The ubiquitin E3 ligase ARIH1 regulates hnRNP E1 protein stability, EMT and breast cancer progression

Breege V Howley,Annamarie Dalton,Toros Dincman,Joseph Karam,Philip H Howe,Bidyut Mohanty,Simon Grelet

doi:10.1038/s41388-022-02199-9

Abstract

The epithelial to mesenchymal transition (EMT), a process that is aberrantly activated in cancer and facilitates metastasis to distant organs, requires coordinated transcriptional and post-transcriptional control of gene expression. The tumor-suppressive RNA binding protein, hnRNP-E1, regulates splicing and translation of EMT-associated transcripts and it is thought that it plays a major role in the control of epithelial cell plasticity during cancer progression. We have utilized yeast 2 hybrid screening to identify novel hnRNP-E1 interactors that play a role in regulating hnRNP-E1; this approach led to the identification of the E3 ubiquitin ligase ARIH1. Here, we demonstrate that hnRNP-E1 protein stability is increased upon ARIH1 silencing, whereas, overexpression of ARIH1 leads to a reduction in hnRNP-E1. Reduced ubiquitination of hnRNP-E1 detected in ARIH1 knockdown (KD) cells compared to control suggests a role for ARIH1 in hnRNP-E1 degradation. The identification of hnRNP-E1 as a candidate substrate of ARIH1 led to the characterization of a novel function for this ubiquitin ligase in EMT induction and cancer progression. We demonstrate a delayed induction of EMT and reduced invasion in mammary epithelial cells silenced for ARIH1. Conversely, ARIH1 overexpression promoted EMT induction and invasion. ARIH1 silencing in breast cancer cells significantly attenuated cancer cell stemness in vitro and tumor formation in vivo. Finally, we utilized miniTurboID proximity labeling to identify novel ARIH1 interactors that may contribute to ARIH1’s function in EMT induction and cancer progression.

Highlights

The epithelial to mesenchymal transition normally occurs during embryonic development, fibrosis and wound healing, and can be aberrantly activated during cancer progression [1, 2]
RESULTS hnRNP E1 is a candidate substrate for ARIH1 Previous research has demonstrated that hnRNP E1 plays an important role in epithelial to mesenchymal transition (EMT) induction and tumor progression in breast cancer. hnRNP E1 mRNA is ubiquitously expressed across multiple cell types, little is known about how this RNA binding
Consistent with this hypothesis, cycloheximide treatment significantly reduced hnRNP E1 protein levels in normal murine mammary gland epithelial (NMuMG) cells, and this effect can be rescued by the proteasome inhibitor MG132 (Supplementary Fig. S1c)

Summary

Introduction

The epithelial to mesenchymal transition normally occurs during embryonic development, fibrosis and wound healing, and can be aberrantly activated during cancer progression [1, 2]. EMT is associated with increased cell migration and invasion, and is linked to the cancer stem cell phenotype, which is thought to occur through the reactivation of embryonic selfrenewal signaling pathways. The tumor suppressor hnRNP E1 (PCBP1) is an RNA binding protein that regulates diverse processes including translation and splicing [5]. HnRNP E1 regulates splicing of EMT-associated genes including CD44 and PNUTS [9, 10]. In normal murine mammary gland epithelial (NMuMG) cells, hnRNP E1 silencing increases migration and invasiveness in vitro and the formation of distant metastases in vivo [7]. HnRNP E1 knockdown (E1KD) cells acquire stem cell-like properties as assessed by mammosphere formation, stemness marker expression, and the ability to reconstitute cleared mammary fat pads in vivo [11]

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Results

Conclusion