RAC1B: A Guardian of the Epithelial Phenotype and Protector Against Epithelial-Mesenchymal Transition.

Rabea Zinn,Hendrik Ungefroren,Hannah Otterbein,Hendrik Lehnert

doi:10.3390/cells8121569

Abstract

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown to potently inhibit transforming growth factor (TGF)-β1-induced cell migration and epithelial-mesenchymal transition (EMT) in pancreatic and breast epithelial cells, but the underlying mechanism has remained obscure. Using a panel of pancreatic ductal adenocarcinoma (PDAC)-derived cell lines of different differentiation stages, we show that RAC1B is more abundantly expressed in well differentiated as opposed to poorly differentiated cells. Interestingly, RNA interference-mediated knockdown of RAC1B decreased expression of the epithelial marker protein E-cadherin, encoded by CDH1, and enhanced its TGF-β1-induced downregulation, whereas ectopic overexpression of RAC1B upregulated CDH1 expression and largely prevented its TGF-β1-induced silencing of CDH1. Conversely, knockdown of RAC1B, or deletion of the RAC1B-specific exon 3b by CRISPR/Cas-mediated genomic editing, enhanced basal and TGF-β1-induced upregulation of mesenchymal markers like Vimentin, and EMT-associated transcription factors such as SNAIL and SLUG. Moreover, we demonstrate that knockout of RAC1B enhanced the cells’ migratory activity and derepressed TGF-β1-induced activation of the mitogen-activated protein kinase ERK2. Pharmacological inhibition of ERK1/2 activation in RAC1B-depleted cells rescued cells from the RAC1B knockdown-induced enhancement of cell migration, TGF-β1-induced downregulation of CDH1, and upregulation of SNAI1. We conclude that RAC1B promotes epithelial gene expression and suppresses mesenchymal gene expression by interfering with TGF-β1-induced MEK-ERK signaling, thereby protecting cells from undergoing EMT and EMT-associated responses like acquisition of cell motility.

Highlights

The human relatedC3 botulinum toxin substrate 1 (RAC1) gene, by alternative splicing, gives rise to two proteins designated Ras-relatedC3 botulinum toxin substrate 1 (RAC1) and related C3 botulinum toxin substrate 1B (RAC1B)
RAC1B Expression in Pancreatic ductal adenocarcinoma (PDAC)-Derived Cell Lines is Associated with a Well Differentiated Epithelial
RAC1B was expressed at higher levels in G1 and G2 cell lines compared to G3 cell lines (Figure 1A)

Summary

Introduction

C3 botulinum toxin substrate 1 (RAC1) and RAC1B. Both proteins belong to the Rho family of monomeric GTPases with RAC1B differing from RAC1 by the presence of an additional exon RAC1B differs from RAC1 by the type of upstream activators, binding partners, and downstream effectors/targets, only few RAC1B-specific target genes have been identified so far. Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with an extremely poor prognosis [2,3]. This is due to its highly metastatic nature and therapy resistance [4] with most patients dying from the consequences of metastatic spread to other organs, to the liver

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Conclusion