Prolyl hydroxylase substrate adenylosuccinate lyase is an oncogenic driver in triple negative breast cancer

Giada Zurlo,Qing Zhang,Xijuan Liu,Marie Zikánová,Jörgen Bierau,Travis Ptacek ,Mingjie Li,Charles M Perou,Cheng Fan,Andrea Robinson ,Jason W Locasale,Jing Zhang,Juan Liu,Alex Von Kriegsheim,Jessica Holler ,Xian Chen,Jeremy M Simon,Ling Xie,Baek Kim,Javier Rodríguez ,Mamoru Takada

doi:10.1038/s41467-019-13168-4

Abstract

Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.

Highlights

Protein hydroxylation affects protein stability, activity, and interactome, contributing to various diseases including cancers
Once the hydroxylation reaction takes place, the enzyme and substrate immediately dissociate, which makes the detection of the prolyl hydroxylase substrates by regular pull-down and mass spectrometry very challenging
The modest number of binding proteins pulled down from mass spectrometry suggests that there may be other ways to enrich for the prolyl hydroxylase substrates

Summary

Introduction

Protein hydroxylation affects protein stability, activity, and interactome, contributing to various diseases including cancers. By developing an enzymesubstrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify adenylosuccinate lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). We show that EglN2 hydroxylase activity is important for mediating the phenotype of EglN2 in TNBC, and EglN2 knockdown does not promote any increase in the protein level of HIF-1α, its known substrate. These observations led to the hypothesis that EglN2 oncogenic behavior in TNBC is mediated by its hydroxylation of another substrate. We develop an orthogonal approach by combining this enzyme-substrate-trapping strategy to GST-EglN2 pull-down followed by mass spectrometry. The ADSL relationship with EglN2, as well as its role in TNBC, is investigated

Methods

Results

Conclusion