DYKDDDDK tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, Immunoprecipitation.