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. 2015 Apr 1;75(7):1181-6.
doi: 10.1158/0008-5472.CAN-14-1726. Epub 2015 Feb 25.

Usp28 counteracts Fbw7 in intestinal homeostasis and cancer

Markus E Diefenbacher  1 , Atanu Chakraborty  1 , Sophia M Blake  1 , Richard Mitter  2 , Nikita Popov  3 , Martin Eilers  4 , Axel Behrens  5
Affiliations

Usp28 counteracts Fbw7 in intestinal homeostasis and cancer

Markus E Diefenbacher et al. Cancer Res. .

Abstract

The stability of several oncoproteins, including c-Myc, is regulated by ubiquitin-dependent degradation mediated by the SCF(Fbw7) ubiquitin ligase. This activity is antagonized by the deubiquitinase Usp28, which is highly expressed in murine and human intestinal cancers. Usp28 was previously shown to interact with its substrates via a "piggyback" interaction with Fbw7, which suggested that Fbw7 is required for Usp28 activity. Unexpectedly, we found that genetic deletion of Usp28 rescued the lethality of Fbw7-deficient primary fibroblasts. Moreover, Usp28 inactivation in the intestine (Usp28(ΔIEC)) ameliorated the hyperproliferation and the impaired goblet and Paneth cell differentiation observed in Fbw7(ΔIEC) mice. The aggressive intestinal tumor formation of APC(Min/+); Fbw7(ΔIEC) mice was restrained when Usp28 was inactivated concomitantly. In both fibroblasts and intestinal cells, Usp28 deficiency corrected the accumulation of SCF(Fbw7) substrate proteins, including NICD1, c-Jun, and c-Myc. These findings suggested that Usp28 function does not depend on the presence of Fbw7, but instead independently recognizes and deubiquitylates the same substrates as SCF(Fbw7). Fbw7 binds to a phosphorylated motif termed the phosphodegron and we found that Usp28 also interacted with this same motif, but only when it is unphosphorylated, offering a mechanistic explanation for identical substrate selection by Fbw7 and Usp28. Our results indicate an unusually direct antagonism between an E3 ligase and a deubiquitinase, Fbw7 and Usp28, in modulating intestinal homeostasis and cancer.

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Figures

Figure 1
Figure 1. Usp28 loss rescues cell proliferation and substrate protein levels in Fbw7-null cells
(A) qRT-PCR analysis of Usp28 and Fbw7 expression in Usp28Δ/Δ, Fbw7Δ/Δ, and Fbw7Δ/Δ; Usp28Δ/Δ MEFs. (B-C) Phase-contrast micrographs (B) and growth curves (C) showing proliferation of MEFs of the above genotypes. *p<0.05; n.s., not significant. Scale bar represents 200μm. Error bars indicate SEM. (D) Western blots showing Usp28 substrate levels in MEFs of the indicated genotypes. Data are representative of results from 3 independent MEF isolations.
Figure 2
Figure 2. Usp28 deficiency partially reverts hyperproliferation and impaired differentiation in Fbw7ΔIEC gut
(A-B) Representative images (A) of murine wild-type (Usp28F/F), Fbw7ΔIEC and Fbw7ΔIEC; Usp28ΔIEC intestines, stained with AB/PAS (goblet cells), Lysozyme (Paneth cells, outlined), BrdU, and SOX9 (stem/progenitor cells). Scale bars represent 100μm (AB/PAS) or 25μm (lower panels). Stained cells are quantified (B). n = 3 animals/genotype (31 villi or 32 crypts counted per mouse). (C) Western blots analyzing isolated crypt cells from mice of the indicated genotypes. (D) qRT-PCR analysis of Notch-1, Hes1, Hes5 and Dll1 mRNA isolated from crypt cells of 3 animals/genotype. *p < 0.05; n.s., not significant. Error bars in (B) and (D) indicate SEM.
Figure 3
Figure 3. Loss of Usp28 ameliorates tumorigenesis in APCmin/+; Fbw7ΔIEC mice
(A) Heat map showing USP28 and FBW7 expression in human intestinal samples from tumors and the corresponding normal tissue. Data from GEO. (B) Loss of Usp28 restores Mendelian distribution of APCmin/+; Fbw7ΔIEC animals. **indicates significant deviation from expected value (Chi-squared test with Yates’ correction). (C) Kaplan-Meier curves showing survival of APCmin/+, APCmin/+; Fbw7ΔIEC and APCmin/+; Fbw7ΔIEC; Usp28ΔIEC animals. (D) Immunohistological analysis of intestinal tumors from mice of the indicated genotypes. Scale bar represents 50μm. Data are representative of at least 5 animals/genotype.
Figure 4
Figure 4. Usp28 binds and deubiquitinates c-Myc in the absence of Fbw7
(A) Co-immunoprecipitation of c-MYC and Flag-USP28 from HCT116 or HCT116ΔFBW7 cells transfected as indicated. (B) Ubiquitin pulldown from HCT116 or HCT116ΔFBW7 cells transfected with c-MYC and His-tagged ubiquitin (His-Ubi) in the absence or presence of USP28. Cells were treated with MG132 for 3hr before lysis. Ubiquitinated complexes were immunoprecipitated using Ni-NTA beads and analyzed by western blotting. (C) Peptide pulldown of Usp28 and Fbw7 from KRasG12D; p53Δ/Δ (KP) or KP Fbw7Δ/Δ cell lines. (D) Co-immunoprecipitation of Flag–c-Myc and HA–Usp28 from HCT116 in the presence of unphosphorylated or p-Thr58/p-Ser62 c-MYC46-74 peptide. Experiments were replicated at least 3 times with similar results.
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