New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.

Klaus Dittmann,Claus Mayer,Stephan M Huber,H Peter Rodemann,Stefan Czemmel,Carol J Wilusz

doi:10.1371/journal.pone.0189087

Abstract

Cell membrane-associated epidermal growth factor receptor (EGFR) translocates into a perinuclear/nuclear location upon stimulation, where it complexes with mRNAs. Treatment with radiation and cisplatin decreases the amounts of mRNAs present within this complex. Gene array analyses of mRNAs in complex with immunoprecipitated nEGFR revealed significant enrichment of different mRNA species compared to the control immunoprecipitation. Functional annotation with help of DAVID Gene Ontology Analysis identified under other terms the HIF-1A/VEGF signaling pathway as one of the top scoring KEGG pathways. RT-PCR and western blots revealed the radiation-induced expression of mRNAs and proteins involved in HIF-1A/VEGF signaling. Simultaneously, the levels of the corresponding validated miRNAs within the complex containing nEGFR and mRNAs were decreased. This finding argues that an mRNA/miRNA/nEGFR complex regulates protein expression. Indeed, we detected the GW182, AGO2, PABPC1 and cNOT1 proteins, which belong to the deadenylase complex, in a complex with nuclear EGFR. Erlotinib-mediated inhibition of EGFR kinase reduced the radiation-induced increase in mRNA expression. In this context, erlotinib reduced AGO2 phosphorylation by the EGFR kinase at residue Y393, which was associated with increased cNOT1 deadenylase activity and reduced mRNA stability. To prove the roles of miRNAs in this context, we transfected cells with an inhibitor of Hsa-mir-1180p5, which targets the NFATC4 mRNA, an mRNA associated with VEGF signaling, or pretreated cells with erlotinib. Indeed, Hsa-mir-1180p5 knockdown increased and the erlotinib treatment decreased the expression of the NFATC4 protein. The expression of the NFATC4 protein controlled the cloning efficiency and radiosensitivity of A549 and FaDu tumor cells. Thus, this study is the first to show that a membrane-located tyrosine kinase receptor, such as EGFR, is internalized to a nuclear/perinuclear location upon exposure to stress and modulates the stability and translation of miRNA-selected mRNAs. This mechanism enables cells to directly express proteins in response to EGFR activation and may contribute to treatment resistance in EGFR-overexpressing tumors.

Highlights

The receptor tyrosine kinase epidermal growth factor receptor (EGFR) (ErbB1) plays a crucial role in both cancer initiation and progression [1,2] and is discussed as a promising target for cancer therapy
We synthesized cDNAs from mRNAs enriched in Nuclear EGFR (nEGFR) complexes that were immunoprecipitated from nuclear extracts to elucidate the role of nEGFR in this process (Fig 1)
Irradiation and cisplatin treatments decreased the relative amount of enriched mRNAs complexed with nEGFR compared to untreated cells (Fig 1A)

Summary

Introduction

The receptor tyrosine kinase EGFR (ErbB1) plays a crucial role in both cancer initiation and progression [1,2] and is discussed as a promising target for cancer therapy. New insights into EGFR biology have established that EGFR signals through two distinct pathways: (i) canonical membrane-associated signaling [3,4] and (ii) non-canonical nuclear signaling to regulate gene expression, DNA replication and DNA damage repair [5,6,7]. Nuclear EGFR (nEGFR) is reported to act as a co-transcriptional activator for cyclin D1 [8, 10]. NEGFR controls proliferating cell nuclear antigen (PCNA) activity during DNA replication [11]. NEGFR regulates the ribonuclease activity of polynucleotide phosphorylase through DNA-PK-mediated phosphorylation [16], and the expression of the c-myc mRNA is increased. The same study reported an inhibitory effect of EGFR-mediated argonaute 2 (AGO2) phosphorylation on miRNA maturation in response to hypoxia [17]

Results

Discussion

Conclusion