Translation Regulatory Factor RBM3 Is a Proto-Oncogene That Prevents Mitotic Catastrophe

Oncogene (2008) 27, 4544–4556 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe

SM Sureban1,6, S Ramalingam1,6, G Natarajan1, R May1, D Subramaniam1, KS Bishnupuri2, AR Morrison2, BK Dieckgraefe2, DJ Brackett3,4, RG Postier3,4, CW Houchen1,4 and S Anant1,4,5

1Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 2Department of Medicine, Washington University School of Medicine, St Louis, MO, USA; 3Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 4OU Cancer Institute, Oklahoma City, OK, USA and 5Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

RNA-binding proteins play a key role in post-transcrip- Introduction tional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory Dysregulated expression of oncogenes and tumor protein, is significantly upregulated in human tumors, suppressors is a critical regulator of tumorigenesis. including a stage-dependent increase in colorectal tumors. Known targets that lead to a tumorigenic phenotype Forced RBM3 overexpression in NIH3T3 mouse fibro- include cyclooxygenase (COX)-2, interleukin (IL)-8 and blasts and SW480 human colon epithelial cells increases vascular endothelial growth factor (VEGF) (Dubois cell proliferation and development of compact multi- et al., 1998; Dixon et al., 2001; Wang et al., 2005). COX- cellular spheroids in soft agar suggesting the ability to 2 is the rate-limiting enzyme in the production of induce anchorage-independent growth. In contrast, down- prostaglandins, an important mediator of various regulating RBM3 in HCT116 colon cancer cells with cellular processes including increased proliferation, specific siRNA decreases cell growth in culture, which was apoptosis resistance and enhanced angiogenesis partially overcome when treated with prostaglandin E2,a (Mukhopadhyay et al., 2003b; Krysan et al., 2005). product of cyclooxygenase (COX)-2 enzyme activity. COX-2 overexpression occurs in multiple tumors, and Knockdown also resulted in the growth arrest of tumor can be observed at various stages of tumorigenesis xenografts. We have also identified that RBM3 knock- (Eberhart et al., 1994). Although transcriptional activa- down increases caspase-mediated apoptosis coupled with tion of COX-2 is an early event, it is also regulated at the nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and post-transcriptional levels of mRNA stability and Chk2 kinases, implying that under conditions of RBM3 translation (Dixon et al., 2000). downregulation, cells undergo mitotic catastrophe. RBM3 Distinct cis-acting AU-rich elements (ARE) sequences enhances COX-2, IL-8 and VEGF mRNA stability and located within the 30untranslated region (30UTR) have translation. Conversely, RBM3 knockdown results in loss been identified in the COX-2, IL-8 and VEGF mRNA in the translation of these transcripts. These data that regulate mRNA stability and translation (Ristimaki demonstrate that the RNA stabilizing and translation et al., 1996; Cokand Morrison, 2001; Dixon et al., regulatory protein RBM3 is a novel proto-oncogene that 2001). Specifically, the first 60 nucleotides in COX-2 induces transformation when overexpressed and is essen- 30UTR encode AREs, which regulate mRNA stability tial for cells to progress through mitosis. and translation (Cokand Morrison, 2001; Mukhopad- Oncogene (2008) 27, 4544–4556; doi:10.1038/onc.2008.97; hyay et al., 2003a). RNA-binding protein HuR interacts published online 21 April 2008 with these ARE sequences to regulate the stability and translation of COX-2 mRNA (Dixon et al., 2000; Cok Keywords: cyclooxygenase-2; RNA stability; transfor- and Morrison, 2001). HuR is also upregulated in mation; mitosis; cell cycle various cancers (Nabors et al., 2001; Erkinheimo et al., 2003; Denkert et al., 2004, 2006). Here, we demonstrate that RBM3 is a proto- oncogene, whose product binds COX-2 ARE and regulates COX-2 mRNA stability and translation (Danno et al., 1997). RBM3 mRNA and protein are upregulated in human tumors. RBM3 induces non- Correspondence: Dr S Anant, Departments of Medicine and Cell transformed cells to grow in an anchorage-independent Biology, University of Oklahoma Health Sciences Center, 970 Stanton manner. In contrast, downregulating RBM3 with short L. Young Boulevard, WP1360, Oklahoma City, OK 73126, USA. interfering RNA (siRNA) decreases HCT116 colon E-mail: [email protected] 6These authors contributed equally to this work. adenocarcinoma cell proliferation. Moreover, there is Received 31 October 2007; revised 15 January 2008; accepted 1 March an increase in apoptosis and activation of checkpoint- 2008; published online 21 April 2008 related proteins that enhance cell cycle progression at RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4545 the level of mitosis suggesting mitotic catastrophe. fied in neurons (Smart et al., 2007). However, these Furthermore, downregulating RBM3 in nude mice isoforms were not observed in the colon cancer cells tumor xenografts decreased angiogenesis. We also (data not shown). HuR was also significantly upregu- demonstrate that RBM3 shuttles between nucleus and lated in the cancers, confirming previous reports (Dixon cytoplasm. et al., 2001; Nabors et al., 2001; Erkinheimo et al., 2003). Details of colorectal adenocarcinoma and specific expression pattern for the three genes are presented in Results Supplementary Table 1. Western blot analyses for protein expression confirmed that both RBM3 and RBM3 is induced in colon cancers HuR are expressed at higher levels in the tumors, with at RBM3 regulates global mRNA translation by interact- least a 10-fold increase in RBM3 (Figure 1b). Immuno- ing with the 60S ribosome (Derry et al., 1995; Danno histochemistry analyses demonstrated RBM3 expression et al., 1997; Dresios et al., 2005). In addition, RBM3 was in a single cell within normal, human colonic crypts identified through its binding to AU-rich sequences in (Figure 1c). However, in the cancer tissues, the COX-2 30UTR (Cokand Morrison, 2001). As COX-2 is expression was widespread, and both nuclear and significantly upregulated in cancers, we first examined cytoplasmic, especially at the later stages of tumorigen- RBM3 expression in colon cancers. There was a stage- esis (Figure 1c). In addition, high levels of RBM3 dependent increase in RBM3 mRNA levels compared to expression was observed in other tumors, including the paired uninvolved tissues, with the highest levels breast, pancreas, colon, lung, ovary and prostate observed in later stages (Figure 1a). Two novel isoforms, (Figure 1d). As a reference, the tumor presented for resulting from alternative splicing were recently identi- colon in Figure 1d is from an adenocarcinoma. High

Figure 1 RBM3 and HuR are overexpressed in tumors. (a) RBM3 and HuR gene expression in tumor and surrounding uninvolved tissues. Significant induction of RBM3 mRNA expression was observed in stages 2–4, whereas HuR was induced only in stage 1. * denote statistically significant differences (*Po0.01). (b) Western blot analyses of total tissue extracts for RBM3 and HuR. Actin was determined as control for gel loading. RBM3 expression is significantly upregulated in the tumors. (c) Immunohistochemistry for RBM3 in normal and colon cancer tissues. Brown stain demonstrates the location of the RBM3 protein in the tissues. (d) Immunohistochemistry for HuR and RBM3 in various human tumors. Brown stain shows the location of the protein.

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4546 levels of HuR expression were also observed in breast, HCT116 cell proliferation. Although the scrambled colon, lung and ovarian tumors (Figure 1d). Thus, siRNA did not affect proliferation, siRNA-mediated expression of RBM3 is significantly induced in cancers downregulation of RBM3 significantly reduced it and is localized in both the nucleus and cytoplasm. (Figure 3d). A 50% reduction in HCT116 cell proliferation was observed with 50 nM siRNA. To determine RBM3 overexpression induces anchorage-independent whether this was due to loss of COX-2, cells were also growth treated with 1 mM of prostaglandin E2 (PGE2), the To determine whether RBM3 overexpression affects product of COX-2 enzyme activity. There were sig- growth rate, NIH3T3 cells stably expressing RBM3 were nificantly higher levels of proliferation in cells also generated. There was a significantly higher level of treated with PGE2 for 24 h, suggesting that the proliferation in the RBM3-transfected cells when decreased proliferation resulting from reducing RBM3 compared to the wild-type, vector-transfected controls levels is, in part, due to decreased COX-2-mediated (Figure 2a). Next, we tested whether RBM3 expressing PGE2 synthesis. Similar results were observed with HT- cells can grow in an anchorage-independent manner, a 29 cells (data not shown). Next, we determined the effect characteristic of transformed cells. All the fast growing of RBM3 downregulation on the growth of HCT116 RBM3-expressing cells show anchorage-independent tumor cell xenografts in nude mice. After the tumors phenotype and grow in 0.3% agar (Figure 2b). More were allowed to develop (15 days), siRNA was injected a importantly, the cells exhibited obvious morphological total of five times at an interval of 3 days. Tumors that differences. The NIH3T3-RBM3 cells formed tight, received either liposome preparation without any densely packed multi-cellular spheroids, where single siRNA or those that included the scrambled siRNA cells could not be distinguished. Moreover, when the continued to grow, with tumor volume reaching 4 103 colony size was compared to that produced by HT-29 and 6 103 mm3, respectively (Figure 3e). On the other colon adenocarcinoma cells, the NIH3T3-RBM3 colo- hand, tumors that received RBM3-specific siRNA were nies were significantly bigger suggesting an aggressive arrested in growth. RBM3 silencing in the tumors was phenotype (Figure 2c). To determine whether RBM3 confirmed by real-time PCR and immunohistochemistry overexpression affected growth of cells that were already analyses (Figures 3f and g). In addition, there was a transformed, SW480 colon cancer cells were stably fivefold decrease in COX-2 mRNA when compared to transfected with RBM3. Cells overexpressing RBM3 the controls in the xenografts where RBM3 was had higher levels of proliferation when compared to knocked down (Figure 3f). Furthermore, although vector transfected controls and formed larger colonies in COX-2 protein was widely expressed in the controls, soft agar (Figures 2e and f). Moreover, the colonies with the expression was significantly reduced in tumors RBM3 overexpression were significantly larger than lacking RBM3 (Figure 3g). These data suggest that those observed with vector transfected or untransfected RBM3 is essential for COX-2 expression in vivo. COX-2 controls (Figures 2f and g). Western blot analyses derived PGE2 regulates expression of angiogenesis demonstrated that the expression of COX-2, VEGF and inducing factors VEGF and IL-8. Real-time PCR cyclin D1 was upregulated in both NIH3T3 and SW480 analyses demonstrated a significant decrease in VEGF cells that had RBM3 overexpression (Figures 2d and h). and IL-8 mRNA in the RBM3 targeted tumors, the Together, these data demonstrate that RNA-binding amounts similar to that seen with COX-2 (Figure 3f). protein RBM3 is a proto-oncogene that induces Furthermore, staining for CD-31, a platelet endothelial anchorage-independent growth when overexpressed. cell adhesion molecule that marks the endothelial cells in blood vessels demonstrated a 70% reduction in micro- RBM3 is essential for tumor growth vessel density in the RBM3-targeted tumors (Figure 3g, Next, we determined the effect of downregulating Supplementary Figure S1). Together, these data suggest RBM3 expression on proliferation. RBM3 protein levels that targeting RBM3 effectively suppressed capillary in HCT116 colon adenocarcinoma cells were signifi- formation through decreased expression of angiogenic cantly downregulated after transfection with an RBM3- factors, resulting in loss of tumor growth. specific, but not scrambled siRNA (Figures 3a and b). Furthermore, COX-2 mRNA and proteins levels were RBM3 is necessary for overcoming mitotic catastrophe decreased in these cells (Figures 3a and c). Next, we To characterize the inhibition of cell growth by RBM3 investigated the effect of RBM3 downregulation on depletion, we next analysed cell cycle progression. While

Figure 2 RBM3 overexpression induces oncogenic transformation. (a) Proliferation of three independent NIH3T3-RBM3 clones were significantly higher than that observed with three independent NIH3T3-vector clones. (b) NIH3T3-RBM3 cells develop large colonies in soft agar, which are bigger than those formed by HT-29 cells. HuR overexpressing cells, on the other hand did not form any colonies in the soft agar. (c) Quantitative estimation of number of colonies formed in soft agar (*Po0.01). (d) Two clones of NIH3T3 cells stably expressing RBM3 were selected based on western blot analyses. Expression of COX-2, VEGF (p21 monomer and p42 dimer) and cyclin D1 increases in the RBM3 overexpressing cells. Clone 1 is the same one shown in b.(e) Proliferation of the SW480-RBM3 clones was significantly higher than that observed with SW480-vector clones. (f) SW480-RBM3 cells develop large colonies in soft agar, when compared to control, untransfected or vector transfected cells. (g) Quantitative estimation of number of colonies formed in soft agar (*Po0.01). (h) Two clones of SW480 cells stably expressing RBM3 were selected based on western blot analyses. Expression of COX-2, VEGF and cyclin D1 increases in the RBM3 overexpressing cells. Clone 2 is the same one as shown in f. *Nonspecific band. COX, cyclooxygenase; VEGF, vascular endothelial growth factor.

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4547 the scrambled siRNA did not affect the cell cycle proﬁle had 19% in this phase (Figure 4a, Supplementary Figure 3 days after transfection, RBM3-speciﬁc siRNA in- S2). However, treatment with 1 mM PGE2 in the setting creased the percentage of cells with 4N DNA content, of RBM3 suppression resulted in only 12.2% of the cells suggesting that RBM3-depleted cells are blocked at in G2/M phase, suggesting that PGE2 can override G2/M (Figure 4a). While transfection of scrambled RBM3-mediated effects on cell cycle. To determine siRNA resulted in 12.7% G2/M, RBM3-targeted cells whether there was apoptosis occurring as well following

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4548 RBM3 knockdown, the levels of TUNEL-positive cells RBM3-depleted cells undergo apoptosis while in the were determined. TUNEL-positive cells following G2/M phase of the cell cycle, which is, in part, due to RBM3 knockdown were significantly higher than that loss of COX-2 derived PGE2. To confirm that the cells seen with the scrambled siRNA (Figure 4b). However, were undergoing apoptosis while in mitosis, the xeno- treatment with 1 mM PGE2 resulted in significantly lower graft cancer tissues were co-stained for phosphorylated number of apoptotic cells, suggesting that PGE2 can histone H3 (Thr-11), a protein that is phosphorylated protect the cells from undergoing apoptosis due to loss during mitosis, and for DNA damage by TUNEL. of RBM3. This further implies that COX-2 expression Many cells in the RBM3 suppressed tumors that were due to the action of RBM3 protects the cells from TUNEL positive also expressed phosphorylated histone apoptosis in an autocrine manner through PGE2. H3 (Figure 4e). There was also an increase in phospho- Further conformation was obtained when western blot H2AX staining in the RBM3-depleted tumors (Supple- analyses were performed, which demonstrated increased mentary Figure S5). Taken together, these data suggest caspase-3 activation in the siRBM3 transfected that the knockdown of RBM3 induces mitotic cata- cells, which was suppressed by PGE2 (Supplementary strophe, where the cells undergo apoptosis while in the Figures S3, S4). To gain further insight into the process of mitosis. mechanism of G2/M cell-cycle-coupled apoptosis upon suppression of RBM3 expression, western blot analyses RBM3 is a nucleocytoplasmic shuttling protein that were performed for Cdc25c, a protein that catalyses the stabilizes COX-2, VEGF and IL-8 mRNA activation of the Cdk1:cyclin B1 kinase complex, which Previous studies have demonstrated that COX-2-derived is believed to be a rate-limiting step for entry into PGE2 induces cells to divide by enhancing mitosis mitosis (Maller, 1991; Millar and Russell, 1992). (Andreis et al., 1981; Munkarah et al., 2002; Wu et al., Suppressing RBM3 resulted in increased Cdc25c protein 2005). Furthermore, treatment of colon cancer cells with levels (Figure 4c). In addition, there was an increase in NS-398, a COX-2 selective inhibitor increased the the level of cyclin B1, which was higher than that number of cells in the G2/M phase, whereas decreasing observed in the control or scrambled siRNA transfected those in the G0/G1 phase (Yamashita et al., 2003). This cells (Figure 4c). Similar results were obtained with suggests that mechanisms to increase COX-2 expression extracts from the tumor xenografts. There was increased would result in protecting the cells from mitotic cyclin B1 in RBM3-lacking tumors (Figure 4c). Further- catastrophe. To identify the mechanism by which more, there were increased nuclear levels of cyclin B1 in RBM3 inhibits mitotic catastrophe, we next determined these tumors (Figure 4d). Two kinases, Chk1 and Chk2, the cellular functions of RBM3. Previous studies which are intermediaries of DNA damage checkpoint identified HuR and RBM3 as being in a complex bound and are activated by phosphorylation on Ser-345/Ser- to AU-rich sequences in COX-2 30UTR (Cokand 317 and Thr-68, respectively have been implicated in Morrison, 2001). Here, we observed that RBM3 inter- Ser-216 phosphorylation of Cdc25c (Canman, 2001; acts with HuR in a yeast two-hybrid analysis (Figure 5a). Walworth, 2001; Bulavin et al., 2002). In addition, Moreover, RBM3 was isolated in a yeast two-hybrid phosphorylation of p53 protein at Ser-15 is critical for screen using HuR as bait (data not shown). To confirm p53 protein stabilization and for activating its apoptotic that the two proteins interact, HuR was generated by 35 function and G2/M checkpoint (Taylor and Stark, in vitro translation in the presence of S-methionine, and 2001). Immunoblots for phosphorylated forms of incubated with recombinant GST-HuR or GST-RBM3 Chk1 (Ser-345), Chk2 (Thr-68) and p53 (Ser-15) showed fusion proteins, followed by affinity purification with a increased phosphorylation of all the three proteins over glutathione-sepharose column. Radiolabeled HuR control in the RBM3 depleted cells in culture and in the bound to RBM3, suggesting that the proteins can tumors (Figure 4c). Collectively, these data imply that interact in solution in the absence of RNA (Figure 5b).

Figure 3 RBM3 is essential for tumor growth. (a) RBM3 specific siRNA (si-RBM3), but not a scrambled siRNA (si-Scr) decreases RBM3 and COX-2 mRNA expression. * denote statistically significant differences (**Po0.01). (b and c) Western blot analysis demonstrated that RBM3 and COX-2 proteins were significantly reduced in the cells treated with RBM3-targeted siRNA. (d) Knockdown of RBM3 expression decreases colon cancer cell proliferation. HCT116 cells were transfected with increasing doses (0–100 nM) of either RBM3-specific or si-Scr. After 48 h, cells were incubated with 1 mM PGE2 for an additional 24 h. Cells transfected with RBM3-specific siRNA demonstrated significant reduction in proliferation, which was partially rescued in the presence of PGE2.* denote statistically significant differences (*Po0.05 and **Po0.01). (e) Antitumor activity of si-RBM3 in mice carrying HCT116 cell tumor xenografts. HCT116 cells were injected into the flanks of Ncr nude mice (five mice per group) and tumors were allowed to develop for 15 days. siRNA was injected directly into the tumors starting on day 15 and every third day for a total of five injections. Tumor sizes with standard error are shown from data collected at the time of every injection. si-Scr treated tumors were larger than the control carrier injected tumors, whereas si-RBM3 treated tumors were smaller. A representative excised tumor at day 28 is shown to the right. * denote statistically significant differences (*Po0.05 and **Po0.01). (f) Decreased gene expression in the si-RBM3 injected tumors. Real-time RT–PCR was performed with total RNA from the tissues and the expression of RBM3, COX-2, IL-8 and VEGF is plotted as relative to control, carrier injected tumors. (*Po0.01). (g) Immunohistochemistry for RBM3, COX-2 and CD31 in HCT116 xenografts. Presence of the protein in each panel is shown by brown stain. The sections were counterstained with hematoxylin (blue stain). There was reduced expression of RBM3 and COX-2 and decreased microvessel density. COX, cyclooxygenase; PGE2, prostaglandin E2; RT–PCR, reverse transcription–polymerase chain reaction; siRNA, short interfering RNA; VEGF, vascular endothelial growth factor.

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4549 To examine the interaction of HuR and RBM3 in be a prognostic marker for cancers (Erkinheimo et al., mammalian cells, immunostaining was performed 2003, 2005; Lopez de Silanes et al., 2003; Heinonen et al., following transient transfection of HeLa cells with 2005; Denkert et al., 2006). Given the strong nuclear N-terminal myc epitope-tagged HuR and N-terminal colocalization of HuR and RBM3, and that there is FLAG epitope-tagged RBM3 plasmids. We found that increased cytoplasmic localization of the protein in the two proteins colocalize, predominantly in the nucleus cancers, we next determined whether RBM3 also (Figure 5c). HuR is primarily nuclear, but can be exhibits shuttling activity. We performed a heterokaryon induced to redistribute from the nucleus to the cyto- assay, where we transfected Flag-tagged RBM3 or HuR plasm (Fan and Steitz, 1998a). Furthermore, cytoplas- in human HeLa cells and fused the cells with the murine mic HuR expression in cancer cells has been suggested to NIH3T3 cells. RBM3, like HuR was found in both the

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4550

Figure 4 RBM3 downregulation results in mitotic catastrophe. (a) siRNA downregulation of RBM3 increased cells in the G2/M phase. HCT116 cells were transfected at the indicated dose of either scrambled (si-Scr) or RBM3-specific (si-RBM3) siRNA for 72 h. Cell-cycle profiles were analysed by flow cytometry following PI staining for DNA content. The percentage of cells in the G2/M phase following si-RBM3 transfection was increased compared to control and si-Scr cells. Addition of PGE2 partially suppressed the RBM3 siRNA-mediated effects. (b) Knockdown of RBM3 leads to apoptosis. HCT116 cells following siRNA transfection were stained by the TUNEL method. Arrows show the TUNEL-positive cells, which were higher in si-RBM3 transfected cells, but less in cells also treated with 1 mM PGE2.(c) Loss of RBM3 induces checkpoint proteins. Lysates from HCT116 cells treated with si-Scr (50 nM) or si-RBM3 (10 and 50 nM), and tumor xenografts from the various treatments were subjected to western blot analyses using specific antibodies for phospho-Ser345 Chk-1, phospho-Thr68 Chk-2, Cdc25C, phospho-Ser15 p53 and cyclin B1. Actin was used as an internal control for loading the gels. (d) Lackof RBM3 increases cyclin B1 translocation to nucleus. Tumor xenografts were subjected to immunohistochemical staining for cyclin B1. The arrows in the si-RBM3 treated tumors indicate cyclin B1-positive cells in the nucleus. Representative photographs are a magnification of 400. (e) RBM3 depletion leads to mitotic catastrophe. Tumors treated with si-RBM3 were stained for TUNEL (green) and phosphorylated histone H3 (red). The cells positive for both are shown in the merged image with yellow stain. DAPI is used to stain the nucleus. COX, cyclooxygenase; PGE2, prostaglandin E2; RT–PCR, reverse transcription–polymerase chain reaction; siRNA, short interfering RNA; VEGF, vascular endothelial growth factor.

Figure 5 RBM3 and HuR interact and enhance stability. (a) Yeast two-hybrid interaction of RBM3 with HuR. RBM3 and HuR expressed as bait and test proteins interact in the yeast by the colonies formed on quadruple dropout media. Breakdown of the X-a-gal results in a blue colony. Tumor suppressor protein p53 and SV40 T antigen (RecT) were used as positive control for interaction, but negative for interaction with either RBM3 or HuR. (b) GST pull-down assay. 35S-methionine labeled in vitro translated HuR (35S-HuR) was incubated with either GST-RBM3 or GST-HuR. The GST-proteins were immobilized on to glutathione sepharose beads. The immobilized proteins were separated by SDS–PAGE and subjected to phosphorimager analyses. Pure GST served as negative control. (c). Colocalization of HuR and RBM3. HeLa cells were transiently transfected with plasmids expressing myc-epitope tagged HuR and FLAG-epitope tagged RBM3. Immunocytochemistry was performed for the myc and FLAG epitopes. Images for the HuR and RBM3 were merged demonstrating colocalization. Nucleus was stained by DAPI. (d) Nuclear-cytoplasmic shuttling of HuR and RBM3. Plasmids encoding FLAG-epitope tagged HuR or RBM3 were transiently transfected into human HeLa cells and subsequently fused with mouse NIH3T3 cells. The proteins were immunostained for the FLAG tag, and the nuclei by Hoescht stain to differentiate human and mouse nuclei. Mouse nuclei, seen as punctuate staining are denoted by an arrow. (e) RBM3 and HuR induce COX-2, IL-8 and VEGF mRNA expression. Ectopic expression of Flag epitope-tagged RBM3 and HuR resulted in signiﬁcant increase in endogenous COX-2, IL-8 and VEGF mRNA in HCT116 cells. There was a trend for even higher levels when proteins were coexpressed (**Po0.01). (f) COX-2 protein increased in cells expressing RBM3 and HuR. COX, cyclooxygenase; GST, glutathione S-transferase; P, GST-bound fraction; S, supernatant; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; T, total input or whole cell extract; VEGF, vascular endothelial growth factor.

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4551 human and murine nuclei implying that RBM3 is a the effect of ectopic transient FLAG-tagged RBM3 and nucleocytoplasmic shuttling protein (Figure 5d). HuR FLAG-tagged HuR on COX-2, IL-8 and VEGF mRNA shuttles to the cytoplasm where it can stabilize certain levels in HCT116 cells. Both RBM3 and HuR alone transcripts such as COX-2 (Fan and Steitz, 1998a, b; signiﬁcantly induced the endogenous expression of Peng et al., 1998; Nabors et al., 2001; Lopez de Silanes COX-2 mRNA (Figure 5e). While the steady-state levels et al., 2003). As knockdown of RBM3 decreased COX-2 of endogenous COX-2 mRNA were increased by mRNA levels, and RBM3 is a nucleocytoplasmic approximately 7-fold in the presence of RBM3 and protein, we next determined whether RBM3 was able HuR, there was further increase to 10-fold when RBM3 to regulate COX-2 expression. For this, we determined and HuR were coexpressed (Figure 5e). Furthermore,

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4552 western blot analyses demonstrated increased COX-2 of COX-2 mRNA via its 30UTR, either alone or in protein expression in the cells that expressed FLAG- partnership with each other. tagged RBM3 (Figure 5f). Similar results were obtained with IL-8 (Figure 5e). However, while both RBM3 and HuR induced VEGF expression when expressed alone, Discussion there was no additive effect when the two proteins were coexpressed (Figure 5e). These data demonstrate that Although, it is apparent that post-transcriptional events RBM3 and HuR coordinate their functions to induce of mRNA stability and translation contribute to the COX-2, VEGF and IL-8 gene expression. development and progression of malignant tumors, the mechanisms that regulate these processes especially in inflammation and cancer are not clearly understood. RBM3 enhances COX-2, VEGF and IL-8 mRNA stability One element that is present in the 30UTR of transcripts and translation encoding oncoproteins, cytokines and transcription HuR is an RNA-binding protein that mediates nucleo- factors is the ARE (Chen and Shyu, 1995; Chen et al., cytoplasmic transport, mRNA stability and translation 1995). However, little is known about the RNA-binding functions following binding to ARE sequences in the proteins that bind these elements. HuR, the best 30UTR of rapidly degraded transcripts such as COX-2, characterized factor regulates the stability of COX-2, VEGF and IL-8. RBM3 also encodes a RNA-binding IL-8 and VEGF mRNAs, resulting in their increased domain suggesting that it might also modulate RNA expression, thereby enhancing tumorigenesis (Myer stability function (Derry et al., 1995; Sutherland et al., et al., 1997; Fan and Steitz, 1998b; Peng et al., 1998; 2005). Electrophoretic mobility shift assays demon- Blaxall et al., 2000; Dixon et al., 2001; Nabors et al., strated that recombinant GST-RBM3 protein binds to 2001; Denkert et al., 2004, 2006; Erkinheimo et al., 2005; ARE sequences located in the first 60 nucleotides of the Heinonen et al., 2005). However, more recently it is COX-2 30UTR (Figure 6a). Furthermore, the coupled believed that cytoplasmic localization, rather than total immunoprecipitation–RT–PCR demonstrated a signifi- amount, governs HuR activity (Blaxall et al., 2000; cantly higher level of RBM3-bound COX-2 mRNA in Erkinheimo et al., 2003, 2005; Denkert et al., 2004, 2006; RBM3 overexpressing cells (Figure 6b). Similar results Heinonen et al., 2005). Here, we have identified RBM3 were obtained with VEGF and IL-8. Next, we deter- as a novel HuR interacting partner, whose expression is mined whether RBM3 is involved in regulating the not only significantly upregulated in tumors, but is also mRNA stability. RBM3 and HuR were transiently localized in the cytoplasm. A more comprehensive study overexpressed in HCT116 cells, following which actino- to determine whether there is a correlation between mycin D was added to inhibit de novo mRNA synthesis. protein localization and tumor aggressiveness is re- In cells transfected with either RBM3 or HuR alone, quired. Nevertheless, our studies demonstrate a similar there was increased stability of COX-2 mRNA, the half- trend in RBM3 and HuR expression, implying a related life increased from 1 h in control cells to 5 h in the if not redundant function. RBM3 or HuR expressing cells (Figure 6c). Further- Nucleocytoplasmic shuttling of RBM3 is similar to more, when RBM3 and HuR were coexpressed, COX-2 that of hnRNP proteins that export mRNA from the mRNA stability increased to 8 h (Figure 6c). Similarly, nucleus (Pinol-Roma, 1997; Shyu and Wilkinson, 2000; the half-life of IL-8 mRNA increased from 0.5 to 1 h Hacker and Krebber, 2004; Carpenter et al., 2006). with either RBM3 or HuR, which was further increased Previously, Steitz and colleagues have speculated to 4 h when the two proteins were coexpressed that HuR may bind to ARE-containing mRNAs and (Figure 6c, middle panel). The half-life of VEGF mRNA remain associated during transit through the nuclear was also increased from 0.5 h to 8 h with either RBM3 or envelope (Fan and Steitz, 1998b). A similar possibility HuR, which was further increased to >8 h with both also exists for RBM3. In addition, as RBM3 is a global proteins (Figure 6c, right panel). These data, taken translation inducer (Dresios et al., 2005), it is tempting together suggest that although RBM3 and HuR are to speculate that RBM3 not only transports the mutually exclusive, they can also synergize to increase mRNAs, but also loads them on to ribosomes to induce the mRNA stability of key oncogenic proteins. translation. To determine whether RBM3-mediated COX-2 The observation that the increase in RBM3 expres- mRNA translation occurs through binding to COX-2 sion is dependent on the tumor stage suggests that it 30UTR, we next determined the levels of a chimeric may play a role in tumorigenesis. Indeed, there is a luciferase-COX-2 30UTR mRNA (Figure 6d). Both significantly higher level of RBM3 expression in RBM3 and HuR significantly increased the steady-state HCT116 cells in the tumor xenograft as compared to levels of luciferase mRNA, which was further increased the cells in culture (data not shown). In addition, RBM3 when both RBM3 and HuR were coexpressed overexpression induced NIH3T3 cells to grow in an (Figure 6e). In addition, there was an increase in anchorage-independent manner. Furthermore, when luciferase activity when RBM3 and HuR were co- RBM3 expression was suppressed, there was a complete transfected (Figure 6f). In contrast, neither RBM3 nor shut down of tumor xenograft growth. Previous studies HuR affected luciferase levels expressed from the from the Gorospe group have shown that knockdown of control transcript (Figure 6f). These data demonstrate HuR results in smaller tumors (Lopez de Silanes et al., the ability of RBM3 and HuR to increase the translation 2003). However, the inhibition was not as pronounced

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4553

Figure 6 RBM3 overexpression increases COX-2 mRNA stability and translation. (a) RBM3 is an ARE-binding protein. The first 60 nucleotides of COX-2 30UTR containing many ARE sequences was transcribed in vitro in the presence of 32P-UTP. Purified recombinant GST-RBM3 was allowed to interact with the radiolabeled RNA and subsequently separated by native PAGE. Presence of the RBM3 bound RNA is shown by a mobility shift as indicated to the right. (b) Increased binding of COX-2, IL-8 and VEGF mRNA to RBM3 following overexpression. Whole cell extracts (T) from vector transfected or RBM3 overexpressing cells were prepared after cross-linking, and subjecting to immunoprecipitation with anti-RBM3 antibody. RNA present in the immunoprecipitate (P) and supernatant (S) were isolated after reversing the cross-linkand subjected to RT–PCR for COX-2, IL-8 and VEGF mRNA. Data demonstrates increased COX-2, IL-8 and VEGF mRNA in the pellet of RBM3 overexpressing cells. (c) RBM3 and HuR increase COX-2, IL-8 and VEGF mRNA stability. HCT116 cells were transfected with Flag epitope-tagged RBM3 and/or HuR and the stability of endogenous transcripts was determined following addition of actinomycin D. Both RBM3 and HuR increased COX-2 (left panel), IL-8 (middle panel) and VEGF (right panel) mRNA stability on their own, which was further increased when the two were coexpressed. (d) Schematic representation of control luciferase mRNA (Luc) and luciferase mRNA containing the full-length COX-2 30UTR (Luc-COX) that is encoded in the plasmid under the control of the CMV promoter. (e) RBM3 and HuR increase the translation of Luc mRNA containing COX-2 30UTR. HCT116 cells transiently overexpressing RBM3, HuR or both were co- transfected with plasmids encoding either the Luc-COX and luciferase activity was measured. Luciferase activity of Luc-COX is shown in blackbars and is relative to control cells. * denote statistically significant differences (** Po0.01). (f) RBM3 and HuR increases the translation of Luc mRNA containing COX-2 30UTR. HCT116 cells transiently overexpressing RBM3, HuR or both were co- transfected with plasmids encoding either the Luc-COX or Luc control mRNA and luciferase activity was measured. Luciferase activity of Luc-COX is shown in blackbars and that of Luc in gray bars. *indicate statistically significant differences (** Po0.01). ARE, AU-rich elements; COX, cyclooxygenase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; RT–PCR, reverse transcription–polymerase chain reaction; VEGF, vascular endothelial growth factor.

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4554 with HuR suppression as that observed here with RBM3 Materials and methods suppression. This might be because HuR levels were only decreased by 35–50% in that study. In this regard, Yeast two-hybrid screening it should be noted that both proteins influence the We cloned full-length HuR cDNA into the yeast vector expression of COX-2, IL-8 and VEGF expression. These pGBKT7 (Clontech, Mountain View, CA, USA) as bait at the EcoR1 and Sal1 restriction sites, and performed yeast two- factors play a major role in tumor cell growth and in hybrid screening with the human cDNA liver library according angiogenesis. Hence, further characterization of the role to the manufacturer’s protocols. For confirmation, we cloned of HuR and RBM3 in tumorigenesis will be useful in the full-length RBM3 cDNA in to yeast vectors pGBKT7 and understanding their contributions to the aggressive pGADT7 at the EcoR1 and BamH1 restriction sites, and HuR phenotype. in pGADT7 at the EcoR1 and Xho1 restriction sites. Importantly, it should be noted that PGE2 not only partially reverses the effects of RBM3 knockdown, but Cell culture and treatment also affects baseline proliferation rate. Previous studies HCT116, SW480 human colon adenocarcinoma, HeLa cervi- have demonstrated that PGE2 induces growth of colon cal carcinoma and NIH3T3 mouse fibroblast cells (all from American Type Culture Collection) were grown in Dulbecco’s cancer cells. PGE2 activated the cyclic AMP/protein kinase A pathway to induce the expression of amphir- modified Eagle’s medium supplemented with 10% fetal bovine egulin, which then exerted a mitogenic effect on the serum. To examine RNA stability, HeLa cells were transiently transfected with pCMV-Tag2B plasmids (Stratagene, La Jolla, cells (Shao et al., 2003). PGE2 has also been shown CA, USA) expressing FLAG-tagged RBM3 and/or FLAG- to activate the phosphatidylinositol 3-kinase/AKT tagged HuR. Twenty-four hours after transfection, the cells pathway resulting in changes in cell motility and colony were treated with actinomycin D (10 mg/ml) to prevent the morphology (Sheng et al., 2001). PGE2 inhibits a de novo mRNA synthesis, and total RNA was isolated using COX-2 inhibitor-mediated apoptosis of colon cancer Trizol reagent. For stable expression, NIH3T3 and SW480 cells by inducing Bcl2 expression (Sheng et al., 1998). cells were stably transfected and colonies isolated following HuR is known to bind to AU-rich sequences in Bcl2 incubation in 800 mg/ml geneticin. mRNA and enhances its expression. This suggests that PGE2 may affect Bcl2 expression by regulating RBM3 Anchorage-independent growth and/or HuR. NIH3T3 and SW480 cells transfected with plasmid vector Not much is known about the pathways that regulate (Vec) or stably expressing RBM3 were suspended in a 0.3% mitotic catastrophe and the role of apoptosis in the Sea Plaque agarose overlay in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum. The process. Previous studies have suggested that apoptosis overlay (1.0 ml), consisting of cells, agarose, and medium, is an early event in the mitotic catastrophic process, in was plated at 2000 cells/well in Nunc 10 cm plates over bottom such cases there was an arrest at the G2/M phase (Wahl layers of soft agarose (0.8%) containing only Dulbecco’s et al., 1996; Chen et al., 1999; Ning and Knox, 1999). modified Eagle’s medium supplemented with 5% fetal bovine G2/M transition is regulated by the mitosis-promoting serum. Plates were incubated at 37 1C for 7 days. Colonies factor, cyclin B and cdc2 kinase (Ohi and Gould, 1999; were counted and photographed. Doree and Hunt, 2002; Starkand Taylor, 2004). Activation of the cdc2 requires cyclin B binding, which Recombinant proteins is positively regulated through dephosphorylation by Recombinant RBM3 and HuR were expressed as N-terminal cdc25. In turn, Cdc25c can be inhibited by phosphor- glutathione S-transferase (GST) fusion proteins from the ylation by Chk1 and Chk2. Activated cyclin B then plasmid pGEX-4T3 (Amersham-Pharmacia, Piscataway, NJ, translocates to the nucleus where it greatly reduces the USA) at BamHI and XhoI. Electrophoretic mobility shift assays were performed as described previously (Deschenes- damage-induced G2 arrest. We observed that there was Furry et al., 2005), using an in vitro transcribed 32P-labeled high level of DNA damage in the RBM3 lacking cells, cRNA encoding the first 60 nucleotides of COX-2 30UTR. based on staining for H2AX. Furthermore, we observed high levels of nuclear cyclin B1. Coupled with this, both siRNA Chk1 and Chk2 were phosphorylated and there was RBM3 siRNA sequence targeting the coding region of increased Cdc25C phosphorylation. At the same time, RBM3 (nucleotides 470–488, Accession no. nm_006743) was there was significant activation of caspase-3 and GGGTATGGATATGGATATG and a scrambled control TUNEL. These data suggest a novel process of mitotic siRNA not matching any of the human genes were obtained catastrophe, where cells lacking RBM3 undergo apop- from Ambion Inc., Austen, TX, USA and transfected using Transfectol (Ambion Inc., Austin, TX, USA). tosis at the same time when they are in G2/M transition, rather than during G2/M arrest. Together, these data imply that the product of the Immunoprecipitation coupled RT–PCR novel RBM3 proto-oncogene is required for preventing HCT116 whole cell lysates were prepared following cross- mitotic catastrophe. RBM3 exerts its effects by increas- linking with formaldehyde, and immunoprecipitated with anti- ing mRNA stability and translation of otherwise rapidly RBM3 IgG using Seize X protein A purification kit (Pierce, Rockford, IL, USA). The pellet and supernatant was subse- degraded transcripts. Furthermore, RBM3 is the central quently incubated at 70 1C for 1 h to reverse the cross-links. regulator of tumorigenesis, depletion of which enhances RNA was isolated and subjected to RT–PCR for COX-2, IL-8 the regression of tumors. Hence, RBM3 may represent a and VEGF. In vitro protein interaction studies were performed potential target for chemotherapeutic and chemopre- as reported earlier with recombinant GST-HuR or GST- ventive strategies. RBM3 (Sureban et al., 2007).

Oncogene RNA-binding protein RBM3 is a proto-oncogene SM Sureban et al 4555 Flow cytometric analysis Cell proliferation assay Procedure for flow cytometric analysis was described in RBM3-targeted siRNA was transfected with 1 105 HCT116 Supplementary procedures. cells and plated simultaneously in a 96-well plate. Cell numbers were estimated after 48 h transfection as described earlier (Landegren, 1984). Real-time PCR analyses Total RNA from cells or colorectal tumor samples was Xenograft tumor model subjected to mRNA analysis for various genes wherever siRNA was administered into the xenografts after incorpora- indicated. b-actin was used as internal control. Primer tion into DOPC (1,2-Dioleoyl-sn-Glycero-3-Phosphocholine) sequences are given in the Supplementary procedures. (Avanti Polar Lipids, Alabaster, AL, USA) (Landen et al., 2005) generated by injecting HCT116 cells (6 106 cells) Western blotting subcutaneously into the flanks of female athymic nude mice Cells and tumor xenograft tissues were lysed and the (NCr-nu) and housed in specific pathogen-free conditions. concentration of protein was determined by BCA protein Tumors were measured with calipers and the volume assay (Pierce). Forty micrograms of protein was subjected to calculated as (length width2) 0.5. The tumors reached western blot analysis. RBM3 antibody was generated in a 1000 mm3 after 15 days of injection of cells. These tumors rabbit against a peptide (Sigma-Genosys, The Woodlands, TX, were injected with 50 ml(5mM) on every third day from day 15 USA). Other antibodies were obtained from Cell Signaling for a total of 5 doses. We included five mice per group for the (Danvers, MA, USA) and Santa Cruz (Santa Cruz, CA, USA). xenograft study and the experiment was repeated thrice. Data Actin was used as an internal control for loading. are represented as mean±s.e.m.

Statistics Immunocytochemistry All the experiments were performed in triplicate. The data was HeLa cells transfected with plasmid encoding FLAG and Myc analysed by Student’s t-test. Where indicated, the data is tags were stained by immunoﬂuoresence. Tumor xenografts presented as mean±s.e.m. and human multiple tissue slides were immunostained as described in the Supplementary section. Acknowledgements

Luciferase reporter gene assay We thankStephen Prescott for helpful suggestions and Joan Luciferase reporter assay was performed with plasmid encod- Steitz for the Flag-HuR plasmid. This workwas supported by ing RBM3 and HuR as described earlier (Sureban et al., 2007). NIH Grants DK-62265 and CA-109269.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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