Suppression of Myc Oncogenic Activity by Nucleostemin Haploinsufficiency

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Oncogene (2012) 31, 3311–3321 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Suppression of Myc oncogenic activity by nucleostemin haploinsufﬁciency

AK Zwolinska1,2,4, A Heagle Whiting3,4, C Beekman1, JM Sedivy3 and J-C Marine1,2

1Laboratory for Molecular Cancer Biology, Department of Biomedical Molecular Biology, VIB-UGent, Technologiepark, Ghent, Belgium; 2Laboratory for Molecular Cancer Biology, Department of Molecular and Developmental Genetics, Campus Gasthuisberg, VIB-K.U.Leuven, Leuven, Belgium and 3Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA

Nucleostemin (NS), a nucleolar GTPase, is highly Introduction expressed in stem/progenitor cells and in most cancer cells. However, little is known about the regulation of its Deregulation of Myc oncoproteins is estimated to expression. Here, we identify the NS gene as a novel contribute to 70% of all human cancers (Dai et al., direct transcriptional target of the c-Myc oncoprotein. We 2004; Dang et al., 2006; Vita and Henriksson, 2006). show that Myc overexpression enhances NS transcription c-Myc overexpression is sufficient to drive quiescent in cultured cells and in pre-neoplastic B cells from El-myc cells into cycle and to accelerate their rate of cell cycle transgenic mice. Consistent with NS being downstream of traverse (Bouchard et al., 1998). Cells respond to this Myc, NS expression parallels that of Myc in a large panel hyperproliferative signal by activating apoptosis (Askew of human cancer cell lines. Using chromatin immunopre- et al., 1991; Evan et al., 1992), at least in part by cipitation we show that c-Myc binds to a well-conserved activating the ARF-p53 tumour suppressor path- E-box in the NS promoter. Critically, we show NS way (Zindy et al., 1998; Eischen et al., 1999) or by haploinsufficiency profoundly delays Myc-induced cancer repressing the expression of anti-apoptotic proteins formation in vivo.NSþ /ÀEl-myc transgenic mice have Bcl-2 and Bcl-XL (Eischen et al., 2001). Bypass of these much slower rates of B-cell lymphoma development, with cell cycle checkpoints and apoptotic pathways is a life spans twice that of their wild-type littermates. hallmark of Myc-driven cancers. Moreover, we demonstrate that NS is essential for the c-Myc is a transcription factor that can either activate proliferation of Myc-overexpressing cells in cultured or repress gene expression. Activation occurs through cells and in vivo: impaired lymphoma development was dimerization with the partner protein Max, and binding associated with a drastic decrease of c-Myc-induced to the consensus DNA sequence CACGTG (E-box). proliferation of pre-tumoural B cells. Finally, we provide Repression occurs through association of c-Myc/Max evidence that in cell culture NS controls cell proliferation dimers with other transcription factors, such as Miz-1 or independently of p53 and that NS haploinsufficiency NF-Y, and interference with their function (Izumi et al., significantly delays lymphomagenesis in p53-deficient 2001; Staller et al., 2001; Seoane et al., 2002; Mao et al., mice. Together these data indicate that NS functions 2003), although it is becoming apparent that c-Myc may downstream of Myc as a rate-limiting regulator of cell also repress transcription through E-boxes (Adhikary proliferation and transformation, independently from its and Eilers, 2005). On the basis of chromatin immuno- putative role within the p53 pathway. Targeting NS is precipitation (ChIP) a number of studies have suggested therefore expected to compromise early tumour develop- that c-Myc has the potential to activate thousands of ment irrespectively of the p53 status. genes, which in turn coordinate a wide range of cellular Oncogene (2012) 31, 3311–3321; doi:10.1038/onc.2011.507; processes including those essentials for cell cycle, growth published online 14 November 2011 and apoptosis but also ribosome biogenesis, metabo- lism, protein folding and self-renewal (Fernandez et al., Keywords: nucleostemin; c-Myc; p53; lymphoma; 2003; Li et al., 2003; Zeller et al., 2006; Chen and transgenic mice Olopade, 2008; Eilers and Eisenman, 2008; Kidder et al., 2008; Kim et al., 2008; Perna et al., 2011). Associating specific targets of c-Myc to its diverse biological effects is therefore a challenge. Although Myc’s ability to promote cellular transformation is well established, a better understanding of the mechanisms through which Correspondence: Dr J-C Marine, Laboratory For Molecular Cancer Myc mediates tumourigenesis is essential for the Biology, Department of Molecular and Developmental Genetics, VIB- development of therapeutic approaches to target this KULeuven, Campus Gasthuisberg, Herestraat 49 box 602, 3000, potent oncoprotein. Leuven, Belgium. Using expression profiling to search for novel Myc E-mail: [email protected] 4These authors contributed equally to this work. targets we have identified nucleostemin (NS), a nucleo- Received 16 May 2011; revised 1 October 2011; accepted 7 October 2011; lar GTP-binding protein that is overexpressed in many published online 14 November 2011 cancer cell lines and in mouse and human primary Suppression of Myc oncogenic activity AK Zwolinska et al 3312 tumours (Tsai and McKay, 2002; Liu et al., 2004; proliferation. We show that NS is haploinsufficient in Sijin et al., 2004; Han et al., 2005; Ye et al., 2008; the control of proliferation of cancer-initiating cells and Malakootian et al., 2010; Zhang and Wang, 2010). tumourigenesis in vivo, in a p53-independent manner. Accumulating evidence indicates that high NS levels Our data indicate that targeting NS may be therapeu- may contribute to the high proliferative capacity of tically efficacious in tumors driven by c-Myc, irrespec- tumour cells and therefore to tumourigenesis: NS over- tive of the p53 status. expression in mouse mammary tumour cells enhances tumourigenesis (Lin et al., 2010). Knocking down (KD) NS expression reduces cell proliferation of cultured osteosarcoma cells (Tsai and McKay, 2002); it also Results decreases the tumourigenicity of HeLa cells on injection Search for novel c-Myc-induced transcriptional targets into nude mice (Sijin et al., 2004). However, it is not known whether decreasing NS function would be Overexpression of the c-Myc oncoprotein is a feature of sufficient to inhibit the tumour development in a spon- a many human cancers. To identify novel transcriptional taneous cancer genetic model. targets that have a critical role in c-Myc-induced Several studies have provided important insights into tumourigenesis, we searched for genes that are differen- the molecular mechanisms of action of NS. NS growth- tially expressed upon c-Myc overexpression using a promoting activity has been proposed to result from its microarray-based approach. As a cellular model, we direct interaction with p53 (Tsai and McKay, 2002; used human lung fibroblast-derived cells (LF1; Brown et al. Bernardi and Pandolfi, 2003). But genetic experiments , 1997) immortalized with the catalytic subunit of in vivo have highlighted p53-independent role of NS in telomerase and the simian virus 40 large T and small T et al. the control cell proliferation: the early embryonic antigens (LF1/TERT/LT/ST; Wei , 2003), referred lethality of NS-deficient embryos, which results from to herein as LF1 c. c-Myc overexpression in this cell line an overall cell proliferation defect is not rescued on a causes a significant increase in cell proliferation and p53-null background (Beekman et al., 2006). In keeping, induction of anchorage-independent growth in soft agar et al. et al. NS is required for the proliferation of normal rat bone (Brown , 1997; Wei , 2003). Global gene marrow stem cells in a p53-independent manner expression was determined in both parental (LF1 c) and (Jafarnejad et al., 2008). NS may also contribute to cell stably c-Myc-expressing (LF1 Myc) cell lines using the proliferation via its role as an integrated component of Affymetrix Human Genome U133 array platform ribosome biogenesis, particularly pre-rRNA processing (Affymetrix, Santa Clara, CA, USA). Of the 44 928 (Romanova et al., 2009). NS forms a large protein probe sets analyzed, 464 showed statistically significant differences in gene expression using a cutoff of more complex that co-fractionates with the pre-60 S riboso- thantwofold between the parental and c-Myc-expressing mal subunit and contains proteins related to pre-rRNA P processing and several ribosomal proteins (RPs). NS cells ( o0.05). Comparison of our list of putative c- Myc-upregulated genes (193 out of the 464 probe sets) KD retards rRNA processing, thereby causing the with the previously reported list of c-Myc-bound accumulation of free RPs and induction of ribosome stress (Romanova et al., 2009). Interestingly, NS role in genomic loci, as determined by a ChIP on chip in HL60 cells (Mao et al., 2003), identified four common ribosome biogenesis might be connected to its contribu- genes, which are likely to be direct c-Myc-transcription to the regulation of the p53 pathway. RPs can tional targets: H2BFG, OIP2, DLEU1 and E2IG3, also indeed directly bind MDM2 and interfere with MDM2- known as NS. In addition, going back to one of our dependent degradation of p53 (Dai and Lu, 2004; Dai previously published microarray experiments, we no- et al., 2004; Jin et al., 2004). Depletion of NS induced ticed that the gene previously referred to as ‘hypothe- the interaction between some RPs (RPL5 and RPL11) tical GTP-binding protein’ (accession no. AA892598) and MDM2, and KD the RPs reversed, at least partly, was in fact NS. According to these data NS expression is p53-dependent arrest caused by NS depletion (Dai et al., significantly elevated in rat fibroblast cell line with 2008). These data therefore provide an alternative explanation for the p53-dependent role of NS in the endogenous c-Myc (TGR) and in c-Myc-null fibroblasts reconstituted with exogenous c-Myc expression (Myc3) control of cancer cell proliferation (Lo and Lu, 2010). compared with c-Myc-null fibroblasts (HO; O’Connell Hence, targeting NS in tumours in which the p53 et al. pathway is intact may offer a way for eradicating , 2003). Hence, the ability of c-Myc to induce NS appears to be evolutionarily conserved. growing cancer cells by cutting off the ribosome supplies for protein production, which is necessary for actively growing cancer cells and/or for activating a p53 tumour NS is a direct Myc target suppression response. We confirmed NS upregulation in Myc-overexpression Little is known about what drives the high NS LF1 c cells and in c-Myc-expressing rat fibroblasts expression in cancer cells. Here, we show that NS is a (TGR and Myc3) using reverse transcriptase–quantita- direct transcriptional target of c-Myc and that it is a tive PCR (RT–qPCR) and western blot analysis critical downstream regulator of Myc’s ability to induce (Figures 1a and b). We also analyzed the expression of accelerated cell proliferation and cancer formation. NS in rat fibroblasts (TGR) under conditions of serum In addition, we address the relevance of NS-dependent starvation, which decreases Myc mRNA and protein inhibition of p53 function in relation to cancer cell expression, and at various time points following serum

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3313 stimulation. c-Myc is rapidly induced on serum addi- Anatomy Project database. Of the 6817 genes in the tion, peaking at around 4 h post-stimulation (Dang database, NS was among top 10 genes whose expression et al., 2006). RT–qPCR confirmed Myc induction upon profile was most similar to c-Myc (Figure 1h). Thus, c- serum stimulation and a concomitant increase in NS Myc and NS are co-expressed in human cancers, which expression was observed, consistent with NS being is consistent with NS being downstream of c-Myc. regulated by c-Myc (Figure 1c). Similarly, NS down- regulation paralleled that of c-Myc at later time points. NS haploinsufficiency protects mice from Myc-induced Cross-species comparison of the rat, mouse and tumourigenesis human NS promoter identified a canonical c-Myc- We next asked whether high levels of NS are required binding site (E-box sequence: 50-CACGTG-30) B60 bp downstream of c-Myc to facilitate c-Myc-induced tumor- upstream of the transcription start site. To determine igenesis. Unlike NS-null mice, NS þ /À mice appear whether c-Myc can directly bind to this E-box we normal, despite a 50% reduction in NS protein levels. To performed ChIP assays in both TGR rat and LF1 c test whether loss of one NS allele affects Myc-induced human fibroblasts. TGR fibroblasts were starved and lymphoma development NS þ /À mice were bred onto a serum stimulated for 4 h. The protein–DNA complexes C57/Bl6 background for more than six generations and were immunoprecipitated with a polyclonal c-Myc were then crossed to C57/Bl6 Em-Myc transgenics. antibody (‘Myc’; Figure 1d) or without antibody Lymphoma development was then followed in (‘NoAb’) as a negative control. The immunoprecipitated NS þ / þ ;Em-Myc and NS þ /À;Em-Myc animals. As DNA was analyzed using qPCR. We observed a 4.5-fold expected, most NS þ / þ ;Em-Myc succumbed to lethal enrichment for c-Myc binding at the NS E-box on serum lymphoma between 90 and 200 days (Figure 2). Strik- stimulation (Figure 1d). A fragment containing the ingly, lymphoma development was profoundly delayed in E-box of the known c-Myc target Nucleolin was used as NS þ /À;Em-Myc mice as compared with NS þ / þ ; a positive control. Fragments devoid of any E-box Em-Myc littermates (Figure 2; P 0.0001, log-rank test). (CHRNB4; Frank et al., 2001) or containing the E-box o The mean survival of NS þ /À;Em-Myc mice was twice of glycine methyltransferase (GNMT), a gene which is that of the mean survival of the NS þ / þ ;Em-Myc not regulated by c-Myc, were used as negative controls. littermates (Figure 2). All NS þ / þ mice died within 40 Sevenfold enrichment in c-Myc binding to the NS E-box weeks (n ¼ 24), whereas 50% (13 out of 26) of the was also observed in c-Myc-overexpressing LF1 Myc NS þ /À animals were still alive at that time. The fibroblasts as compared with the parental LF1c cells lymphomas that developed in NS þ /À;Em-Myc animals (Figure 1e). Fragments containing the E-box of the were typical pre-B/B-cell lymphomas that arise in CAD gene and PAPT, a gene that is not regulated by Em-Myc mice. We conclude that NS haploinsufficiency Myc, were used here as positive and negative controls, markedly compromises c-Myc-mediated tumourigenesis. respectively (Fernandez et al., 2003; Brodsky et al., 2005). Together these data indicate that c-Myc is directly recruited to the E-box present in the NS NS is a critical regulator of Myc-induced cell promoter, implying that NS is a direct transcriptional proliferation target of c-Myc. Having established the relevance of NS in c-Myc- To confirm that c-Myc can induce NS expression induced tumourigenesis, we next investigated the under- in vivo we analyzed NS expression in the Em-myc lying mechanism by dissecting the role of NS in c-Myc transgenic mice. These mice express high levels of functions. We knocked down NS by siRNA in an c-Myc in the B-cell lineage and develop pre-B and/or established embryonic rat fibroblast Rat1A cell line B-cell lymphoma with mean survival of about 11 weeks expressing an inducible form of c-Myc, the c-Myc/ (Adams et al., 1986). Although NS protein level is low in estrogen receptor (ER) fusion protein (Mai et al., 1996). wild-type bone marrow and spleen, which mainly NS expression increased as a result of c-Myc induction contain early progenitors and mature B-cells, respec- (c siRNA þ 4-hydroxytamoxifen) and transfection of tively, it is significantly elevated in spleen and in tumors NS siRNAs completely prevented this upregulation of the Em-myc tumor-bearing mice (Figure 1f). As (Figure 3a). In agreement with its oncogenic nature, expected, NS protein levels in the spleens of NS þ /À increased levels of c-Myc drive cellular proliferation. mice are approximately half of that in wild-type (NS þ / þ ) NS KD had only a marginal effect on cell prolifera- mice (Figure 1f). NS mRNA, as measured by tion in the absence of c-Myc induction in this experi- RT–qPCR, is significantly elevated in the spleen of the mental setting; however, preventing c-Myc-induced Em-myc tumor-free mice and perfectly parallels the level NS upregulation by siRNA profoundly decreased cell of Myc in spleen and lymphoma from the Em-myc proliferation upon 4-hydroxytamoxifen-mediated c-Myc tumor-bearing mice (Figure 1g). These data further induction (Figure 3b). As expected, in addition to confirm that that NS transcription is directly induced by stimulating cell proliferation c-Myc-induction promoted Myc in vivo. spontaneous apoptosis (Meyer et al., 2006). However, We next tested whether there is a correlation between we did not observe any effect of NS KD on the extent of c-Myc and NS expression in human cancers. To this Myc-induced apoptosis in this experimental setting end, we searched for genes whose expression profiles are (data not shown). most similar to c-Myc in a large panel of human Induction of c-Myc in Rat1A Myc/ER cells causes cell lines (NCI60) using the NCI’s Cancer Genome anchorage-independent growth in soft agar (Qi et al.,

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3314

Figure 1 NS is a direct transcriptional target of c-Myc. (a) Expression of NS in human LF1 (LF1 c) and LF1 cells with c-Myc overexpression (LF1 Myc) relative mRNA of NS in these cells assessed by RT–qPCR. (b) Expression of NS in rat fibroblasts wild-type for c-Myc (TGR), c-Myc À/À (HO) and in c-MycÀ/À reconstituted with ectopic c-Myc overexpression (Myc3). NS mRNA expression, confirmed by qPCR (middle panel), corresponds to the expression of NS protein (right panel). (c) NS and c-Myc show similar patterns of mRNA expression under conditions of serum starvation and stimulation in TGR cells. (d) c-Myc binds directly to the NS promoter in rat cells. ChIP was performed with a polyclonal c-Myc antibody (Upstate), or with no antibody (negative control), in TGR cells that were serum starved to prevent c-Myc expression (0 h), or serum stimulated for four hours to induce c-Myc expression (4 h). qPCR was performed using primers spanning a NS E-box, a Nucleolin E-box (positive control), a promoter with an E-box that has been previously shown not be bound by c-Myc (GNMT, negative control) and a promoter with no E-box (CHRNB4, negative control). (e) c-Myc binds directly to the NS promoter in human cells. ChIP was performed in LF1 control cells (LF1 c) and in LF1 cells with c-Myc overexpression (LF1 Myc), using c-Myc antibody (Upstate), or no antibody (negative control). qPCR primers were spanning: NS E-box, CAD E-box (positive control) and a gene not expressed in LF1 cells, testis specific poly(A) polymerase b (PAPT; negative control). (f) NS is highly overexpressed in all c-Myc-driven lymphoma-related tissues but not in healthy spleens. Western blot analysis on spleens (S) and tumours (T) derived from NS þ / þ ;Em-Myc and NS þ /À;Em-Myc. Lysates from healthy spleens from NS þ /À and WT C57BL/6 mice were used as controls. Vinculin served as a loading control. Two splicing variants of NS are detected with anti- NS antibody (Malakootian et al., 2010). S – healthy spleen, IS, lymphoma-infiltrated spleen; T, tumour. (g) NS mRNA expression strongly correlates with c-Myc expression. As expected, NS mRNA levels in spleens (S) from non-c-Myc-overexpressing N þ /À animals are about half of the ones detected in the wild-type (NS þ / þ ) mice. In spleens from Em-Myc animals (Em-Myc) the difference becomes less pronounced because of strong c-Myc-driven transactivation of NS expression. IS, lymphoma-infiltrated spleen; S, healthy spleen; T, tumour. Relative expression levels are presented as averages from three mice per each genotype. (h) Expression of NS correlates with c-Myc expression in the NC60 cancer cell lines. Analysis of a data set available from the National Cancer Institute’s Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/) reporting expression profiles for 6817 genes across 60 cancer cell lines. Red, high expression; blue, low expression. NS was included as one of the 10 genes with expression profiles most similar to c-Myc.

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3315

Figure 2 NS haploinsufﬁciency delays c-Myc-induced B-cell lymphoma, (a) Kaplan–Meier curves showing the disease-free survival of Em-Myc mice of the indicated NS status. ‘n’ designates the number of mice in each group (Po0.001, log-rank test). (b) The graph represents the average life spans of NS þ / þ ;Em-Myc and NS þ /À;Em-Myc mice (***Po0.001).

2004). Strikingly, cells transfected with NS siRNAs of spontaneous apoptotic cells were comparable in grew very few small colonies (Figure 3c); there was a NS þ /À;Em-Myc and NS þ / þ ;Em-Myc animals 3.6-fold reduction (as compared with control siRNA) (Figure 3f). Hence, consistent with our in vitro data, in the ability of these cells to form colonies as a NS þ /À mice do not have a defect in DNA-damage result of NS KD. Together these results indicate that response signaling or p53-dependent apoptotic response NS is dispensable for c-Myc-induced apoptosis but on a sensitized Em-Myc background. functions as a critical mediator of c-Myc-induced proliferation. To confirm these findings in vivo we assessed cell NS is required for cell proliferation and tumourigenesis proliferation in response to Myc overexpresion in the independently of p53 Em-Myc transgenic mice using BrdU incorporation Previous transfection studies indicated that NS dampens assays and fluorescence-activated cell sorting (FACS) p53-mediated growth suppression, through a direct analyses. As expected, we observed an increase in the physical interaction with p53 (Tsai and McKay, 2002). rate of cell cycle progression, as monitored by BrdU We therefore tested whether the critical role for NS in incorporation, both in bone marrow and splenic pre- the regulation of cell proliferation and tumourigenesis tumoral B cells from the Em-Myc mice (Figure 3d). uncovered in our study is linked to its ability to regulate Consistently, the percentage of Em-Myc-positive cells in p53 function. We established stable NS KD lines in the S phase of the cell cycle was markedly increased human colon cancer cells lines HCT116 and isogenic compared with wild-type cells (Figures 3e and f). Impor- HCT116 p53KO. c-Myc and NS were expressed at tantly, a decrease in cell proliferation was observed high levels in both parental HCT116 and HCT116 in normal pre-B cells of NS þ /À mice compared with p53KO cell lines (data not shown). A robust NS KD NS þ / þ mice, and this difference was more pro- was observed in both of these cell lines upon infection nounced in the pre-tumoral B cells that expressed Myc with two different lentiviral NS-shRNA constructs (Figures 3d–f). We therefore conclude that NS is a rate- (Figure 4a). Strikingly NS KD decreased the ability of limiting factor in pre-B-cell proliferation and that NS these cells to grow in vitro irrespective of p53 status as haploinsufficiency limits the ability of Myc to efficiently assessed using growth curve assays, FACS analysis and drive their proliferation and expansion in vivo. soft agar assays (Figures 4b–d). The FACS analysis Myc overexpression induces p53-dependent apoptosis indicated that NS KD resulted in a decreased percentage partly by inducing expression of the Mdm2-antagonist of cells in S-phase and an increased proportion of cells Arf (Dominguez-Sola et al., 2007) and partly by in the G2/M phase of the cell cycle. The cell cycle block promoting a DNA-damage response (Vafa et al., 2002; was accompanied by an increase in the sub-G1 fraction, Ray et al., 2006; Wang et al., 2008). Accordingly, which likely result from increased cell death. Impor- caspase3/7 glow assays and FACS analysis revealed a tantly, these growth inhibitory effects were observed in significant increase in apoptosis in pre-tumoral B cells of both p53 competent and p53KO cells. the Em-Myc mice as compared with their wild-type To assess a putative p53-dependent role of NS in counterparts (Figure 3e and data not shown). As tumour formation, NS heterozygous mice were crossed expected, we observed stabilization of p53 and induction with p53-null mice (Jacks et al., 1994). T-cell develop- Ser-18 phosphorylation of p53 in pre-tumoral and ment proceeds normally in p53-deficient mice although tumoral B cells of the Em-Myc mice, an event that they eventually succumb to thymic lymphomas between occurs specifically in response to DNA-damage response 15 and 25 weeks of age (Donehower et al., 1992) and (Chao et al., 2000). However, the extent of p53 only few of them survive past week 30 (Hursting et al., stabilization and phosphorylation and the percentage 1994). Strikingly, partial reduction of NS expression

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3316

Figure 3 NS is essential for c-Myc-induced proliferation. (a) Knockdown of NS mRNA by RNA interference in Rat1A Myc/ER cells. Cells were transfected with either non-targeting control oligos (c siRNA) or siRNA oligos targeting NS mRNA (NS siRNA). Approximately 4 h after the cells were seeded; c-Myc overexpression was induced by addition of 200 nM 4-hydroxytamoxifen (OHT). (b) Growth curves of the Rat1A Myc/ER cells after NS knockdown. Cells were transfected with either non-targeting control oligos (c siRNA) or siRNA oligos targeting NS mRNA (NS siRNA). After 2 days plating, c-Myc was induced in some cells were with addition of OHT ( þ OHT). (c) Soft agar growth after NS knockdown. Cells were transfected with either non-targeting control oligos (c siRNA) or siRNA oligos targeting NS mRNA (NS siRNA). (d) Flow-cytometric analysis of in vivo 5-bromodeoxyuridine (BrdU) incorporation in B220 þ cells from bone marrow (BM, gray bars) and spleen (black bars) isolated of 6–7 week-old mice of indicated genotypes. Data represent the mean (±s.d.) of biological replicates; ‘n’ designates the number of animals in each group. (e) and (f) cell cycle distribution (e) and quantification of the percentage of cells in S phase (f). Data represent the mean (±s.d.) of biological replicates; ‘n’ designates the number of animals in each group. (g) Apoptosis measured using caspase-3/7 Glo luminescence assays (Promega). Pre-tumoural B220 þ cells were isolated from bone marrow (BM) and spleens of 6–7 week old mice of indicated genotypes. The graph shows average luminescence values from independent biological replicates (±s.d.). ‘n’ designates the number of animals in each group. (h) Western blot of protein lysates from tumours and healthy or lymphoma-infiltrated spleens. IS, lymphoma-infiltrated spleen; S, healthy spleen; T, tumour.

was sufficient to significantly delay tumor formation in all 6 NS þ / þ controls died within 30 weeks. Thus, NS p53-null mice and extended their mean survival twofold haploinsufficiency delays lymphomagenesis in a manner (log-rank test: Po0.001; Figure 5). In all, 4 out of 19 that is independent of its role in the regulation of p53 NS þ /À; p53-null mice survived past 40 weeks wherease function.

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3317

Figure 4 NS depletion impedes cell cycle progression independently of p53. (a) Knockdown of NS mRNA in HCT116 cells (p53 þ / þ or p53À/À), using lentiviral vectors expressing NS shRNA1, NS shRNA2 or EV control. (b) Growth curves of HCT116 p53 þ / þ and p53À/À cells following NS knockdown. (c) Cell cycle analysis of HCT116 p53 þ / þ and p53À/À cells following NS knockdown. Left panel shows cell cycle proﬁles of HCT116 cells expressing EV, NS shRNA1 or NS shRNA2. The right panel represents the distribution of the cells throughout the cell cycle as determined by the analysis of the cell cycle proﬁles. The upper section shows HCT116 p53 þ / þ cells, whereas the lower one the HCT116 p53À/À cells. (d) Soft agar growth assay of the HCT116 of indicated genotypes, expressing the indicated RNAs.

Figure 5 NS haploinsufﬁciency delays tumourigenesis in absence of p53. (a) Kaplan–Meier curve of p53-deﬁcient transgenic mice (C57Bl/6 genetic background), WT or heterozygous for NS. ‘n’ designates the number of mice in each group (Po0.001, log-rank test). (b) The mean survival of animals of the indicated genotypes (***Po0.001).

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3318 Discussion ability to stimulate ribosome biogenesis and protein synthesis, at least in part via induction of NS expression. The mechanisms leading to the widespread upregulation However, we also show that the function of NS in of NS in various human cancer cell lines and primary tumourigenesis is independent of its role in the regula- tumours are poorly understood. Previous reports have tion of the p53 tumor suppressor pathway. Indeed, pointed at a correlation between NS expression and reducing NS dose by half (NS þ /À) was sufficient to the presence of the active c-Myc (Rosenwald et al., 2002; significantly delay lymphomagenesis on a p53-null Dave et al., 2006; Shaffer et al., 2006). In this manu- background. This observation contrasts with the finding script we extend this correlation to a large panel of that Rpl24 or Rpl38 haploinsufficiencies do not have human cancer cell lines (NC60) and show dramatic any effect on tumor formation in the context of the p53- accumulation of NS in c-Myc-driven B-cell lymphomas null background (Barna et al., 2008). Together these isolated from Eu-Myc mice. Importantly, by identifying data raise the possibility that while RP and NS NS as a novel direct c-Myc-transcriptional target we haploinsufficiencies similarly affect tumourigenesis they provide for the first time direct mechanistic insights might still do so, at least partly, via distinct mechanisms. underlying the correlation between c-Myc and NS. Finally, our data indicate that in contrast to strategies As c-Myc is frequently activated in human malignancies targeting MDM2/MDMX, the efficacy of which relies our findings also provide an explanation for the upregu- on the presence of an intact p53 pathway (Toledo and lation of NS in human cancers, although we cannot Wahl, 2007; Marine, 2010), targeting NS could represent exclude that additional mechanisms might contribute to an alternative valuable therapeutic approach in the the high NS expression. management of cancers with a disabled p53 pathway. Critically, our data indicate that NS is not just another The presence of two putative GTP-binding sites within component of the complex transcriptional networks NS makes it a potential target for molecules that mimic induced by c-Myc; instead we show that it is a critical or compete for GTP binding. It has recently been regulator of tumor development and a key mediator of c- reported that AVN-944, a potent specific inosine Myc-induced tumourigenesis in vivo. Decreasing NS monophosphate dehydrogenase inhibitor, currently in levels by 50% (haploinsufficiency) is sufficient to drasti- Phase I trials for the treatment of hematologic cally limit c-Myc-induced lymphoma development in vivo. malignancies and in Phase II trials for solid tumors, Our data indicate that this delay is not a consequence of leads to the rapid MDM2-dependent degradation of an impaired DNA-damage response or p53-dependent NS in tumor cell lines (Huang et al., 2009). The effect apoptotic response but rather of an altered ability of c- is a consequence of targeting of the GTP-uncoupled Myc to accelerate cell cycle progression in the tumor- NS for proteasomal degradation by MDM2 and initiating cells. Although the exact biochemical mechan- appears to be specific as it was not observed with the ism underlining the role of c-Myc-induced NS in cell two other nucleolar proteins nucleophosmin and proliferation awaits further studies it could be linked to its nucleolin. Modulation of NS levels using AVN-944 previously recognized role in ribosome biogenesis. could therefore be particularly effective in tumours Depletion of NS indeed retards rRNA processing, overexpressing MDM2. thereby causing free RPs accumulation, accompanied by a substantial decrease in overall protein synthesis (Romanova et al., 2009). Interestingly, c-Myc is known Materials and methods to regulate ribosome biogenesis and translation by upregulating the transcription of rRNA and RPs and Cell lines and culture conditions auxiliary factors that are required for rRNA processing, Human LF1 (Brown et al., 1997) expressing the catalytic ribosome assembly and export of mature ribosomal subunit of telomerase (hTERT), simian virus 40 LT and simian subunits from the nucleus to the cytoplasm (van Riggelen virus 40 ST, and c-Myc (Wei et al., 2003) were cultured in Ham’s F10 nutrient mixture supplemented with 15% fetal et al., 2010). The ability of c-Myc to regulate the þ / þ À/À expression of yet another regulator of this process, NS, bovine serum. The pair of p53 and p53 human colon cancer cells HCT116 was obtained from Bert Vogelstein (Johns is entirely consistent with this view. Importantly, an Hopkins University, Baltimore, MD, USA). The cells were increasing number of observations indicate that c-Myc’s cultured in Dulbecco’s modified Eagle’s medium supplemented contribution to tumourigenesis is at least partly connected with 10% fetal bovine serum. Immortalized embryonic Rat-1 to its ability to modulate ribosome biogenesis. In Eu-Myc fibroblast lines including TGR-1 (TGR; Prouty et al., 1993), mice, the same model system utilized in the study herein, TGR-1-bearing homozygous c-myc deletion HO15.19 (HO; loss of one allele of Rpl24 or Rpl38 decreased the Mateyak et al., 1997) HO cells reconstituted with c-Myc incidence of lymphoma by 20% and delayed tumour expression (Myc3; Mateyak et al., 1999) and Rat1A expressing onset by over 100 days (Barna et al.,2008).Notably, the Myc/ER fusion protein (obtained from S McMahon, similarly to what we have observed for NS, decreased Kimmel Cancer Center, Philadelphia, PA, USA) were cultured Rlp24 gene dosage impeded the ability of c-Myc to in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum (Hyclone, Waltham, MA, USA). For the stimulate cell cycle progression independently from the experiments requiring quiescent cultures, cells were grown to cell cycle program established at the transcriptional level confluence and serum starved in media containing 0.1% serum by c-Myc hyperactivation. By analogy it is therefore for 48 h. The cells were stimulated on addition of media with tempting to speculate that c-Myc-induced cell prolifera- 10% calf serum. All cells were incubated at 371 Cinan tion and tumourigenesis might dependent on c-Myc’s atmosphere of 5% CO2.

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3319 Lentiviral and retroviral vectors CTGA-30 and 50-GCCATGCCAATGTTGTCTCTTAT-30; The lentiviral vector pLKO.1-puro expressing NS shRNA1 mouse TATA-box-binding protein: 50-TCTACCGTGAATC (targeting all variants of human NS at 50-GCTAAACTGT TTGGCTGTAAA-30 and 50-TTCTCATGATGACTGCAGC TCTCTGTATAA-30) or NS shRNA2 (targeting all variants AAA-30; mouse Rlp13a: 50-CCTGCTGCTCTCAAGGTTG of human NS at 50-CAGCAAGTATTGAAGTAGTAA-30) TT-30 and 50-TGGTTGTCACTGCCTGGTACTT-30. Gene was obtained from the RNAi Consortium at the Broad expression levels and errors on the gene expression levels were Institute. Lentivirus production was achieved according calculated using qBasePLUS 1.0 analysis software (Vande- to the protocol available through the RNAi Consortium, sompele et al., 2002). using 293T packaging cells and FuGENE 6 transfection reagent (Roche, Basel, Switzerland). HCT116-infected cells Chromatin immunoprecipitation were selected in 1 mg/ml puromycin. The ChIP protocol was carried out using the ChIP Assay kit according to the manufacturers’ instructions (EMD RNA interference (RNAi) Millipore, Billerica, MA, USA). qPCR reactions were carried siRNAs were transiently transfected using nucleofection out using the following : rat ChIP primers: NS E-box (Amaxa Biosystems, Gaithersburg, MD, USA). A total of at position -67: 50-ACTTTCCGGCAGCTTCTTGA-30 and 1 Â 106 cells per each sample were combined with 100 ml 50-GTCTGGAGCAGAAGAGGTCTCAG-30; control pri- Nucleofector solution (Amaxa Biosystems) and 1.5 mg siRNA. mers targeting E-box for nucleolin at position 574:50- Following nucleofection, cells were transferred directly to the CGCGTCCGAGGCAGTG-30 and 50-TCCATCTACCGTC culture dish. For HCT116 cells, Kit V and the D-32 setting was ACGGTCAG-30; an irrelevant E-box not bound by c-Myc, used for nucleofection. For TGR and Rat1A Myc/ER cells Kit glycine methyltransferase (GNMT): 50-CAAGCGCCGTC R and the T-16 setting was used for nucleofection. The AGGATG-30 and 50-CAGTACGGCTGCGGGTG-30; and a following siRNAs were used: promoter without an E-box, neuronal acetylcholine receptor NS: rat NS siRNA sense/antisense: 50-GACAUUGUGU b4 (CHRNB4): 50-TGCCTCGGGTGAACTAAGATG-30 and UAGAAGUUUtt-30/50-AAACUUCUAACACAAUGUCtg-30 50-GCCTCATTCGTCTTGGGAACT-30; as reported in (Silencer pre-designed siRNA, Ambion, Carlsbad, CA, USA); (Frank et al., 2001) human ChIP primers: NS E-box at Human NS siRNA1 sense/antisense: 50-GGUGAUUGAAGC position -55: 50-CTCGTCAGTGGCTTCAGTTCAC-30 and CUCCGAUdTdT/AUCGGAGGCUUCAAUCACCdTdT-30 50-GACCCCACTTACTAGGCCTTTTC-30; The following (Dharmacon, Lafayette, CO, USA); Human NS siRNA2 control primers targeting E-box for carbamoyl-phosphate sense/antisense: 50-GGCUUACAAGGAGCAUGCAAGUU synthetase 2 (CAD) at position -454: 50-ACGTGGTTC GU/ACAACUUGCAUGCUCCUUGUAAGCC-30 (Stealth CAGTGGAGTTTG-30 and 50-CGACCCGTCCTCCAACA RNAi, Invitrogen, Carlsbad, CA, USA). CTA-30; and a gene not expressed in LF1 cells, testis-specific poly(A) polymerase (PAPT): 50-CGTTGCACCAAGTCAT 0 0 0 Microarray analysis GTGG-3 and 5 -CCTCCTCGACAGCCCTTAAA-3 ; as re- Total RNA was extracted using Trizol reagent (Invitrogen). ported in (Fernandez et al., 2003; Brodsky et al., 2005). Synthesis of double-stranded cDNA, followed by conversion to target cRNA, and hybridization to the Affymetrix Human Immunoblot analysis Genome U133 array set was carried out by the manufacturer’s Protein lysates from exponentially growing rat fibroblasts were instructions (Affymetrix). The probe arrays were washed and prepared using Laemmli buffer. Proteins were separated by stained using the GeneChip Fluidics Station 400 (Affymetrix), 10% sodium dodecyl sulfate–PAGE and transferred to and scanned using the Agilent GeneArray scanner (Agilent, Immobilon-P membrane (EMD Millipore). Antibodies used: SantaClara,CA,USA).Datawerenormalizedtoatarget anti-NS (R&D Systems, Minneapolis, MN, USA); anti- intensity of 1500, and analysis was carried out using Microarray GAPDH (Ambion); Secondary antibodies were horseradish Suite 5.0, MicroDB 3.0 and Data Mining Tool 3.0 software peroxidase-conjugated (Jackson Immunoresearch, West (Affymetrix). Grove, PA, USA). Lysis of mouse organs and immunoblotting were performed Quantitative real-time PCR (qPCR) as described before (De Clercq et al., 2010). The primary Total RNA was extracted using Trizol reagent. RNA samples antibodies used were: anti-vinculin (clone hVIN-1, Sigma, were treated with DNase I, and 2 mg of total RNA from each St Louis, MO, USA); anti-CD45R (B220; clone RA3-6B2, sample were reverse transcribed with a high-capacity cDNA Santa Cruz, Santa Cruz, CA, USA); anti-NS (EMD Milli- archive kit (Applied Biosystems, Carlsbad, CA, USA). pore); anti-myc (clone 9E10, Abcam, Cambridge, UK), anti- Quantitative RT–qPCR assays were performed using Fast p53 (clone 1C12, 1:1000, Cell Signaling, Beverly, MA, USA); SYBR Green 2 Â Master Mix, following the manufacturer’s anti-caspase-3 cleaved (Cell Signaling) and anti-gH2AX (Cell instructions (Applied Biosystems). Primer pairs used are Signaling). The secondary antibodies used were horseradish following: rat NS: 50-AGGATGGTGATGATCAAGAACA peroxidase-conjugated (Amersham, Amersham, UK). TG-30 and 50-GATGGTTTACTTGCTGTTGATTGC-30; rat GAPDH: 50-ACCACCAACTGCTTAGCCCC-30 and 50-TTC Flow cytometry TGAGTGGCAGTGATGGC-30; rat c-Myc: 50-CCAGCAGC As previously reported (Schorl and Sedivy, 2003), flow GACTCTGAAGAAG-30 and 50-GATGACCCTGACTCG cytometric analysis was performed using a BD Biosciences GACCTC-30; human NS (amplifies all isoforms): 50-TCGG FACS-Caliber instrument (BD Biosciences, Franklin Lakes, GTTGGAGTAATTGGTTTC-30 and 50-TGTAAGCCCC NJ, USA) and CellQuest and Modfit software. Exponentially ATGGATACACC-30; human actin: 50-AAGGATTCCTAT growing cells were fixed in ethanol and stained with propidium GTGGGCGA-30 and 50-TCCATGTCGTCCCAGTTGGT-30; iodide (Sigma) or sulforhodamine 101 (Molecular Probes, c-Myc: 50-AGTGCTGCATGAGGAGACAC-30 and 50-GGT Eugene, OR, USA). For flow-cytometric analysis of BrdU TTGCCTCTTCTCCACAG-30; mouse NS: 50-TGCAGCAGT incorporation in mouse B220 þ cells, bone marrow and CATGAAGAAGG-30 and 50-CCGAAGACCGAAAAAGG spleens were isolated from 6–7 week-old mice. BrdU injection ATT-30; mouse actin-b:50-GCTTCTAGGCGGACTGTTA of mice, magnetic sorting-based isolation of B220 þ from

Oncogene Suppression of Myc oncogenic activity AK Zwolinska et al 3320 organs (MACS, Miltenyi Biotec, Cologne, Germany), fixation et al., 1994; Beekman et al., 2006). All mice were backcrossed and indirect immunofluorescent BrdU labeling, were per- to C57Bl/6 strain for a minimum of 10 generations. Animals formed as described previously (De Clercq et al., 2010). displaying signs of reduced vitality or with visible tumours were euthanized. Luminescence cell apoptosis assay B220 þ cells were isolated from bone marrow and spleens of 6–7 week-old mice using magnetic sorting (MACS, Miltenyi Conflict of interest Biotec), as described previously (De Clercq et al., 2010). Directly after the isolation, the cells were counted and The authors declare no conflict of interest. apoptosis was measured using Caspase-3/7 Glo luminescence assays (Promega, Fitchburg, WI, USA) according to the manufacturers protocol. Acknowledgements

Soft agar assay This work was supported in part by the Geconcerteerde The soft agar assay was performed as described (Zhang et al., Onderzoeksacties (GOA, University Ghent, Belgium). A K 2005). Colony growth was assessed after 2 weeks. Zwolinska is a recipient of a FWO fellowship. Work in the Sedivy lab was supported by grant R01 GM041690 from the Mouse strains NIH. A Whiting was supported in part by training grant T32 NS þ /À,Em-Myc and p53À/À mouse strains and their PCR GM007601. The Genomics Core Facility at Brown University genotyping were described earlier (Adams et al., 1986; Jacks was supported in part by the COBRE grant P20 RR015578.

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