Expression of DDX3 Is Directly Modulated by Hypoxia Inducible Factor-1 Alpha in Breast Epithelial Cells

Mahendran Botlagunta,Farhad Vesuna,Balaji Krishnamachary,Paul T Winnard,Arvind H Patel,Venu Raman,Guus M Bol,S K Batra

doi:10.1371/journal.pone.0017563

Abstract

DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

Highlights

Human DDX3 is a member of the DEAD-box family of RNA helicases and is located on the X chromosome [1]
Hypoxia regulates DDX3 expression in breast cancer cells To investigate whether the expression of DDX3 is regulated by hypoxia, we compared the mRNA levels of DDX3 in cells cultured under normoxic (20% O2) and hypoxic (1% O2) or hypoxia-mimetic conditions
Characterization of human DDX3 promoter in MCF 7 cell line We investigated whether regulation of DDX3 by hypoxia occurs at the transcriptional level

Summary

Introduction

Human DDX3 is a member of the DEAD-box family of RNA helicases and is located on the X chromosome [1]. DEAD-box RNA helicases have been shown to function in RNA metabolism including translation, ribosome biogenesis, pre-mRNA splicing, and nucleo-cytoplasmic RNA transport [2,3,4]. In humans DDX3 has a function in folliculogenesis as its deletion or dysfunction represents an important genetic cause of primary amenorrhea or impairment of female fertility [9]. DDX3 has been the focus of a great deal of research because of its involvement in the replication of the human immunodeficiency virus, hepatitis C virus, and poxviruses [10,11,12,13,14,15,16]

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Results

Conclusion