Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7

Abstract

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Keywords: EGFR; FBXW7; colorectal cancer; organoids.

Fig. 1.

Establishment of FBXW7 mutant colon organoids. (A) Bar graph displaying the incidence of FBXW7 mutations across different tumor types with at least 500 cases (systematic analysis of all cases reported in Cell BioPortal). (B) Descriptive flowchart of the experimental setup. (C) DNA traces of hotspot mutations introduced in colon organoids; the original aa sequence of the locus is reported above the DNA traces while the changed aa is reported below. PAM and sgRNA sequences are reported in red and blue, respectively, the red box indicates the desired mutation. (D) Representative bright-field and histological pictures of FBXW7-WT and mutant organoids. (Scale bar: 100 µm.) (E) Western blot showing the expression of CCNE1.

Fig. 2.

FBXW7 mutations reduce the Egf dependency in human colon organoids. (A) Representative bright-field pictures of FBXW7 WT and mutant colon organoids cultured in the presence of different concentrations of Egf. (Scale bar: 100 µm.) (B) Bar graph showing the expansion potential of the organoids in different Egf concentrations (arrows indicate continued growth, x indicates the absence of growth). (C) Representative pictures of Ki67 staining on FBXW7 WT and mutant organoids cultured in high (50 ng/mL) and low Egf (0.5 ng/mL). The Insets show higher magnification of cell nuclei. (Scale bar: 100 µm.) (D) Bar graph summarizing the ki67 quantification in high and low Egf, n = 10, ** stands for P-value < 0.01, ***P-value < 0.001 vs. WT in low Egf; (E) Heatmap showing the expression of MAPK target genes between WT and mutant organoids. Genes have been extracted from the differentially regulated ones showing a Log2 FC > 1.5. Colors range from low (yellow) to high (dark blue) reads count. (F) GSEA showing increased activity of proliferative E2F target genes in FBXW7 mutant organoids.

Fig. 3.

Impact of FBXW7 mutations on EGFR. (A and B) Western blot showing differential expression of EGFR (A) and CCNE1 (B) in FBXW7 WT and mutant organoids cultured in the presence of 0.5 ng/mL of Egf and after it has been removed for 24 h and 48 h. (C) Whole-mount staining for EGFR and KI67. (Scale bar: 100 µm.) (D) FACS plot showing increased cell membrane localization of EGFR in FBXW7 WT and mutant organoids, n = 2. (E) Bar graph reporting the quantification of EGF intensity at the cell membrane in different FBXW7 mutant as measured by FACS with a Cetuximab-647 antibody, n = 2. (F and G) Bright-field pictures of WT and mutant organoids cultured in the presence of different ERBBs activators, scale bar 100 µm (F) and quantification of their ability to propagate in vitro in the presence of the indicated growth factor (G) (arrows indicate continued growth, x indicate the absence of growth). (H) Volcano plot highlighting the enrichment of phosphorylated proteins between WT and FBXW7 mutant organoids, n = 3.

Fig. 4.

EGFR contains two phosphodegron sites. (A) Alignment of the EGFR sequence among orthologs showing the conserved presence of multiple phosphodegron-like motifs. (B) DNA traces of T693A mutation; the original aa sequence of the locus is reported above the DNA traces while the changed aa is reported below. PAM and sgRNA sequences are reported in red and blue, respectively. (C) Whole-mount staining of EGFR in EGFR WT and mutant organoids. (Scale bar: 100 µm.) (D) Western blot showing EGFR expression in WT and EGFR mutant organoids. (E) FACS plot showing the expression of membrane-bound EGFR in WT and phosphodegron mutant organoids. (F and G) Bright-field pictures (F) and quantification (G) of the clonal outgrowth of WT and phosphodegron mutant organoids in limiting Egf concentrations, * stands for P-value < 0.05. (H) Bar graph showing the long-term culture potential of WT, FBXW7 and EGFR degron mutant organoids (arrows indicate continued growth, and x indicates the absence of growth), results from two individual clones are represented in the bar graph.

Fig. 5.

FBXW7 mutations reduce the sensitivity toward anti-EGFR therapies. (A) Bright-field pictures of FBXW7 WT and mutant organoids treated with decreasing concentrations of Cetuximab. (Scale bar: 500 µm.) (B) Dose–response curve indicating the Cetuximab sensitivity of different organoid lines. (C) Heatmap indicating the IC50s of Cetuximab on FBXW7 WT and mutant lines. (D) Dose–response curves indicating the sensitivity to Cetuximab of FBXW7 WT and mutant CRC-derived organoids. (E) Heatmap indicating the IC50s of different inhibitors tested on CRC-derived FBXW7 WT and mutant lines, color range from low (blue) to high (red) IC50. (F) Bar graph showing the number of days required to progress in our patients’ cohort. (G) Kaplan–Meier survival analysis describing the faster progression of FBXW7-Mutant patients treated with the anti-EGFR treatment. (H) Bar graph showing the percentage of patients showing SD or PR as the first response to treatment with anti-EGFR antibodies; SD, PR, and PD represent stable disease, partial response, and progressive disease, respectively.