Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8)

Neha Tiwari,Erik Van Nimwegen,Helena Antoniadis,Mikhail Pachkov,Phil Arnold,Gerhard Christofori,Nathalie Meyer-Schaller,Elad Katz

doi:10.1371/journal.pone.0057329

Neha Tiwari, Erik Van Nimwegen + Show 6 more

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https://doi.org/10.1371/journal.pone.0057329

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Abstract

We have identified the zinc-finger transcription factor Kruppel-like factor 4 (Klf4) among the transcription factors that are significantly downregulated in their expression during epithelial-mesenchymal transition (EMT) in mammary epithelial cells and in breast cancer cells. Loss and gain of function experiments demonstrate that the down-regulation of Klf4 expression is required for the induction of EMT in vitro and for metastasis in vivo. In addition, reduced Klf4 expression correlates with shorter disease-free survival of subsets of breast cancer patients. Yet, reduced expression of Klf4 also induces apoptosis in cells undergoing TGFβ-induced EMT. Chromatin immunoprecipitation/deep-sequencing in combination with gene expression profiling reveals direct Klf4 target genes, including E-cadherin (Cdh1), N-cadherin (Cdh2), vimentin (Vim), β-catenin (Ctnnb1), VEGF-A (Vegfa), endothelin-1 (Edn1) and Jnk1 (Mapk8). Thereby, Klf4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. Specifically, increased expression of Jnk1 (Mapk8) upon down-regulation of its transcriptional repressor Klf4 is required for EMT cell migration and for the induction of apoptosis. The data demonstrate a central role of Klf4 in the maintenance of epithelial cell differentiation and the prevention of EMT and metastasis.

Highlights

Epithelial to mesenchymal transition (EMT) involves the loss of epithelial-cell markers and gain of mesenchymal-cell markers at the invasive front of various solid tumors and constitutes a central step during carcinogenesis [1,2,3,4,5]
Decreased expression of Kruppel-like factor 4 (Klf4) during TGFb-induced EMT was confirmed in the murine breast cancer cell line Py2T, which has been established from a breast tumor of MMTV-PyMT transgenic mice [27], as well as in EpRas cells (Figure 1B–C, Figure S1B)
Klf4 expression was found reduced in human breast cancer cell lines, such as in SKBR3 upon EGF-induced EMT and in MCF7 cells which undergo EMT upon stable depletion of E-cadherin (Figure 1D, E) [9]

Summary

Introduction

Epithelial to mesenchymal transition (EMT) involves the loss of epithelial-cell markers and gain of mesenchymal-cell markers at the invasive front of various solid tumors and constitutes a central step during carcinogenesis [1,2,3,4,5]. We have previously established a list of genes that change in their expression during the consecutive morphological states of TGFb-induced EMT in normal mammary epithelial cells (NMuMG) [9]. This analysis identified Kruppel-like factor 4 (Klf4) as a transcription factor that is reduced in its expression during TGFb-induced EMT. Klf is required to maintain the cell morphology of mammary epithelial cells: while its loss induces EMT-like morphological changes, forced expression of Klf in invasive breast cancer cells induces epithelial differentiation by directly repressing the expression of Snail, a potent repressor of Ecadherin gene expression, and by directly binding to the Ecadherin promoter and up-regulating E-cadherin expression [26]. Other direct Klf target genes and the molecular mechanisms underlying Klf4’s functional contribution to EMT and metastasis have remained elusive

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