UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis

Abstract

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC, and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes.Consistent with the tumor suppressor function of UBR5 (Hyd) in Drosophila, Hyd suppressed dMyc-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were co-amplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC co-amplification. Further, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYCmediated apoptosis priming and in protection from drug-induced apoptosis. Significance:Findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells.Research.

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

“…Western blot quantification as well as flow cytometry analysis revealed significant MYC protein stabilization upon UBR5 knockout, particularly during early time points of CHX treatment. These findings are in agreement with a previously published study 19 .

Figure 4 Validation of UBR5 as a regulator of MYC protein stability and interaction.

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“…In this study and in accordance with the previous study from Qiao et al 19 , we propose a novel mechanism of MYC regulation through UBR5 that provides a potential therapeutic target in MYC-amplified cancers. As a HECT E3 ligase, UBR5 has its own catalytic cysteine to accept ubiquitin from the E2 enzyme before ligating it to its substrate (MYC), providing a starting point for the development of specific therapeutic interventions.…”

“…We validated these findings in various cell lines, and could show that depletion of UBR5 reduces cellular viability in cell lines with high MYC expression level through the increase of MYC protein level. This observation further confirms previous studies indicating that high level of MYC protein can drive the cell into apoptosis 16,19 . Finally, we show that in cancer cell lines there is a strong correlation between MYC and UBR5 amplification, suggesting a selection pressure for co-amplification and a potential vulnerability in this subpopulation towards loss of function of UBR5.…”

“…Having shown cellular evidence for UBR5-mediated MYC ubiquitination, we sought to investigate if this is mediated through direct interaction using a biochemical in vitro ubiquitination approach. Qiao et al 19 used cellular immunoprecipitation assays with MYC deletion constructs to propose amino acids 181–189 of MYC as a potential candidate for UBR5 interaction. Here, we show ubiquitination catalyzed by a C-terminal UBR5 construct spanning amino acids 2463–2792 (including the HECT domain, referred to as UBR5 (2463–2792)) onto two lysines we attached to the C-terminus of a MYC peptide spanning amino acids 174–196 (Fig.…”

“…FBXW7) and further identifies UBR5 as a novel MYC E3 ligase. During manuscript preparation, Qiao et al published their findings of UBR5 as a novel regulator of MYC stability using an orthogonal siRNA approach in a U2OS MYC-T58A mutant model 19 , thereby both independent studies increase the confidence in the role of UBR5 as a regulator of MYC protein stability.…”