Proteogenomic analysis identifies EGFR as the top candidate protein down-regulated by ZNRF3/RNF43 in cancers.

A. Volcano plot of proteins associated with ZNRF3 mRNA expression in human adrenal cortical carcinoma (ACC), using the TCGA dataset (n = 92).

B. Boxplot of EGFR protein levels in human adrenal cortical carcinomas with different ZNRF3 gene copy number alteration, using the TCGA dataset (n = 92).

C, The top ten proteins negatively correlated with ZNRF3/RNF43 mRNA levels, ranked by Stouffer’s method, using the TCGA colorectal adenocarcinoma (CO/READ) (n = 629) and the CPTAC colon adenocarcinoma (COAD) (n = 110) datasets.

D. Boxplot of EGFR protein levels in human colon adenocarcinomas expressing RNF43 WT or G659Vfs*41 mutant, using the CPTAC dataset.

E. Scatterplot of EGFR protein level versus RNF43 mRNA expression using microsatellite stable (MSS) colon adenocarcinoma in the CPTAC dataset.

F. Bar graph of significant associations between RNF43/ZNRF3 mRNA expression and EGFR protein level in cancer datasets from TCGA PanCancer Altas Study. *, insignificant associations were not shown.

ZNRF3/RNF43 downregulates EGFR protein level.

A. Overexpression of RNF43, ZNRF3, or CBL decreases EGFR protein level compared to GFP control in MDA-MB-231 cells, shown by representative Western blot images (left panel) and quantification results (right panel). Cells were infected with lentivirus expressing GFP, or E3 ligases. Means ± SEMs are shown. p-values were calculated by one-way ANOVA uncorrected Fisher’s LSD test.

B. Znrf3 knockout increases P-EGFR and total EGFR levels in MEFs upon EGF stimulation (50 ng/ml, 10 min).

C. RNF43 knockout increases P-EGFR and total EGFR levels in HT29 cells untreated or treated with recombinant EGF (50 ng/ml, 10 min).

D. RNF43 knockout increases the level of mutated EGFRL858R protein in HT29 cells, as shown by representative Western blot images (left panel) and quantification results (right panel). Means ± SEMs are shown. p-values were calculated by Student’s t-test.

E, F. RSPO2 treatment (50 ng/ml, 2-4 hours) enhances EGFR protein levels in HT29 (E) and LS180 (F) cells.

G. Overexpression of RSPO2 WT or F105A/F109A mutant but R65A/Q70A mutant enhances EGFR protein level, shown by representative Western blot images (left panel) and quantification results (right panel). Means ± SEMs are shown. p-values were calculated by one-way ANOVA uncorrected Fisher’s LSD test. n.s., not significant.

Loss of ZNRF3/RNF43 delays EGFR protein degradation.

A. Knockout of Znrf3 has no impact on Egfr mRNA level in MEFs. Means ± SEMs are shown. p-values were calculated by Welch’s t-test. n.s., not significant.

B. Knockout of RNF43 has no impact on EGFR mRNA level in HT29 cells. Means ± SEMs are shown. p-values were calculated by Welch’s t-test.

C. RNF43 knockout inhibits EGF-induced EGFR protein degradation in HT29 cells. Cells were stimulated with EGF (50 ng/ml) for indicated times. Representative Western blot images (left panel) and quantification results (right panel) were shown. Means ± SEMs are shown. p-values were calculated by two-way ANOVA uncorrected Fisher’s LSD test.

D. Znrf3 knockout increases the cell surface level of EGFR protein in MEFs unstimulated or stimulated with EGF (50 ng/ml, 10 min).

E. RNF43 knockout increases the cell surface level of EGFR protein in HT29 cells unstimulated or stimulated with EGF (50 ng/ml, 10 min). The cell surface EGFR levels were measured by flow cytometry. The bars mark the relative peak shifts after EGF stimulation in WT (black) or KO (red) cells.

ZNRF3/RNF43 enhances EGFR ubiquitination through the RING domain

A. Overexpression of RNF43 enhances EGFR ubiquitination level upon EGF (50 ng/ml) stimulation in MDA-MB-231 cells. CBL serves as a positive control. Cells were co-infected with lentivirus expressing EGFR and GFP, RNF43, or CBL.

B, C. Knockout of RNF43 decreases EGFR ubiquitination in HT29 cells. (B) EGFR ubiquitination was examined by Ub IP followed by EGFR IB; (C) HT29 cells were pretreated with 20 µM MG132 and 100 nM Bafilomycin A1 for 4 hours. EGFR ubiquitination after EGF treatment (50 ng/ml, 30 min) was examined by EGFR IP followed by Ub IB.

D, E. ZNRF3/RNF43 downregulates EGFR protein level through the RING domain. 293T cells were co-transfected with EGFR and Vector, ZNRF3 WT or ΔRING mutant (D), RNF43 WT or ΔRING mutant (E).

F. ZNRF3 regulates EGFR ubiquitination through the RING domain. 293T cells were co-transfected with EGFR and Vector, ZNRF3 WT or ΔRING mutant. EGFR ubiquitination after EGF treatment (50 ng/ml, 10 min) was examined by EGFR IP followed by Ub IB. Cells were pretreated with 20 µM MG132 and 100 nM Bafilomycin A1 for 4 hours.

ZNRF3/RNF43 interacts with EGFR through the extracellular domain

A. Ectopically expressed EGFR is co-immunoprecipitated with Myc-tagged RNF43 and ZNRF3 in MDA-MB-231 cells.

B. Representative images of proximity ligation assay in HCT116 cells co-transfected with EGFR and Myc-ZNRF3ΔRING. Red, PLA signals; Blue, DAPI nuclei staining; Scale bar=20 µm.

C. Schematic diagram of tagged ZNRF3 proteins. SP, signal peptide; FL, full-length; ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain; RING, E3 ligase RING domain.

D. Immunofluorescence staining for ZNRF3 in 293T cells expressing Myc-tagged ZNRF3 FL, ECD-TM, TM-ICD. Scale bar=40 μm.

E. ZNRF3 extracellular domain is required for ZNRF3 interaction with EGFR. 293T cells were co-transfected with EGFR and Myc-tagged ZNRF3 constructs and the lysate amounts were adjusted to achieve comparable levels of EGFR protein in each IP system (Input, left panel). EGFR interaction with ZNRF3 FL, ECD-TM, or TM-ICD were examined by Myc-tag IP followed by EGFR IB (middle panel) or by EGFR IP followed by Myc-tag IB (right panel). *, IgG heavy chain.

ZNRF3/RNF43 inhibits EGFR-mediated cell growth

A. Knockout of Znrf3 enhances MEF cell growth, as measured by cell counting at the indicated time points. p-values were calculated by two-way ANOVA uncorrected Fisher’s LSD test.

B. qPCR analysis for WNT target genes in WT and Znrf3 KO MEFs. Means ± SEMs are shown for this and other graphs. p-values were calculated by Welch’s t-test. n.s., not significant.

C-E. Supplementing RSPO1 promotes Apcmin mouse intestinal tumor organoid growth. The equal number of single cells from Apcmin mouse intestinal tumor organoids were embedded in Matrigel and cultured without or with 10% RSPO1 conditioned medium for 8 days. Representative images (C), and quantification of the size (D) and number (E) of formed Apcmin mouse intestinal tumor organoids are shown. Scale bar = 500 μm. p-values were calculated by Welch’s t-test.

F. Supplementing RSPO1 enhances EGFR protein level in Apcmin mouse intestinal tumor organoids. Representative images (left panel) and quantification (right panel) of EGFR protein level are shown. p-values were calculated by Welch’s t-test.

G. qPCR analysis for Egfr and WNT target genes in Apcmin mouse intestinal tumor organoids cultured with or without RSPO1 supplements. Genes with no significant changes after RSPO1 treatment were plotted in grey, genes significantly down-regulated after RSPO1 treatment were plotted in blue. p-values were calculated by Welch’s t-test. **, p-value < 0.01.

H. Overexpression of ZNRF3 inhibits HT29 cell growth. HT29 cells stably overexpressing GFP or ZNRF3 were seeded in equal numbers and measured by confluence percentage using Incucyte. p-values were calculated by two-way ANOVA uncorrected Fisher’s LSD test.

I. Overexpression of ZNRF3 reduces EGFR protein level in HT29 cells.

J. Erlotinib treatment blocks EGFR phosphorylation in WT and RNF43 KO HT29 cells. Cells were treated with 5 μM erlotinib for 48 hours.

K. Erlotinib treatment inhibits cell growth in RNF43 KO HT29 cells. p-values were calculated by one-way ANOVA uncorrected Fisher’s LSD test.

L. Erlotinib treatment inhibits cell growth in Znrf3 KO MEF cells. Znrf3 KO and WT MEF cells were treated with 0.5 μM erlotinib, or 0.1 μM WNT-C59 or both for 96 hours, and the growth was determined by CCK-8 assay. Means ± SEMs are shown. p-values were calculated by two-way ANOVA, and the significance is presented by compact letter display. Columns marked by the same letter exhibit significant differences.

ZNRF3/RNF43 loss enhances EGFR signaling and promotes tumorigenesis.

A. Overexpression of ZNRF3 suppresses HT29 tumor growth in vivo. Representative bioluminescence images (top panel) and quantification (bottom panel) of flank-injected HT29 cells expressing either GFP or Myc-tagged ZNRF3. p-values were calculated by two-way ANOVA uncorrected Fisher’s LSD test.

B. Overexpression of ZNRF3 inhibits P-EGFR and total EGFR levels in HT29 tumors. Representative images (left panel) and quantification of P-EGFR (right-top panel) and total EGFR (right-bottom panel) protein levels are shown. Means ± SEMs are shown. p-values were calculated by Welch’s t-test (B)

C. Representative H&E images of prostate tissues from WT or prostate-specific Znrf3/Rnf43 knockout mice. Prostate tissue or tumor samples were collected at 1-year-old. Scale bar=300 μm.

D. Total pathological scores of prostate tissues from WT or prostate-specific Znrf3/Rnf43 knockout mice. n=6 mice per group. p-values were calculated by Mann-Whitney.

E. Representative images of immunochemistry staining for EGFR, P-EGFR, and active-β-Catenin in prostate tissues from WT or prostate-specific Znrf3/Rnf43 knockout mice. Scale bar=40 μm.