Oncogene (2009) 28, 4053–4064 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The DEAD-box protein p72 regulates ERa-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERa-positive breast cancer NC Wortham1,6, E Ahamed2,6, SM Nicol1, RS Thomas2, M Periyasamy2, J Jiang2,3, AM Ochocka2, S Shousha3, L Huson4, SE Bray5, RC Coombes2, S Ali2 and FV Fuller-Pace1 1Centre for Oncology and Molecular Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK; 2Department of Oncology, Imperial College London, Hammersmith Hospital Campus, London, UK; 3Department of Histopathology, Imperial College London, Charing Cross Hospital, London, UK; 4Statistical Advisory Service, Imperial College London, South Kensington Campus, London, UK and 5Tayside Tissue Bank, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK The DEAD-box RNA helicases p68 (DDX5) and p72 Introduction (DDX17) have been shown to act as transcriptional co- activators for a diverse range of transcription factors, The DEAD-box subfamily of RNA helicases, originally including oestrogen receptor-a (ERa). Here, we show that, so named on the basis of the presence of a conserved although both proteins interact with and co-activate ERa motif having the sequence Asp-Glu-Ala-Asp, have been in reporter gene assays, small interfering RNA-mediated implicated in cellular processes involving the regulation knockdown of p72, but not p68, results in a significant of RNA structure, including pre-mRNA processing, inhibition of oestrogen-dependent transcription of endo- RNA export, RNA degradation, ribosome assembly, genous ERa-responsive genes and oestrogen-dependent translation and miRNA maturation (Linder et al., 1989; growth of MCF-7 and ZR75-1 breast cancer cells. Tanner and Linder, 2001; Fuller-Pace, 2006). The p68 Furthermore, immunohistochemical staining of ERa-posi- RNA helicase (DDX5) and the closely related p72 RNA tive primary breast cancers for p68 and p72 indicate that helicase (DDX17) are two key members of the DEAD- p72 expression is associated with an increased period of box RNA helicases that are essential for viability; relapse-free and overall survival (P ¼ 0.006 and 0.016, p68À/À mice die in utero and p72À/À mice die shortly respectively), as well as being inversely associated with after birth, with mice for both knockouts exhibiting Her2 expression (P ¼ 0.008). Conversely, p68 shows no severe developmental defects (Fukuda et al., 2007). association with relapse-free period, or overall survival, but Recent findings have shown that p68 and p72 act as it is associated with an increased expression of Her2 transcriptional co-regulators required for the action of (P ¼ 0.001), AIB-1 (Po0.001) and higher tumour grade diverse transcription factors. Evidence for this was first (P ¼ 0.044). Our data thus highlight a crucial role for p72 forthcoming through the demonstration that p68 in ERa co-activation and oestrogen-dependent cell growth associates with oestrogen receptor-a (ERa), with pre- and provide evidence in support of distinct but important ferential interaction being observed for ERa phosphory- roles for both p68 and p72 in regulating ERa activity in lated at serine 118 (Endoh et al., 1999). p72 has also breast cancer. been shown to be an ERa co-activator (Watanabe et al., Oncogene (2009) 28, 4053–4064; doi:10.1038/onc.2009.261; 2001). p68 also acts as a transcriptional co-activator of published online 31 August 2009 the ERa-related androgen receptor (Clark et al., 2008), the tumour suppressor p53 (Bates et al., 2005) and Keywords: p68 RNA helicase; p72 RNA helicase; Runx2, a regulator of osteoblast differentiation (Jensen oestrogen receptor-a; gene regulation; breast cancer; et al., 2008). Both p68 and p72 have also been shown to tamoxifen co-activate MyoD, acting as regulators of muscle differentiation (Caretti et al., 2006). Although the mechanisms of p68 and p72 action as transcriptional co-activators remain to be defined, their interaction with Correspondence: Professor S Ali, Department of Oncology, Imperial other co-activators, including the histone acetyltrans- College London, Hammersmith Hospital Campus, London W12 ferases, CBP/p300 and P/CAF (Rossow and Janknecht, ONN, UK. E-mail: [email protected] or Dr FV Fuller-Pace, Centre for 2003; Shin and Janknecht, 2007), and the steroid Oncology and Molecular Medicine, University of Dundee, Ninewells receptor co-activator (SRC) family of nuclear receptor Hospital and Medical School, Ninewells Drive, Dundee, Tayside DD1 co-activators (Watanabe et al., 2001), which themselves 9SY, UK. interact with CBP/p300 and P/CAF (Goodman and E-mail: [email protected] 6These authors contributed equally to this work. Smolik, 2000), is suggestive of roles for p68 and p72 in Received 23 March 2009; revised 6 July 2009; accepted 30 July 2009; the regulation of histone modification by histone published online 31 August 2009 acetyltransferases. A potential role for the RNA p68, p72, ERa co-activation and breast cancer NC Wortham et al 4054 binding/helicase activity of p68/p72 has been suggested before the next cycle of transcription begins (Shang by the finding that they co-immunoprecipitate with et al., 2000; Metivier et al., 2003). In the cycles of co- ERa, SRC and the RNA co-activator SRA (Watanabe regulator recruitment, p68 is one of the earliest ERa co- et al., 2001), although RNA helicase activity per se does activators to be recruited, suggestive of a particularly not appear to be required for co-activation in reporter important role for p68 in the regulation of oestrogen- assays (Endoh et al., 1999). Finally, p68 and p72 also responsive gene expression. have potential as co-repressors through association with In this study, we have characterized further the histone deacetylase HDAC1 (Wilson et al., 2004), the importance of p68 and p72 in gene regulation by ERa, association being promoted by sumoylation of p68 in particular using RNA interference-mediated down- (Jacobs et al., 2007). regulation of p68 and p72 in the MCF-7 breast cancer Expression of ERa is one of the key features of breast cell line. Furthermore, we have investigated the expres- cancer, the most frequently observed cancer in women in sion of both proteins in a cohort of human breast industrialized nations, with ERa expression being found cancers and correlated their expression with a number of in approximately 70% of tumours. Its presence is both prognostic markers. These studies have identified p72 as prognostic and predictive of response to endocrine a key factor in the regulation of oestrogen-dependent therapies. Determination of ER status of tumours is cell growth and have identified associations with therefore crucial in the management of ER-positive breast improved patient survival, indicating that it has an cancers by treatment with endocrine agents, such as important role in breast cancer pathogenesis. tamoxifen, that inhibit ERa activity (Ali and Coombes, 2002), or aromatase inhibitors that reduce the levels of circulating estrogens. However, following an initial response to tamoxifen, a significant proportion of patients Results relapse. These tumours often remain ERa positive and p68 and p72 co-activate ER in synergy with SRC-1 may respond to an alternative endocrine agent, showing a To examine p68/p72 co-activator function, we transfected ERa-dependence for the continued growth of these COS-1 cells with an oestrogen-responsive firefly luciferase tumours (Buzdar and Howell, 2001; Morris and Wakel- ing, 2002), and indicating altered ERa function as a reporter plasmid and combinations of full-length ERa or mutants lacking either AF1 or AF2 (Figure 1a), together possible mechanism underlying treatment failure. with p68, p72 and the well-characterized steroid receptor Oestrogen receptor-a is a member of the nuclear co-activator SRC-1. In agreement with previous reports receptor superfamily of ligand-activated transcription (Endoh et al., 1999; Watanabe et al., 2001), ERa activity factors (Mangelsdorf et al., 1995). Activation of gene was stimulated by co-transfection with p68 and p72 expression by ERa is mediated by two transcription (Figure 1b); this co-activation was synergistically en- activation domains, AF1 and AF2, which act in a promoter- and cell-specific manner. AF1, located hanced by SRC-1. As has also previously been described (Endoh et al., 1999), the RNA helicase activity of p68 is N-terminal to the DNA-binding domain (DBD), functions dispensable as co-transfection with a helicase-inactive p68 in a ligand-independent manner and its activity can be stimulated through growth factor-regulated signalling mutant did not prevent the stimulation of ERa activity by p68 (Figure 1c), nor was co-activation of ERa blocked cascades in the absence of oestrogen (Bunone et al., by a similar mutation in p72. 1996). AF1 activity is enhanced by activating phospho- p68 was originally identified as an ERa co-activator rylation of serines 104, 106 and 118 through the following in vitro purification of proteins that interacted mitogen-activated protein kinase (MAPK) pathway preferentially with ERa phosphorylated at ser-118 by (Ali et al., 1993; Le Goff et al., 1994; Thomas et al., MAPK (Endoh et al., 1999). In reporter assays using 2008), and by cyclin-dependent protein kinases CDK2 truncated ERa lacking the ligand-binding domain/AF2, (Ser-104/Ser106) (Trowbridge et al., 1997) and CDK7 p68 and p72 stimulated AF1 activity (Figure 1d). (Ser-118) (Chen et al., 2000). In addition, these Although mutation of Ser-104 or Ser-106 did not phosphorylation sites are substrates for GSK-3 (Me- dunjanin et al., 2005; Grisouard et al., 2007). AF2 is prevent co-activation by p68 or by p72, mutation of Ser-118 gave little or no stimulation of ERa activity by integral to the ligand-binding domain and its activity p68 and p72, with mutation of all three residues requires oestrogen binding by the ligand-binding domain, which results in a conformational change completely preventing co-activation of AF1 by p68 and p72.