Hippo/Mst1 Stimulates Transcription of the Proapoptotic Mediator NOXA in a FoxO1-Dependent Manner

Karel Valis,Stephen J Ralph,Jiri Neuzil,Jaromira Chladova,Lubomir Prochazka,Evzen Boura,Tomas Obsil,Jakub Rohlena,Jaroslav Truksa,Lan-Feng Dong

doi:10.1158/0008-5472.can-10-2203

Abstract

The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.

Highlights

The total number of cells forming individual organs is determined by the balanced act of cell proliferation and cell death
Since Noxa is the only Bcl-2 family protein regulated by a-tocopheryl succinate (a-TOS) [25], we studied here its regulation in the context of apoptosis induced by the agent
To determine which factors are involved in the regulation of NOXA transcription, we performed ab initio analysis of the NOXA promoter and found a constrained DBE, which is conserved in a number of mammalian species

Summary

Introduction

The total number of cells forming individual organs is determined by the balanced act of cell proliferation and cell death. Epithelial cells exhibit contact inhibition arising from cell–cell and cell–substratum interactions [1]. Tumors feature impaired mechanisms of contact inhibition, resulting in rampant cell proliferation. Specific reactivation of these control mechanisms in cancer cells can promote inhibition of tumor progression associated with induction of apoptosis. Authors' Affiliations: 1Molecular Therapy Group, Institute of Biotechnology, and 2Protein Structure Group, Institute of Physiology, Academy of Sciences of the Czech Republic; 3Faculty of Science, Charles University, Prague, Czech Republic; 4Veterinary Research Institute, Brno, Czech Republic; and 5School of Medical Science, Griffith University, Southport, Queensland, Australia. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). This work forms a part of the PhD thesis of K.

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