Regulation of TP73 transcription by Hippo-YAP signaling

https://doi.org/10.1016/j.bbrc.2020.07.132Get rights and content

Highlights

  • The transcription of TP73 is repressed by YAP, the major effector of Hippo pathway.

  • The repression of TP73 transcription by YAP is WW domain-dependent.

  • YAP repressed TP73 expression leads to cancer cell resistance to chemotherapeutic drugs.

Abstract

Yes-associated protein (YAP) is a key downstream effector of the highly conserved Hippo signaling pathway, which regulates organ size, regeneration and tumorigenesis. Known classically to function as a transcriptional co-activator, YAP interacts with TEA domain transcription factors (TEAD1-4) to induce expression of target genes. However, a number of genes are repressed upon YAP activation, suggesting a transcriptional repressor role of YAP. Here, we report that TP73 is a direct target gene of YAP, and its transcription is repressed by YAP in a TEAD-independent manner. On the other hand, WW domains of YAP are indispensable for the regulation of TP73 expression, which may recruit YAP to TP73 gene though interaction with ZEB1 and/or RUNX2, two transcriptional repressors. Moreover, YAP-mediated repression of TP73 promotes cancer cell survival in the presence of chemotherapeutic agents, suggesting YAP-TP73 signaling as a mechanism for cancer cell resistance to chemotherapies.

Introduction

The Hippo pathway was first discovered in Drosophila as a crucial regulator of organ size and is evolutionarily conserved in mammals [1]. The central components of the Hippo pathway include a kinase cascade in which MST1/2 (Mammalian sterile 20-like 1/2) or MAP4Ks (Mitogen activate protein kinase kinase) phosphorylate and activate LATS1/2 (Large tumor suppressor homolog 1/2), which in turn phosphorylates YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ binding motif). YAP and TAZ, two homologous transcriptional co-activators, serve as major effectors of the Hippo pathway. When Hippo signaling is on, phosphorylation of YAP and TAZ results in their cytoplasmic sequestration. On the other hand, when Hippo signaling is off, YAP and TAZ translocate to the nucleus where they interact with TEAD transcription factors to induce transcription of target genes involved in cell proliferation, apoptosis, differentiation, and stemness [[2], [3], [4], [5], [6], [7]]. YAP and TAZ are also known to interact with other transcription factors, including RUNX2 (runt-related transcription factor 2), ErbB4 (erb-b2 receptor tyrosine kinase 4) and SMAD family proteins to regulate gene expression [[8], [9], [10]].

Beyond its role in organ size control, the Hippo pathway is recognized to play key roles in tumorigenesis, metastasis, and drug resistance. In humans, amplification of the chromosome region containing the YAP gene (11q22) has been reported in different tumor types and cancer cell lines [11]. Mutations of the Hippo pathway upstream regulator NF2 (Neurofibromin 2) are frequently observed in malignancies such as Neurofibromatosis type 2, Mesothelioma, and Ependymoma, and YAP activation is primarily responsible for tumorigenesis driven by NF2 loss of function [12,13]. Additionally, YAP protein is elevated and nuclear-enriched in several human cancers, including ovary, lung, colon, liver, and prostate cancers [14]. High YAP expression has also been shown to correlate with reduced survival in lung cancer and liver cancer patients [15]. Furthermore, YAP confers resistance against several cytotoxic agents such as 5-Fiuorouracil (5-FU), cisplatin, and doxorubicin [16]. However, the key genes and mechanisms involved in YAP-dependent drug resistance are poorly defined.

In this study, we identify TP73, a member of the p53 tumor suppressor family, as a gene transcriptionally repressed by YAP. TP73 repression is dependent on the WW domains of YAP, whereas TEAD binding is dispensable. Functionally, YAP-mediated TP73 repression promotes cancer cell survival in the presence of doxorubicin, suggesting that TP73 downregulation is important YAP-induced drug resistance.

Section snippets

Cell culture, transfection, and lentivirus infection

MCF10A cells were cultured as previously described [17]. HEK239A, U2OS, Caco2, and H1299 cells were cultured in DMEM (Hyclone) containing 10% FBS (Gibco) and P/S. RKO and HCT116 cells were cultured in RPMI-1640 (Hyclone) containing 10% FBS and P/S. All cell lines were maintained at 37 °C with 5% CO2. Transfection, and lentivirus infection were previously described [17].

Plasmids

TAp73, RUNX2 and ZEB1 were cloned into pLVX vector. Plasmids encoding YAP, YAP-2SA, YAP-S94A, YAP-WW1-mut, YAP-WW2-mut,

YAP represses TP73 transcription

To uncover the genes regulated by YAP, we performed RNA-sequencing (RNA-seq, GSE70506) using control and YAP-overexpressing MCF10A cells. Comparative analysis revealed that 2616 genes (fold change>2, p < 0.05) were differentially regulated by YAP (Table S3). The top 200 genes with the most significant changes were selected for clustering and Gene Ontology (GO) analysis (Figs. S1A and S1B). A number of YAP target genes were found to be involved in cell proliferation, differentiation and

Discussion

As a key effector of the Hippo signaling pathway, YAP is conventionally labeled as a “transcription co-activator”, but it also represses transcription as evidenced by the large number of genes downregulated in YAP-overexpressing cells (Fig. S1). However, such YAP-repressed genes and their associated functions have not been extensively explored. Several studies have reported that YAP could repress the expression of LGR5 [25], DDIT4, and Trail [30], indicating a critical effect of YAP repressed

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study is supported by grants from the National Natural Science Foundation of China (81772965), the National Key R&D program of China (2018YFA0800304), Science and Technology Commission of Shanghai Municipality (19JC1411100), and Shanghai Municipal Commission of Health and Family Planning (2017BR018) to FXY.

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  • 1

    These authors contributed equally to this work.

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