Talks: ShT-01.1–2 Proteasomal degradation of the transcription factor SHARP1 via the SCFßTrCP Ubiquitin Ligase Complex

A recently identified suppressor of the metastatic behavior of triple negative breast cancer (TNBC) is the bHLH transcription factor SHARP1. SHARP1 blocks the invasive phenotype of TNBC by inhibiting hypoxia-inducible factors. Moreover, loss of SHARP1 expression correlates with poor survival of breast cancer patients and represents a prognostic marker for TNBC. Here we show that SHARP1 is an unstable protein that is targeted for proteasomal degradation by an SCF ubiquitin ligase. To identify the specific F box protein interacting with SHARP1, we immunopurified SHARP1. Mass spectrometry analysis of immunoprecipitated SHARP1 identified the F-box protein ßTrCP and the SCF subunits SKP1 and CUL1. To confirm the ßTrCP-SHARP1 interaction and test its specificity, we immunoprecipitated various F-box proteins and examined their binding to SHARP1. ßTrCP was the only F-box protein interacting with SHARP1. Most proteins recognized by ßTrCP contain a DSGXX(X)S motif, known as phosphodegron, in which the phosphorylated serine residues recruit ßTrCP. SHARP1 has a similar motif in which the second serine is replaced by glutamic acid. A SHARP1 mutant, in which Ser240 and Glu245 were mutated to Ala, was not able to immunoprecipitate ßTrCP, indicat ing that Ser240 and Glu245 are required for SHARP1 binding to ßTrCP. Our results raise the possibility that ßTrCP-mediated degra dation of SHARP1, a suppressor of TNBC metastasis, may con tribute to the metastatic property of TNBC. To test this hypothesis, TNBC cells expressing non-degradable SHARP1 were injected into the mammary fat pad of female mice. Kaplan–Meier curves for tumor-free survival indicated that cells expressing SHARP1(S240A/ E245A) generated fewer tumors. Accordingly, a genetic screen employing an shRNA library against ubiquitylation-related genes identified ßTrCP as a potential target of tumor cell invasion. Indeed, silencing of ßTrCP by short hairpin RNAs in TNBC cells reduced their migratory and invasive potential in vitro.