Β-Arrestin-1 Is a Nuclear Transcriptional Regulator of Endothelin-1-Induced

ORIGINAL ARTICLE b-arrestin-1 is a nuclear transcriptional regulator of endothelin-1-induced b-catenin signaling

L Rosano` 1, R Cianfrocca1, P Tocci1, F Spinella1, V Di Castro1, F Spadaro2, E Salvati3, AM Biroccio3, PG Natali1 and A Bagnato1

Despite the fundamental pathophysiological importance of b-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that b-arrestin-1 (b-arr1) is an epigenetic regulator of endothelin (ET)-1-induced b-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ETAR) activation by ET-1, b-arr1 increases its nuclear translocation and direct binding to b-catenin. This in turn enhanced b-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant b-arr1 incapable of nuclear distribution. b-arr1–b-catenin interaction controls b-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by b-arr1 silencing or by mutant b-arr1, as well as by b-catenin or p300 silencing, confirming that nuclear b-arr1 forms a functional complex capable of regulating epigenetic changes in b-catenin- driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of b-arr1 or mutant b-arr1 expression, as well as ETAR blockade, inhibits metastasis. In human EOC tissues, b-arr1–b-catenin nuclear complexes are selectively enriched at b-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo b-arr1–b-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a b-arr1-mediated epigenetic mechanism controls b-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.

Oncogene (2013) 32, 5066–5077; doi:10.1038/onc.2012.527; published online 3 December 2012 Keywords: beta-arrestin; beta-catenin; endothelin A receptor; ovarian carcinoma; chromatin remodeling

INTRODUCTION consequently increasing nuclear factor-kB transcriptional Epithelial ovarian cancer (EOC) often features endothelin (ET)-1 responsiveness.21 However, the underlying mechanisms of and ET A receptor (ETAR) axis, which signals through the cytosolic b-arr1-mediated transcription regulation in pathophysiological scaffold proteins b-arrestins (b-arrs).1,2 b-arrs, consisting of b-arr1 conditions, such as cancer, still remain to be elucidated. Given that and b-arr2, are involved in G-protein-coupled receptor signaling, b-arr1 does not contain a discernible DNA-binding domain,22 it is and guide the receptor signals also in malignant cells.3–12 A global important to identify the nuclear interactions of b-arr1 and their proteomics analysis of b-arr1-interacting proteins demonstrated functional implications in cancer progression. that b-arr1 interactions take place not only in the cytoplasm, but b-arr was also reported to regulate Wnt/b-catenin signaling, also in the nucleus,13 suggesting that b-arr1 has a role in which is aberrantly activated in human cancer and is required for transcriptional regulation in different human cells.14–16 Previous tumor progression and stem cell regulation.23–27 In this context, studies indicate that under d-ork-opioid receptor stimulations, we previously demonstrated that downstream to ETAR, barr1 nuclear b-arr1 specifically accumulates at p27 and c-fos promoters serves as a cytosolic scaffold for the stabilization of b-catenin.5,28 through scaffolding cyclic adenosine monophosphate response- One feature of b-catenin signaling activation is its presence in the element (CRE) binding protein and histone acetyltransferase p300, nucleus, where b-catenin initiates transcription as a member of thus enhancing local histone acetylation and gene transcription.17 multimeric complexes acting as the central transcriptional A similar mechanism was also observed during T-cell activation, in activator. Although several studies extensively depict many which nuclear b-arr1 increases Bcl-2 expression and contributes to molecular mechanisms controlling b-catenin-mediated trans- CD4 þ T-cell survival.18 Nuclear b-arr1 is also essential for the cription, they do not completely explain how b-catenin gets mitogenic and antiapoptotic activity of nicotine in human non- into the nucleus to control transcription during meta- small cell lung cancer cells, through regulating the binding to E2F statization. In this regard, we hypothesized that b-arr1, if indeed family of transcription factors.19 Moreover, b-arr1 regulates nuclear capable of nuclear function, would be endowed with a novel signaling during hypoxia to promote survival of breast cancer cells epigenetic mechanism by recruiting b-catenin to specific target by facilitating hypoxia-inducible factor-1-dependent vascular gene promoters to activate transcription. Therefore, in an attempt endothelial growth factor expression.20 Recent results show that to identify b-arr1-interacting transactivators, which bind to those b-arr1, via the interaction with importin b1, is imported in the promoters involved in tumor progression, we examined the nucleus where it is able to recruit protein modifiers to p65/RelA, nuclear interaction that connects b-arr1 to b-catenin signaling in

1Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Rome, Italy; 2Section of Experimental Immunotherapy, Department of Haematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita`, Rome, Italy and 3Laboratory of Experimental Chemotherapy, Regina Elena National Cancer Institute, Rome, Italy. Correspondence: Dr L Rosano` or Dr A Bagnato, Laboratory of Molecular Pathology, Regina Elena National Cancer Institute, Via delle Messi D’Oro 156, Rome 00158, Italy. E-mail: [email protected] or [email protected] Received 29 May 2012; revised 16 October 2012; accepted 16 October 2012; published online 3 December 2012 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5067

Figure 1. b-arr1 localizes in the nucleus on activation of ETAR by ET-1. HEY, A2780 and OVCA 433 cells expressing endogenous b-arr1 (a)orHEY cells expressing b-arr1–FLAG (b) were treated with ET-1 for the indicated times, and cytoplasmic and nuclear extracts were subjected to immunoblotting (IB) for b-arr1 or FLAG. The average of nuclear b-arr1 in the graphics showed in (a, b) corresponds to the quantiﬁcation of protein bands in images from three independent experiments, normalized to proliferating cell nuclear antigen (PCNA) content ±s.e.m. b-arr1 localization by confocal laser scanning microscopy (CLSM) examination (three-dimensional reconstruction images) in HEY cells expressing b-arr1–FLAG (c) stimulated for 15, 30 or 60 min with 100 nM ET-1 and/or 1 mM zibotentan for 15 min, or expressing b-arr1–Q394L–FLAG (d), stimulated for 15 min with ET-1, and stained with an anti-FLAG Ab. Nuclei are reported in blue (40,6-diamidino-2-phenylindole (DAPI)). Scale bars ¼ 20 mm.

EOC. In this study, we report that the nuclear transcriptional Supplementary Figure S1B). Nuclear accumulation of both function of b-arr1 in response to ETAR activation represents a endogenous b-arr1 (Figure 1a) and exogenous b-arr1–FLAG novel epigenetic mechanism involved in controlling b-catenin (Figure 1b) increased by twofold after 15 min of ET-1 challenge, activity to promote epithelial–to-mesenchymal transition (EMT) demonstrating that ET-1 promotes nuclear accumulation of b-arr1 and tumor metastasis, offering the possibility to develop new in EOC cells and that this translocation is not an artifact of FLAG- strategies for inhibiting ETAR/b-arr1/b-catenin-mediated tumor fusion or overexpression of b-arr1. Confocal laser scanning progression. microscopy analysis in HEY and OVCA 433 cells stably expressing b-arr1–FLAG confirmed these results and revealed that ET-1 stimulation induced a strong nuclear translocation of b-arr1 after RESULTS 15 min (Figure 1c and Supplementary Figure S1C), and that was ET-1 induces nuclear translocation of b-arr1 in ovarian cancer cells abrogated by treatment with zibotentan, a specific ETAR As previously reported,5 b-arr1 was able to shuttle from the antagonist, indicating that b-arr1 nuclear translocation was cytoplasm to the cell membrane (Supplementary Figure S1A) to mediated by ETAR (Figure 1c and Supplementary Figure S1C). 5 interact with ETAR within a few minutes of ET-1 treatment. In order As EOC cells express both b-arr1 and b-arr2, we further to investigate whether ET-1 stimulation induces also the analyzed the nucleocytoplasmic shuttling of both b-arr1 and translocation of b-arr1 to the nucleus, we analyzed the sub- b-arr2 on ET-1 treatment in HEY cells transiently expressing cellular localization of b-arr1 in the cytosolic and nuclear extracts b-arr1–green fluorescent protein (GFP) or b-arr2–yellow fluorescent from HEY, A2780 and OVCA 433 cells, endogenously expressing protein (YFP). Both b-arr1 and b-arr2 are able to move between b-arr1. Immunoblotting analysis clearly showed that b-arr1, which the cytoplasm and nucleus but b-arr2 possesses a strong nuclear in quiescent cells is localized predominantly in the cytosolic export signal in its C terminus, which hinders its retention in the extracts, translocated to the nuclear compartment after ET-1 nucleus.17,29,30 Before ET-1 stimulation, b-arr1–GFP fluorescence stimulation in a time- and dose-dependent manner (Figure 1a and was distributed preferentially in the cytoplasm, although a faint

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5068 fluorescence could be detected in the nucleus, and b-arr2–YFP and Supplementary Figures S3A and B), confirming that ETAR- was confined solely to the cytoplasm. Whereas, ET-1 treatment driven nuclear b-arr1 binds to b-catenin. This interaction is induced a strong nuclear accumulation of b-arr1–GFP but not b- necessary for inducing b-catenin/TCF4 transcriptional activity. arr2–YFP (Supplementary Figure S1D). Then, we transfected HEY and OVCA 433 cells with b-arr1–Q394L mutant, in which the ETAR-mediated nuclear translocation of b-arr1 regulates the nuclear export signal of b-arr2 was introduced into b-arr1, by a transcription of selected b-catenin target genes 17,29,30 single point (Q394L) mutation. and we observed that the To further analyze the functional role of b-arr1 and b-catenin nuclear distribution of b-arr1 was inhibited on ET-1 challenge nuclear interaction, we analyzed how b-arr1 could influence the (Figure 1d, Supplementary Figures S1C and S1F). Moreover, the transcription of a set of b-catenin/TCF4-target genes, such as ET-1 treatment with 10 nM Leptomycin B, an inhibitor of nuclear export (Supplementary Figures S4A–E), Cyclin D1, Axin 2, Matrix metallo- signal-mediated active nuclear export, induced a strong nuclear proteinase (MMP)-2 and -9.32–35 As shown in Figure 3a, quantitative accumulation of b-arr2–YFP in both basal and ET-1-treated PCR showed that silencing b-arr1 in HEY cells, downregulated the conditions but not appreciable effects regarding the subcellular expression of Axin 2, ET-1, MMP-2 and Cyclin D1, but not MMP-9. distribution of endogenous or exogenous b-arr1 (GFP or FLAG) This indicated that b-arr1-dependent transcriptional regulation of were observed (Supplementary Figures S1D, S1E and S1G), b-catenin might occur in a defined set of genes. These effects indicating that ET-1 controls the nuclear trafficking of b-arr1 most were rescued by overexpression of b-arr1–FLAG, but not by b- likely to modulate nuclear signaling of ETAR in EOC cells. arr1–Q394L (Figure 3a and Supplementary Figure S3B), demonstrating that b-catenin-dependent gene transcription is linked to the presence of nuclear b-arr1. b-arr1 interacts with b-catenin in the nucleus and regulates its Chromatin IP (ChIP) assays in HEY and OVCA 433 cells transcriptional activity expressing endogenous b-arr1/b-catenin showed that ET-1 To investigate whether b-arr1 could act as a nuclear scaffold to induced a time-dependent in vivo recruitment of b-arr1, TCF4, regulate b-catenin nuclear accumulation, we analyzed their and b-catenin to ET-1, Cyclin D1, Axin 2 and MMP-2, but not at potential physical interaction in the nucleus. Confocal laser MMP-9 promoters, which was inhibited by treatment with scanning microscopy analysis of HEY and OVCA 433 cells zibotentan (Figures 3b and c and Supplementary Figure S5A), expressing b-arr1–FLAG showed that within 15 min of ET-1 indicating that in response to ETAR activation, b-arr1 is selectively stimulation b-arr1 nuclear translocation was associated with enriched with b-catenin at selected gene promoters. Moreover, we b-catenin. This colocalization was impaired by zibotentan, as well found that the ChIP signals of b-catenin at the ET-1 promoter (ETP) as by b-arr1–Q394L expression (Figure 2a and Supplementary were lost by b-arr1–Q394L expression (Figure 3d). Figure S2A). Moreover, as determined by co-immunoprecipitation Altogether, these results indicate that b-arr1 influences the (IP) assays, endogenous b-arr1 bound to endogenous b-catenin in transcriptional activity of b-catenin/TCF4 and that this function the nuclei of ET-1-treated HEY cells (Figure 2b), and this interaction correlates with its presence in the nucleus as well as its was inhibited by zibotentan (Supplementary Figure S2B). More- recruitment with b-catenin on selective promoter target genes. over, when HEY cells overexpressed FLAG-tagged b-arr1, but not b-arr1–Q394L, b-arr1 interacted with endogenous b-catenin in both the cytoplasm (5 min) and the nucleus (15 min), and ET-1 HDAC1 dissociated from the b-catenin target gene promoters in the presence of b-arr1 activation increased the translocation of both proteins to the nucleus (Figure 2c and Supplementary Figure S2C). To further In the absence of b-catenin binding, the promoters of b-catenin target genes are occupied by TCF/LEF family proteins and co- explore the nuclear functions of b-arr1, we used four b-arr1 36 deletion mutants tagged with an AU5 epitope at their carboxyl repressors of Groucho/TLE1 and histone deacetylase 1 (HDAC1), termini,21,31 and expressed them in HEY cells. As shown in rendering the chromatin more compact and transcriptionally Figure 2d, of the four b-arr1 mutants, three were found to be inactive. To determine whether b-arr1 has a role in regulating the correlated with b-catenin. The b-arr1–1-180S mutant, deleted of function of the b-catenin/TCF4 complex at the target promoters, the region required for its nuclear localization,21 was not co-IP we first probed the association of TCF4 and HDAC1 with b-catenin with the b-catenin, suggesting that this truncated b-arr1 construct target gene promoters. ChIP assays showed that, in untreated was unable to interact with it. Given that both b-arr1–1-180S and cells, HDAC1 was associated with the ET-1 and MMP-2 promoters, b-arr1–1-90N were found to associate with b-catenin, these results whereas ET-1 treatment led to a substantial decrease in HDAC1 suggested that the deletion of a region consisting of amino acids and ET-1 and MMP-2 promoter interactions (Figure 4a). Interest- 1-180 of b-arr1, containing the nuclear localization signal, leads to ingly, b-arr1 knockdown dramatically enhanced HDAC1 associa- a loss of b-arr1–b-catenin interaction. Altogether, these findings tion with both promoters (Figure 4a). This supports the hypothesis that b-arr1 promoted dissociation of TCF4-bound HDAC1 from indicate that b-arr1 functions as a chaperone of ETAR in regulating b-catenin nuclear localization. b-catenin target gene promoters and accumulation of nuclear Furthermore, the endogenous TCF4 protein, a well-known b-catenin/TCF4 complex competent to activate the expression of binding partner of b-catenin, was detected in the b-arr1 and b- downstream target genes. catenin nuclear IPs, and its levels increased following ET-1 treatment (Figure 2e and Supplementary Figure S2E). In these p300 interacts with b-arr1 and b-catenin to mediate histone H3 nuclear IPs, the silencing of TCF4 reduced the ET-1-induced and H4 acetylation on b-catenin-dependent transcription nuclear interaction between b-arr1 and b-catenin, demonstrating Given the role of histone acetyltransferase p300 in b-arr1- that the b-catenin–TCF4 complex interacts with b-arr1 in the mediated gene expression,17 we assessed whether b-arr1 could nucleus of EOC cells (Supplementary Figure S2F). form a multimeric complex with b-catenin and with p300 in the To analyze whether this nuclear association might be directly nucleus by combinatorial IP. In HEY cells, ET-1 treatment promoted associated with the transcriptional activity of b-catenin/TCF4 by the association of b-arr1 with p300 and b-catenin in the nucleus, the recruitment on its target genes, we assessed the effects of as detected in b-arr1, p300 and b-catenin IPs (Figure 4b). Further gain–loss function of b-arr1 on b-catenin transactivating function experiments showed that silencing p300 prevented the associa- by using the TopFlash or FopFlash luciferase reporter. The tion of b-arr1 with b-catenin (Figure 4b and Supplementary Figure transcriptional activity of b-catenin induced by ET-1 was inhibited S5B), indicating that the presence of p300 is required together by silencing b-arr1 and rescued by the re-expression of b-arr1– with b-arr1 and b-catenin for the epigenetic regulation promoted FLAG, but not by b-arr1–Q394L, or b-arr1-1-180S mutant (Figure 2f by the ET-1/ETAR axis. Similarly, b-arr1 or b-catenin silencing

Oncogene (2013) 5066 – 5077 & 2013 Macmillan Publishers Limited b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5069

Figure 2. b-arr1 links b-catenin in the nucleus and regulates its transcriptional activity. (a) b-arr1 and b-catenin colocalization by confocal laser scanning microscopy (CLSM) examination in HEY cells expressing b-arr1–FLAG or b-arr1–Q394L–FLAG, stimulated for 15 min with 100 nM ET-1 and/or 1 mM zibotentan, stained with an anti-FLAG (red) and anti-b-catenin (green) Abs. Nuclei are reported in blue (40,6-diamidino-2- phenylindole (DAPI)). Colocalization of b-arr1 and b-catenin was represented in the merge images. Scale bars ¼ 20 mm. (b) HEY cells expressing endogenous b-arr1 were treated with 100 nM ET-1 for 15 min and nuclear fractions were IP with irrelevant immunoglobulin G (IgG) (negative control), anti-b-arr1 or anti-b-cat Abs and immunoblotting (IB) with anti-b-catenin and anti-b-arr1 Abs. (c) HEY cells expressing b-arr1–FLAG or b-arr1–Q394L–FLAG were treated with 100 nM ET-1 for 15 min and nuclear fractions were IP with irrelevant IgG (negative control), or anti-b-cat Ab and IB with anti-FLAG, and anti-b-catenin Abs. (d) Upper panel: schematic representation of full-length and 4 deletion mutants of b-arr1- AU5. Lower panel: expression vectors coding for the four deletion mutant and the full-length b-arr1-AU5 were individually expressed in HEY cells. Cells were treated with 100 nM ET-1 for 15 min and interaction between b-catenin and AU5-tagged b-arr1 constructs was determined by IP with anti-AU5 and IB with anti-b-cat and anti-AU5 Abs. (e) Nuclear fractions of HEY cells treated as in (b) were IP with anti-b-arr1 or anti-b- catenin Abs or irrelevant IgG and IB with anti-TCF4, anti-b-arr1 and anti-b-catenin Abs. (f) b-Catenin transcriptional activity evaluated as in HEY cells transfected with scramble (SCR) or b-arr1 shRNA (sh-b-arr1) and then co-transfected with TOP/Flash or FOP/Flash reporter, and empty (Mock), or b-arr1–FLAG, or b-arr1–Q394L–FLAG or AU5–b-arr1–1-180N mutant expression vectors and stimulated with ET-1. Results were expressed as the ratio of TOP/Flash over FOP/Flash activity and represent values is mean±s.e.m. of results of six independent experiments.

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5070

Figure 3. b-arr1 is involved in b-catenin-dependent target gene expression. (a) Quantitative reverse transcriptase (qRT)–PCR analysis of Axin 2, ET-1, MMP-2, MMP-9 and Cyclin D1 expression in HEY cells transfected with scramble (SCR) or sh-b-arr1, and rescued with Mock or b-arr1–FLAG or b-arr1–Q394L–FLAG expression vectors. Bars indicate average of values from duplicate qRT–PCR reactions ±s.e.m. (b) HEY cells were treated with ET-1 and/or zibotentan for indicated times and the binding of b-arr1, b-catenin and TCF4, to ET-1, Cyclin D1, Axin 2 and MMP-2 promoter regions was measured by ChIP assays followed by PCR. Nonspeciﬁc immunoglobulin G (IgG) was used as the irrelevant antibody (IRR) for all ChIP reactions. The input DNA lane represents one-twentieth of the precleared chromatin used in each ChIP reaction. (c) HEY cells were treated with ET-1 for indicated times and the binding of b-arr1 and b-catenin to MMP-9 promoter region was measured by ChIP assays. (d) HEY cells stably expressing b-arr1–Q394L–FLAG were treated with ET-1 for indicated times and the binding of b-arr1 and b-catenin to ETP region was measured by ChIP assays.

induced a loss in the ability to bind between the other two b-arr1 (Figure 5c). Consistent with these results, the silencing of components of the transcriptional complex (Figure 4b), suggest- either b-catenin or p300 (Figure 5d, Supplementary Figures S4C ing that b-arr1 may provide a nuclear anchor for p300 and and S5B) or b-arr1 (Figure 3a), strongly inhibited the expression of b-catenin as a prerequisite for b-catenin-mediated transcription. b-catenin target genes, ET-1, MMP-2 and Cyclin D1, as analyzed by ChIP assays showed that ET-1 treatment induced a time- quantitative reverse transcriptase–PCR. Altogether, these results, dependent recruitment of p300, as well as b-catenin, on ET-1, showing the depletion of one component of the transcriptional MMP-2, and Cyclin D1 promoters (Figure 4c). b-arr1 silencing, or complex impairs the binding of the others to the target gene b-arr1–Q394L expression, significantly inhibited the recruitment promoters, identify p300 and b-arr1 as two new components of both p300 and b-catenin on these promoters (Figure 4d). required for nuclear b-catenin function. The human ETP sequence Moreover, on ET-1 stimulation, b-arr1, b-catenin and p300 co- contains four functional TCF-binding elements (TBEs).32,33 To occupied the ETP (Figure 4e), confirming that nuclear b-arr1 is determine the presence of the multimeric complex on each TBE involved in recruiting p300 and b-catenin to the target genomic site on the ETP together with acetylated H3 and H4, we performed regions of b-catenin, as demonstrated by ChIP-re-ChIP assays. ChIP assays using pairs for three specific promoter fragments, Altogether, these results reveal that b-arr1 might promote the from À 1798 to À 22-bp surrounding the transcriptional initiation dissociation of HDAC1 from b-catenin/TCF4 nuclear complex while site of the ET-1 gene, containing TBE 1, 2, 3–4, or for a promoter increasing p300 binding to the b-catenin target gene promoters. fragment not containing TBE. ET-1 enhanced acetylation of H3 and The potential role of b-arr1 in p300-mediated gene-specific H4 and recruitment of b-arr1, b-catenin and p300 on ETP in all four acetylation was then investigated. ET-1 stimulation enhanced TBE regions, but not in the not containing TBE region time-dependent recruitment of H3 and H4 acetylated and of (Supplementary Figure S5C), indicating that that b-arr1 is critically b-catenin, b-arr1 and p300 on ET-1 and MMP-2 promoters involved in promoting histone acetylation of H3 and H4 at the (Figure 5a). Moreover, in ET-1-treated cells silenced for b-arr1, or ET-1 locus containing the binding elements for b-catenin. At a for b-catenin, or expressing b-arr1–Q394L, or in cells transfected functional level, we tested the role of nuclear b-arr1 in the activity with p300DHAT, carrying a deletion of p300 acetyltransferase of a reporter gene with a human ETP sequence spanning À 1300 domain, no acetylation of histone H3 and H4 was observed to þ 230-bp surrounding the transcriptional initiation site and (Figure 5b). Among the conserved lysine residues susceptible to containing only a functional TBE located at À 73 to À 67 bp and a acetylation by p300 in histone H3,37 ET-1 treatment increased the mutant reporter construct (muETP) generated by deleting the levels of both lysine 18 and 27 of histone H3 (H3K18Ac and functional TBE from the ETP.32 Deletion of the functional TBE H3K27Ac) on ET-1, MMP-2 and Cyclin D1 promoters. Moreover, completely abolished the ETP activity (Figure 5e). More impor- these acetylations were significantly reduced in cells silenced for tantly, the overexpression of exogenous b-arr1, but not b-arr1–

Oncogene (2013) 5066 – 5077 & 2013 Macmillan Publishers Limited b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5071

Figure 4. ET-1 stimulates the displacement of HDAC1 and the recruitment of b-arr1, b-catenin, and p300 on the promoter regions of speciﬁc b-catenin target genes. (a) HEY cells were transfected with scramble (SCR) or sh-b-arr1 and treated with ET-1 for 15 min and the binding of b-arr1, b-cat, TCF4 and HDAC1, to ET-1 and MMP-2 promoter regions was measured by ChIP assay. (b) HEY cells were transfected with SCR, or si-p300, or sh-b-arr1, or si-b-catenin, treated with ET-1 for 15 min and nuclear fractions were IP with anti-b-arr1, or anti-b-cat, or anti-p300 Abs, or IRR immunoglobulin G (IgG), and immunoblotting (IB) with anti-p300, anti-b-cat, and anti-b-arr1 Abs. (c) HEY cells were transfected with SCR or sh-b-arr1 and treated with ET-1 for indicated times and the binding of b-arr1, b-catenin and p300 to ET-1, Cyclin D1 and MMP-2 promoter regions was measured by ChIP assay. (d) HEY cells transfected with b-arr1–FLAG or b-arr1–Q394L–FLAG expression vectors, were treated with ET-1 for indicated times and the binding of b-arr1, b-catenin and p300 to Cyclin D1 promoter region was measured by ChIP assay. (e) HEY cells were treated with ET-1 for 15 min and the co-occupancy of b-arr1/b-catenin, p300/b-arr1 and b-cat/p300 to ETP region was measured by ChIP- re-ChIP assays. b-arr1, p300 and b-cat Abs were used for the ﬁrst ChIP and then b-catenin, b-arr1 and p300 Abs were used, respectively, for the second IP.

Q394L, was able to enhance the ETP activity, which was not b-catenin (Figure 5f, Supplementary Figures S5D and S5E). These recovered when b-arr1 was overexpressed in HEY cells transfected effects were rescued by the re-expression of exogenous b-arr1, with muETP (Figure 5e). To further evaluate the role of nuclear but not by the mutant b-arr1–Q394L, indicating that b-arr1 b-arr1–b-catenin interaction in the promoter activities, we specifically binds to b-catenin and transactivates the specific gene performed reporter assays. A significant decrease in the ET-1, promoters (Figure 5f and Supplementary Figure S5F). MMP-2 and Cyclin D1 promoter activity and expression was Altogether, these results showed that recruitment of p300 and observed in cells silenced for b-arr1, as well as in cells silenced for b-arr1 to b-catenin-responsive promoters was a specific event

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5072

Figure 5. p300 interacts with b-arr1 and b-catenin to mediate H3 and H4 acetylation on b-catenin-dependent gene transcription. (a) HEY cells were treated with ET-1 for indicated times and the binding of b-catenin, b-arr1, p300 and the levels of acetylated (Ac) H3 and H4 histones to ET-1 and MMP-2 promoter regions was measured by ChIP assay. (b) HEY cells, transfected with scramble (SCR), or sh-b-arr1, or si-b-cat, or rescued with p300DHAT, or b-arr1–Q394L, were treated with ET-1 for 15 min and the binding of b-arr1, b-catenin, p300, AcH3 and AcH4 to ETP region was measured by ChIP assay. (c) HEY cells, transfected with SCR, or sh-b-arr1, were treated with ET-1 for 15 min and the binding of speciﬁc H3K18Ac and H4K27Ac to ET-1, Cyclin D1 and MMP-2 promoter regions was measured by ChIP assay. (d) Quantitative reverse transcriptase (qRT)–PCR analysis of ET-1, MMP-2 and Cyclin D1 expression in HEY cells transfected with SCR, or si-p300, or si-b-catenin. Bars indicate averages of values from duplicate qRT–PCR reactions ±s.e.m. (e) HEY cells transfected with Mock, or b-arr1, or b-arr1–Q394L expression vectors were co-transfected with a reporter plasmid containing wild-type (ETP) or mutant ETP (muETP). Values are mean±s.e.m. from triplicate samples. (f) HEY cells transfected with SCR or sh-b-arr1, and rescued with Mock or b-arr1 or b-arr1–Q394L expression vectors were co-transfected with ET-1 or Cyclin D1 or MMP-2 promoter sequences and treated with 100 nM ET-1. Values are mean±s.e.m. for triplicate samples from six independent experiments.

induced by ET-1/ETAR in EOC cells. The formation of this nuclear Nuclear b-arr1 is required for ETAR-mediated invasive behavior

complex regulates histone acetylation in speciﬁc chromosomal To examine whether nuclear b-arr1 was essential for ETAR- regions, which is most likely the mechanism upregulating ET-1, mediated b-catenin regulated genes involved in cell motility, we MMP-2 and Cyclin D1 expression. performed migration and invasion assays in HEY cells. Cell

Oncogene (2013) 5066 – 5077 & 2013 Macmillan Publishers Limited b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5073

Figure 6. ETAR-dependent b-arr1-mediated transcriptional complex induces invasion and EMT behavior. (a) Cell migration and invasion assays were performed in HEY cells, transfected with scramble (SCR) or sh-b-arr1, and rescued with Mock or b-arr1 or b-arr1–Q394L expression vectors. Serum-free medium (C) or ET-1 (100 nM) was used as chemoattractants. The quantifications of transwell migration and invasion experiments from three independent experiments ±s.e.m. each performed in triplicate are shown. (b) Representative images of cells allowed to invade in a transwell invasion chamber after treatment as in (a), then fixed and stained with crystal violet. (c) E-cadherin, Snail, Slug, Twist and N-cadherin expression in cells transfected as in (a) evaluated by quantitative reverse transcriptase (qRT)–PCR. Bars indicate averages of values from duplicate qRT–PCR reactions ±s.e.m. migration and invasion induced by ET-1 was significantly inhibited b-arr1, but not b-arr1–Q394L, reverted the expression of EMT by b-arr1 silencing (Figures 6a and b). Most importantly, the re- markers, confirming the role of nuclear b-arr1 in inducing invasive expression of b-arr1, but not mutant b-arr1–Q394L, was able to behavior and EMT in these cells. rescue the ability of these cells to migrate and invade (Figures 6a and b), confirming that nuclear b-arr1 is a check-point of invasive signals in EOC cells. Given the role of ET-1/ETAR axis in inducing Loss of b-arr1 inhibits metastatic potential of ovarian cancer EMT in EOC cells previously demonstrated, we analyzed xenografts whether nuclear b-arr1 was also involved in ETAR-driven molecular To demonstrate that nuclear b-arr1 is required for metastatization changes consistent with EMT,38,39 a key mechanism that in vivo, we implanted parental HEY cells or HEY cells stably induces invasion and metastasis.40 To this end, we examined depleted of b-arr1 by short hairpin RNAs (shRNA) and/or re- the expression of the epithelial marker E-cadherin and its expressing b-arr1 or mutant b-arr1–Q394L into the peritoneal transcriptional regulators, Snail, Slug and Twist, and other cavity (orthotopic site) of female nude mice and followed their mesenchymal markers, such N-cadherin. Downregulation of metastatization pattern on the peritoneal surface, omentum, mRNA expression levels for Snail, Slug, Twist and N-cadherin and small bowel, mesentery, and in both ovaries (Figure 7a and in parallel upregulation of E-cadherin was observed in HEY cells Supplementary Figure S6A). Two groups of mice were injected silenced for b-arr1 (Figure 6c). Moreover, the re-expression of with parental HEY cells, and after 1 week were treated with vehicle

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5074

Figure 7. Loss of b-arr1 inhibits metastatic potential of ovarian cancer xenografts. (a) Female nude mice were intraperitoneally injected with HEY cells or HEY cells expressing sh-scramble (SCR) þ Mock or sh-b-arr1 (clones #3 or #5) þ Mock or sh-b-arr1 (#3) þ b-arr1 or sh-b-arr1 (#3) þ b- arr1-Q394L. One week after tumor cell injection, two groups of mice injected with untransfected HEY cells were treated with vehicle control (C), or zibotentan for 4 weeks. After 5 weeks, all mice were euthanized and intraperitoneal organs were examined for visible metastases. Values represent the average ±s.e.m. of 10 mice for group from three independent experiments. (b) Binding of b-arr1 and b-catenin to ET-1, MMP-2 and Cyclin D1 promoter regions was measured by ChIP assays on metastatic nodules from mice described in (a). (c) The occupancy of b-arr1 and b-catenin to ET-1, Cyclin D1 and MMP-2 promoter regions was measured by ChIP assays in 20 human EOC tissues and in 2 non- tumoral ovarian tissues. The results show 10 representative samples of 20 EOC tissues.

or zibotentan (21 mg/kg/day intraperitoneally) for 4 weeks. Wild- DISCUSSION type and scramble-silenced HEY cells produce a comparable The b-catenin signaling pathway is aberrantly activated in human number of metastases (Figure 7a). At the end of 5 weeks, b-arr1 cancers and its activation results in dramatic changes in chromatin silencing significantly decreased the number of visible metastases remodeling.23 A key feature of this signaling activation is b-catenin (Figure 7a and Supplementary Figure S6A), in a manner that nuclear localization, for which the underlying molecular mimicked the effects of zibotentan treatment. Moreover, con- mechanisms remain elusive. Here, we show a unique function of sistent with in vitro results, the re-expression of b-arr1 was b-arr1 required for nuclear b-catenin activity in promoting tumor sufficient to induce metastasis, whereas the expression of b-arr1– metastasis. We demonstrated that in EOC cells, ETAR activation by Q394L reduced the metastatic spread (Figure 7a). ET-1 induces nuclear translocation of b-arr1. In the nucleus, b-arr1 To assess whether b-arr1 and b-catenin were selectively interacts with b-catenin and p300 to form a functional complex enriched at the ET-1, MMP-2 and Cyclin D1 promoters in vivo,we with TCF4 on target-gene promoters, such as Axin 2, ET-1, Cyclin performed ChIP assays in metastatic nodules from different D1 and MMP-2, which have a key role during cell invasion. This groups of mice. We detected binding between these promoters functional platform is necessary for epigenetic modification, such and b-arr1 and b-catenin in metastases from scramble Mock- as histone acetylation, and gene expression. Thus, in human EOC transfected cells but not in metastases from sh-b-arr1-Mock samples a tight association between nuclear b-arr1 and b-catenin (Figure 7b) demonstrating in vivo that the recruitment of nuclear occurs on ET-1, Cyclin D1 and MMP-2 promoters. Therefore, the b-arr1 and b-catenin to b-catenin-responsive promoters was a b-arr1–b-catenin interaction represents an important mechanism critical event induced by ETAR in promoting metastastic diffusion for controlling EOC progression. during EOC progression. Recent results have highlighted a new role for b-arr1 by which G-protein-coupled receptor may regulate gene transcription, b-arr1–b-catenin nuclear complexes in human ovarian carcinomas suggesting that this mechanism might mediate new biological To more directly probe the in vivo association between b-arr1 and functions of b-arr1 in pathophysiological conditions, such as b-catenin to its responsive promoters in human ovarian cancer cancer.17–20 Although proteomic analysis has revealed that b-arr1 tissues, ChIP assays were performed on 20 advanced-stage EOC may directly interact with many proteins,13 no data have been tumors and on two non-tumoral ovarian tissues. This analysis previously reported regarding the interaction of b-arr1 with showed increased levels of b-arr1 and b-catenin associated with b-catenin. Apart from the role of b-arr1 into regulating b-catenin ET-1, Cyclin D1 and MMP-2 promoters in 70%, 80% and 80%, signaling in the cytosolic compartment,5,25–27 our findings unveil respectively, of tumor tissues analyzed compared with normal a novel role of b-arr1 as a nuclear platform in b-catenin ovarian tissues (Figure 7c, Supplementary Table S1 and transcriptional activity, depending on its subcellular nuclear Supplementary Figure 6B). Notably, increased levels of b-arr1 distribution thus accounting for the epigenetic modification and and b-catenin were associated with ET-1 (77%), Cyclin D1 (88%) gene transcription of selected target genes. In particular, our and MMP-2 (88%) promoters in grade 3–4 tumors, whereas no findings identify b-arr1 as a new interacting partner of b-catenin in grade 2 tumors co-expressed b-arr1 and b-catenin on these the nucleus, where it displaces HDAC1 and recruits histone promoters. Together, these data provide further support for a acetylase p300 to specific gene promoters increasing local direct and functional association between b-arr1 and b-catenin in acetylation of H3 and H4 histones and transcription of genes human EOC samples at specific set of genes, correlating with critical in promoting invasive behavior. In this context, b-arr1 tumor grade, and suggest that regulation of ETAR-driven gene appears to control two critical and related aspects of b-catenin expression by b-arr1 and b-catenin contributes to human EOC nuclear function: nuclear accumulation and assembly of a progression. transcription activation complex. Hence, our study provides

Oncogene (2013) 5066 – 5077 & 2013 Macmillan Publishers Limited b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5075 novel mechanistic insights by which b-arr1–b-catenin represents (Macclesfield, UK) and added 30 min before the ET-1. Leptomycin B was the initial scaffold on which transcriptional regulatory complexes purchased from Sigma-Aldrich (Sigma-Aldrich Srl, Milan, Italy). could be built to regulate different biological outcomes. Distinct b-arr1 complexes in fact can recruit or interact with factors that Fluorescence microscopy analysis modulate the transcription of specific target genes orchestrating a Cells transfected with b-arr1–FLAG were fixed in 2% formaldheyde, network that regulates cell migration, EMT and metastasis permeabilized with 0.25% Triton-X 100 and then immunostained with formation. the primary Ab to FLAG (Sigma-Aldrich) and to b-catenin (Santa Cruz Despite the wealth of information documenting several aspects Biotechnology Inc., Santa Cruz, CA, USA) and counterstained with 40,6- of ET-1 signaling in cancer, deciphering the complex signaling diamidino-2-phenylindole. Confocal laser scanning microscopy observa- networks that are governed by ET-1 and that influence ET-1 tions were performed with a Leica TCS SP2 AOBS apparatus, using 63 Â / autocrine circuit, is still incomplete. As ET-1 elicits pleiotropic 1.4 NA objective and excitation spectral laser lines at 405, 488 and 594 nm activities, it would be intriguing to speculate whether these and using the Leica Confocal Software (Leica Lasertechnik version 2.6 rel. activities are mediated by chromatin remodeling in the regulation 1537, Heidelberg, Germany) and Adobe Photoshop software programs (Adobe system Incorporated, Rome, Italy). Signals from different fluor- of transcription. Here, we demonstrated that ET-1/ETAR axis escent probes were taken in sequential scan mode (three-dimensional modulates the self-amplifying positive feedback loop of reconstruction images) and colocalization was visualized in merge images. b-catenin-ET-1 through the nuclear function of b-arr1. Thus, ET-1 Further details are given in Supplementary Methods. signaling through b-arr1 promotes the formation of a b-arr1/ b-catenin/p300 transcription complex on ETP region. Hence, one can envisage a reciprocal relationship in which ET-1 stabilizes Immunoblotting and IP b-catenin38,39 resulting in the autoregulatory b-catenin-mediated Cells were lysed in lysis buffer (250 mM NaCl, 50 mM HEPES (pH 7.4), 1 mM transcription of ET-1 itself. EDTA, 1% Nonidet P-40, protease inhibitors). NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Illkirch-Cedex, Given the importance of b-catenin signaling in human cancers 41,42 France) were used to separate cytoplasmic and nuclear fractions. Further and stem cell regulation, our findings establish that ETAR- details are given in Supplementary Methods. mediated nuclear import of b-arr1 might allow fine-tuning of responses during tumor progression through its interaction with b-catenin. b-arr1 plasmid and transfection constructs For exogenous expression of b-arr1, we used pcDNA3–b-arr1–FLAG (wild- Of note, the in vivo results showing the active role of ETAR- driven nuclear b-arr1 in metastatic dissemination, as well as the type) plasmid construct, a ‘wobble’ mutant construct encoding rat b-arr1 presence of b-arr1–b-catenin nuclear complexes in the metastatic sequences resistant to small interfering RNA targeting kindly provided by Dr Robert Lefkowitz (Howard Hughes Medical Institute, Duke University, nodules indicate that ETAR, in an b-arr1–b-catenin-dependent way, Durham, NC, USA), b-arr1–GFP construct kindly provided by Dr Dirk may engage a distinct set of genes, enabling EOC cells to invade Roosterman (Innere Medizin D, Universita¨tsklinikum Mu¨nster, Mu¨nster, and metastasize. Moreover, the present results also suggest that Germany) and b-arr2–YFP, kindly provided by Dr Michel Bouvier (Institut de co-occupancy of b-arr1 and b-catenin on selected gene promoters Recherche en Immunologie et en Cance´rologie, Universite´ de Montreál, in human EOC tissues is associated with advanced-stage tumors. Montreal, Quebec, Canada). Mutation of Gln-394 in Leu of b-arr1–FLAG The present findings, which are consistent with our previous construct was done by using QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). All constructs were verified observations reporting that co-expression of ETAR and b-arr1 in human EOC tissues may be indicative of the malignant by sequencing. For transient expression of b-arr1–FLAG or b-arr1–Q394L– 5 FLAG, constructs were transfected in cells using LipofectAMINE 2000 phenotype, and that ETAR overexpression was associated with 38 reagent (Life Technologies Italia, Monza, Italy) following the manufacturer’s the switch of cadherin expression, highlight the pathobiological instructions. Cells transfected with the empty vector pCDNA3 were used as relevance of ETAR/b-arr1/b-catenin in the regulation of EMT and control (Mock transfected). For their stable expression, transfected cells aggressive behavior. In conclusion, we propose a model in which were selected by using 500 mg/ml G418 sulfate (Calbiochem-Novabiochem ET-1 initiates direct interaction between b-arr1 and b-catenin to Corporation, San Diego, CA, USA). G418-resistant cells were pooled 3 regulate epigenetic modifications driving EMT and tumor weeks after transfection and thereafter cells were maintained in media progression through forming a multiprotein complex. Therefore, containing 500 mg/ml of G418. For transient expression of full-length or four different b-arr1 deletion b-arr1 promotes the compartmentalization of ETAR signaling, which mediates qualitative signal-dependent histone modification mutants, we used constructs tagged with an AU5 epitope at their and gene transcription. Thus, ET R blockade, which by preventing carboxyl termini kindly provided by Professor Richard D Ye (Department of A Pharmacology, College of Medicine, University of Illinois, Chicago, IL, nuclear b-arr1–b-catenin association inhibits transcription of USA) and transfected using LipofectAMINE 2000 reagent (Life Technologies genes that trigger EMT and metastatic process, may represent a Italia). targeted therapeutic approach that warrants exploration in EOC To evaluate the role of p300, we used a full-length p300 construct, kindly treatment.43,44 provided by Professor Vittorio Sartorelli (Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA) and a MATERIALS AND METHODS p300DHAT construct (carrying a deletion of p300 acetyltransferase domain 1603–1658), kindly provided by Dr A Felsani (CNR, Institute of Neurobiol- Cell lines and reagents ogy and Molecular Medicine, Rome, Italy). Established human ovarian serous adenocarcinoma cell lines, HEY and OVCA 433, obtained by Professor Giovanni Scambia (Catholic University School of Medicine, Rome, Italy), and A2780, purchased from European RNA silencing Collection of Cell Cultures (Salisbury, UK), cultured as previously The small inhibitor duplex RNAs (small interfering RNA) were from described,42,45 were passaged in our laboratory for o3 months after Dharmacon (Fisher Scientific, Illkirch Cedex , France) and were human ON- resuscitation. All cells were tested routinely for cell proliferation (3H- TARGET plus SMART pool si-b-catenin #L-003482-00-0050, si-p300: #L- thymidine incorporation) as well as mycoplasma contamination, and they 003486-00-0050, si-ETAR: #L-005485-00-0050. The siCONTROL Nontarget- showed similar growth rate and negative mycoplasma during the ing Pool (#D-001810-10-20) was used as negative control. Cells were experiments. Cells were serum starved by incubation 24 h in serum-free transfected with 100 nmol/l small interfering RNAs using LipofectAMINE medium (RPMI for HEY cells and Dulbecco’s modified Eagle’s medium for reagent (Life Technologies Italia). After 48 h, total proteins were isolated OVCA 433 and A2780 cells) before each experiment. ET-1 was purchased and analyzed by immunoblotting analysis. from Peninsula Laboratories (San Carlos, CA, USA) and clinical grade The lentiviral-based shRNAs plasmids (pLKO.1 plasmids) used to zibotentan N-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl] knockdown b-arr1 were purchased from Sigma-Aldrich. Five plasmid- phenyl) pyridine-sulfonamide, was kindly provided by AstraZeneca clones were tested for their knockdown efficiency (TRCN0000230149;

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077 b-arrestin-1 is an epigenetic regulator of b-catenin L Rosano` et al 5076 TRCN0000230147; TRCN0000005160; TRCN0000005161; TRCN0000230150). Statistical analysis Non-Target shRNA Control Vector (SHC002) was used as negative control. Each experiment was repeated at least three times with comparable Transient transfection was performed by adding shRNA plasmid along with results, unless indicated otherwise. Results are expressed as mean±s.e.m. LipofectAMINE 2000 (Life Technologies Italia) in six-well plates for 48 h Differences between group were studied using analysis of variance (Supplementary Methods). followed by a Bonferroni post test. Statistical analysis was done with SPSS The silencer selected pre-designed and validated siTCF4 (#4392421) and software (SPSS 11, SPSS Inc., Chicago, IL, USA). silencer selected negative control #1 (Life Technologies Italia) were used to silence TCF4 and as negative control, respectively. CONFLICT OF INTEREST Reporter assay The authors declare no conflict of interest. Reporter activity was measured using the Luciferase assay system (Promega Corporation, Fitchburg, WI, USA) in cells co-transfected with pTOP/Flash or pFOP/Flash (Upstate Biotech, Billerica, MA, USA) or ETP ACKNOWLEDGEMENTS reporter sequence and 100 ng pCMV–b-galactosidase (Promega Corpora- We gratefully acknowledge Valentina Caprara and Aldo Lupo for excellent technical tion) vectors using LipofectAMINE 2000 reagent (Life Technologies Italia). assistance, Maria Vincenza Sarcone for secretarial assistance, Dr Maurizio Fanciulli for Luciferase assay were carried out according to standard procedures valuable comments, Dr Robert Lefkowitz (Howard Hughes Medical Institute, Duke (Supplementary Methods). University) for his insightful comments and for kindly providing b-arr1 expression vector; Dr Zhuohua Zhang (University of California San Diego School of Medicine, La Jolla, CA, USA) for ET-1 promoter reporter and its mutant control, and Dr Tania Chromatin IP Merlino for the formal revision of the manuscript. This work was supported by the Chromatin was extracted from cells and ChIP was done as described in grant funded by the Associazione Italiana Ricerca sul Cancro. Supplementary Methods. REFERENCES Enzyme-linked immunosorbent assay 1 Nelson J, Bagnato A, Battistini B, Nisen P. The endothelin axis: emerging role in The release of ET-1 in the conditioned media of serum-starved cells was cancer. Nat Rev Cancer 2003; 3: 110–116. measured in triplicate on microtiter plates by using an ET-1 ELISA kit 2 Rosano` L, Bagnato A. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

& 2013 Macmillan Publishers Limited Oncogene (2013) 5066 – 5077