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Post‐transcriptional gene expression control by NANOS is up‐regulated and functionally important in pR b‐deficient cells

2014; Springer Nature; Volume: 33; Issue: 19 Linguagem: Inglês

10.15252/embj.201488057

1460-2075

Wayne Miles, Michael Korenjak, Lyra Griffiths, Michael A. Dyer, Paolo Provero, Nicholas J. Dyson,

Ocular Oncology and Treatments

Article7 August 2014free access Source Data Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells Wayne O Miles Wayne O Miles Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Michael Korenjak Michael Korenjak Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Lyra M Griffiths Lyra M Griffiths Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michael A Dyer Michael A Dyer Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Paolo Provero Paolo Provero Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Nicholas J Dyson Corresponding Author Nicholas J Dyson Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Wayne O Miles Wayne O Miles Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Michael Korenjak Michael Korenjak Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Lyra M Griffiths Lyra M Griffiths Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Michael A Dyer Michael A Dyer Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Paolo Provero Paolo Provero Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Nicholas J Dyson Corresponding Author Nicholas J Dyson Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA Search for more papers by this author Author Information Wayne O Miles1, Michael Korenjak1, Lyra M Griffiths2, Michael A Dyer2, Paolo Provero3,4 and Nicholas J Dyson 1 1Massachusetts General Hospital Cancer Center and Harvard Medical School, Laboratory of Molecular Oncology, Charlestown, MA, USA 2Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA 3Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy 4Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy *Corresponding author. Tel: +1 617 726 7800; Fax: +1 617 726 7808; E-mail: [email protected] The EMBO Journal (2014)33:2201-2215https://doi.org/10.15252/embj.201488057 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE). Synopsis Despite of lost control over E2F transcriptional programs, cells with pRb mutations can contribute relatively normally to tissues in mice and flies. Complete gene expression deregulation is prevented by the post-transcriptional regulator NANOS, which is normally repressed by pRb but upregulated in its absence. pRb activity increases the binding of repressor complexes such as dREAM to the NANOS promoter. Elevated NANOS levels promote proliferation and survival of pRb-deficient cells, as well as homeostasis of RBF1-depleted tissues. NANOS upregulation reduces cellular stress associated with pRb inactivation in part by post-transcriptionally silencing stress-response genes, including MAP kinases that act upstream of p53. Repression of NANOS expression by E2F/pRb is conserved from flies to humans. Introduction Cell proliferation and apoptosis are fundamental cellular processes that are essential for development, differentiation, and tissue homeostasis. Each cell within eukaryotic organisms has built-in safeguards that limit the tumorigenic potential of cells that lose their normal controls. The family of E2F transcription factors plays a central role in the regulation of both proliferation and apoptosis. E2F proteins control the expression of genes involved in cell cycle progression, checkpoint activation, and senescence. The term "E2F" is the integrated activity of a family of proteins that contains both activators of transcription (dE2F1 (flies), E2F1-E2F3 (humans)) and repressors of transcription (dE2F2 (flies), E2F4-8 (humans)) (Chen et al, 2009). An additional layer of regulation is provided at cell cycle genes by the pocket protein family of transcriptional repressors (RBF1, RBF2 (flies) pRb, p107, and p130 (humans)) (Burkhart & Sage, 2008; Dick & Rubin, 2013). The pocket proteins bind directly to activator E2F's and act as molecular scaffolds to repress E2F-mediated transcription (Dimova et al, 2003). Dynamic fluctuations between the activities of E2F and pRb proteins regulate normal cell proliferation (van den Heuvel & Dyson, 2008). pRb is functionally inactivated in the majority of tumors, and its activity can be compromised by several different types of events that include E2F amplification (Feber et al, 2004), viral infection (E6/E7) (Dyson et al, 1989), CDK4/6 amplification (Khatib et al, 1993), p16 mutation/silencing (Okamoto et al, 1994) or by mutations within the Rb1 gene (Friend et al, 1986). Although pRb inactivation is widespread in cancer, it is evident the loss of pRb function also generates a series of cellular stresses. For example, pRb loss causes dramatic and widespread changes in transcriptional profiles (Herschkowitz et al, 2008), leads to changes in chromatin architecture (Zhang et al, 2012) and undermines genomic integrity (Longworth et al, 2008; Manning et al, 2010). The mechanism(s) that counteract these stresses and enable the oncogenic growth of pRb-deficient cells remains poorly understood. However, elucidating the mechanisms that allow cells to cope with the pressures associated with pRb loss is important, since this may reveal points of vulnerability that can be exploited therapeutically to target cancer cells. Although pRb is frequently inactivated in cancer cells, analysis of chimeric animals has shown that Rb1 null cells (or rbf1 null cells in Drosophila) typically do not overproliferate and contribute significantly to differentiated tissues that are relatively normal in appearance (Maandag et al, 1994; Du, 2000). There are likely to be multiple reasons why Rb loss, or E2F deregulation, does not typically promote cell proliferation or cell death. Genetic studies show that in some contexts, related proteins may compensate for pRb loss (Bremner et al, 2004) and that other cdk regulators provide redundant levels of control (Park et al, 1999). In this study, we have explored the idea that there may be additional types of control that act in pRb-deficient cells to counterbalance the changes in gene transcription. To identify novel candidate genes which may counterbalance E2F dys-regulation, we searched for genes that were directly regulated by E2F/pRb in both Drosophila and mammalian cells and were up-regulated in both species following Rb/RBF inactivation. One of the most intriguing genes that met these criteria was the RNA-binding protein, NANOS. NANOS is a conserved and essential single-stranded RNA-binding protein which functionally cooperates with its obligate binding partner, Pumilio (Pum) (Wharton & Struhl, 1991). Together they form the core of the Pumilio post-transcriptional repressor complex and suppress the translation of mRNAs containing a Pumilio Regulatory Motif (PRE) (UGUAXAUA) within their 3′ untranslated regions (UTR) (Asaoka-Taguchi et al, 1999; Sonoda & Wharton, 1999). The PUM complex activity prevents the translation of its substrates via a number of mechanisms including, 5′ decapping (Cao et al, 2010), ribosome stalling (Friend et al, 2012), miRNA recruitment (Nolde et al, 2007; Kedde et al, 2010; Friend et al, 2012; Miles et al, 2012), and de-adenylation (Van Etten et al, 2012). RNA immunoprecipitation experiments of PUM complexes in multiple systems have identified a significant number of conserved substrates involved in regulating important oncogenic processes including cell cycle progression, differentiation, and apoptosis (Gerber et al, 2004, 2006; Galgano et al, 2008). In support of these findings, tissue-specific disruption of Pumilio/Nanos activity in a variety of tissues and systems has implicated the post-transcriptional regulation of Pum/Nanos as essential for tissue differentiation (Deshpande et al, 1999), stem cell maintenance/pluripotency (Tsuda et al, 2003; Chen et al, 2012; Lai et al, 2012), and preventing p53-mediated apoptosis (Chen et al, 2012; Lai et al, 2012). Here, we show that NANOS, a fundamental component of the Pum complex, is a direct target of pRb regulation and that NANOS expression is strongly induced following pRb inactivation. This elevation in NANOS levels is seen in multiple experimental systems and, as a result, NANOS gains in importance in pRb- or RBF1-deficient cells. One of the consequences of Nanos upregulation is that it suppresses p53-mediated growth. As a result, the elevated levels of NANOS are particularly important for cancer cell lines that retain a functional p53. Results To identify conserved E2F/pRb targets genes, we conducted RBF1 and E2F (E2F1 and E2F2) ChIP-chip experiments from wild-type (w1118) Drosophila larvae and compared the results with the lists of classic E2F/RB targets identified in human cells (Bieda et al, 2006). In addition to the expected E2F targets that we have characterized previously (Korenjak et al, 2012), we noted that the novel E2F2 and RBF1 targets included all three components of the Drosophila Pumilio post-transcriptional repressor complex: pumilio, nanos, and brat (Fig 1A). The Pumilio complex is an interesting target of E2F/RBF regulation because it, in turn, reduces the activity of activator E2F's in both flies (E2F1) and humans (E2F3) (Miles et al, 2012). To confirm our ChIP-chip results, we conducted ChIP-RT-PCR experiments using antibodies targeting RBF1, E2F1, and E2F2 from Drosophila larvae. This analysis confirmed that the promoter of nanos is strongly bound by RBF1 and the repressive E2F (E2F2), but not by the activator E2F (E2F1), (Fig 1B, Supplementary Fig S1A). The remaining components of the complex, pumilio and brat, are weakly bound by RBF1 and E2F2 (Fig 1B, Supplementary Fig S1A). These data suggest that RBF1 and E2F2 directly constrain the activity of the Pum complex by repressing the expression of the rate-limiting component, Nanos, rather than by regulating the expression of all of the components of the Pum complex. Figure 1. E2F/pRB regulate the expression of the Pumilio complex ChIP-Chip from Drosophila larvae of RBF1, E2F2, E2F1, and IgG controls on the nanos, pumilio, and brat promoters. RT-PCR from ChIP of IgG, E2F1, Rbf1, and E2F2 from wild-type (w1118) Drosophila larvae surrounding the transcription start site of actin, brat, pumilio, and nanos (mean ± SD, n = 3). RT-PCR of pumilio (pum), brat, and nanos expression from adult females expressing UAS-RNAi constructs targeting Gal4, e2f1, dp, e2f2, and rbf1 (mean ± SD, n = 3). RT-PCR results of PUM components (PUM1, PUM2, NANOS1, NANOS2, and NANOS3), E2F target (Cyc A) and non-E2F target (E2F3) from BJ fibroblast cells transfected with siRNA pools targeting the pocket proteins (Rb1, p107, and p130) (mean ± SD, n = 3). Correlation of the expression of the PUM components with Rb1 in cancer cell lines from the Sanger cancer cell line encyclopedia database. RT-PCR results of NANOS1 expression in normal human retina, primary retinoblastoma tumors, and retinoblastoma tumor cells grown as orthotopic xenografts (mean ± SD, n = 3). Download figure Download PowerPoint RBF1 and E2F2 are components of the Drosophila, Rb, E2F, and Myb-associated protein (dREAM) complex, a transcriptional silencing complex that represses many E2F target genes (Korenjak et al, 2004). To determine whether components of the Pum complex are targets for dREAM-mediated repression, we analyzed datasets of published genome-wide dREAM ChIP experiments from Drosophila Kc cells (Georlette et al, 2007) and found a strong ChIP enrichment for all of the dREAM components (E2F2, Myb, Mip120, Mip130, and Lin-52) on the pumilio, nanos, and brat genes (Supplementary Fig S1B). To establish the functional significance of E2F2/RBF1 binding to these promoters, we assayed gene expression levels from Drosophila S2 cells and flies containing dsRNA or RNAi sequences targeting E2F/RBF family members. Depletion of RBF1 or E2F2 (but not E2F1) strongly induced the expression of nanos and modestly elevated the levels of pum and brat (Fig 1C, Supplementary Figs S1C and S2A and B). To further assess the contribution of the dREAM complex to the regulation of these targets, we analyzed the levels of the Pum complex in E2F2 homozygous mutant flies and microarray studies from Kc cells treated with dsRNA targeting dREAM components (Georlette et al, 2007). E2F2 mutant flies (Supplementary Fig S1D) and dsRNA-treated Kc cells (Supplementary Fig S2C) display elevated expression of the Pum components, suggesting that dREAM activity regulates the expression of the Pum complex. To confirm that these changes in gene expression were due to direct regulation by the dREAM complex, the promoters of the pumilio, nanos, and brat genes were cloned upstream of a luciferase reporter gene. Depletion of RBF1 or E2F2, but not E2F1, by dsRNA in S2 cells strongly up-regulated the expression from the nanos promoter. It also weakly increased the luciferase production from the pumilio and brat promoters (Supplementary Fig S2D). We conclude that the E2F2/RBF1/dREAM complex in Drosophila directly binds the promoters of nanos, pumilio, and brat and that this regulation is important in repressing the expression of the rate-limiting component of the Pum complex, Nanos. To investigate the role of E2F/pRb regulation of the PUM complex in human cells, we examined the capacity of each pocket protein [pRb, p107 (Rb like 1 (RBL1)), and p130 (RB like 2 (RBL2))] to regulate PUM/NANOS expression in human fibroblasts. The pocket proteins were depleted from BJ cells using siRNAs, and the effects on expression and protein levels of the PUM complex were measured. As shown in Fig 1D and Supplementary Fig S3A, reducing the levels of the pocket proteins produced a strong up-regulation in the expression of the NANOS1 and NANOS3 genes, akin to that of the more conventional E2F target, Cyclin A (Cyc A) (Takahashi et al, 2000). Depletion of the pocket proteins induced only slight changes in PUM1 and PUM2 expression and did not affect NANOS2 levels (Fig 1D). Reducing pocket protein function using siRNAs led to elevated levels of PUM1, PUM2, and NANOS1 (NOS1) proteins (Supplementary Fig 3B and C). These findings suggest that NANOS1 protein levels are elevated due to transcriptional up-regulation upon loss of pocket protein activity and that the changes in PUM protein levels are likely due to increased stabilization of the PUM complex, in agreement with previous studies (Sonoda & Wharton, 1999). Consistent with the idea that the dREAM complex represses NANOS1 expression, chromatin immunoprecipitation (ChIP) experiments using antibodies targeting the dREAM complex components, E2F4, p107, and p130, confirmed that all three proteins bind directly to the promoter of NANOS1 (NOS1) in human fibroblasts (BJ cells) (Supplementary Fig S4A). Interestingly, ChIP experiments showed that E2F4 and p107 were completely absent from the NANOS1 promoter in Y79 retinoblastoma cells that completely lack pRb (Supplementary Fig S4B), and the binding of these dREAM components to the NANOS1 promoter was dramatically reduced by knockdown of pRb from BJ cells (Supplementary Fig S4A). These observations suggest that pRb stabilizes dREAM-binding to the NANOS1 promoter, a conclusion that agrees with previous studies linking pRb function to dREAM-mediated repression (Tschop et al, 2011). The functional inactivation of Rb family members is a widespread phenomenon in cancer as these proteins regulate important oncogenic pathways including cycle cell progression, senescence, differentiation, and apoptosis (for review (Di Fiore et al, 2013)). Commonly, cancer cells constitutively inactivate the pocket proteins by overexpressing the cyclin-dependent kinases which target pRb (Khatib et al, 1993) or by disrupting the upstream regulators of CDK activity (p16INK4A) (Okamoto et al, 1994). To determine how these regular oncogenic events modify pRb's capacity to regulate the expression of the PUM complex, we depleted p16 from BJ cells or treated HCT116 cells which lack p16, with CDK4/6 inhibitors. Knockdown of p16 in BJ cells stimulated the expression of the entire PUM complex except NANOS2 (Supplementary Fig S5A). Conversely, re-activating pRb by treating HCT116 cells with the CDK4/6 inhibitor (PD0332991) reduced cell number and NANOS expression (Supplementary Fig S5B and C). To examine whether there is a link between pocket proteins and the expression of PUM complex components in cancer cells, we compared the expression of each PUM and NANOS gene to that of each pocket protein (Rb1, Rbl1, and Rbl2) across a broad panel of tumor cell lines (Barretina et al, 2012). In agreement with our Drosophila data linking RBF1 to the repression of Nanos, this analysis revealed a strongly significant anti-correlation between pRb and NANOS1 expression (P = 1.03 × 10−13) and a weaker anti-correlation between pRb and NANOS3 levels (P = 1.22 × 10−5) (Fig 1E, Supplementary Fig S6A and B). Consistent with our fly experiments, we did not find a negative correlation between pRb and PUM1, PUM2, or the poorly characterized Nanos homolog, NANOS2 (Fig 1E). Previous studies have identified a gene expression signature associated with pRb loss in tumors (Herschkowitz et al, 2008). When we compared the pRb loss signature with the expression pattern of the PUM complex components, we found that NANOS1 expression is correlated with the Rb1 loss signature (Supplementary Figs S6C–F and S7). As an additional test of pRb's role in regulating NANOS1 expression, we compared the expression profiles of the PUM complex in primary retinoblastoma tumors (that which contain homozygous mutations in the Rb1 gene) with control retina tissue. NANOS1 expression is up-regulated in the primary retinoblastoma tumor cells (3/3) and retinoblastoma tumor cells grown (2/3) as orthotopic xenografts in mice (Fig 1F). We conclude that NANOS1 expression is up-regulated in cells deficient for pRb activity. Collectively, these data show that RBF1/pRb controls Nanos/NANOS1/3 expression and that this regulation is conserved between Drosophila and humans. Next, we investigated the importance of this interaction. To examine how elevated levels of the Pumilio complex contributed to the cellular homeostasis of tissue with reduced dREAM activity, we tested how reducing the expression of the Pum complex affected Drosophila wings sensitized by RNAi transgenes that depleted E2F2/RBF1/dREAM (Mip120/Mip130) components (Dietzl et al, 2007). Expression of the Pum/Nos/Brat RNAi transgenes alone produced no visible phenotype (Fig 2A, Supplementary Fig S8). Reducing the levels of the Pumilio complex using RNAi in the wing pouch of Drosophila sensitized by depletion of dREAM proteins caused mis-shaped and blistered wings (Fig 2A and B, Supplementary Fig S8, and Supplementary Table S1). A detailed description of how this assay was scored can be found in the Materials and Methods section. These results show that Nanos levels are not only up-regulated when dREAM function is reduced, but that the elevated activity of the Pum complex is also important in Drosophila tissues with compromised E2F/RBF regulation. Figure 2. The Pumilio and dREAM complexes genetically interact Phenotypes produced from genetic interaction experiments using RNAi driven by Nub-Gal4 to reduce the levels of dREAM components (e2f2, mip120, mip130, and rbf1) and the pumilio complex members (pum, nos, and brat) in the Drosophila wing pouch. RNAi constructs used in this experiment were PUM RNAi (36676), NANOS RNAi (28300), BRAT RNAi, and luciferase RNAi as a control. Genetic interaction analysis of wings was scored as follows: no phenotype (−), variable minor phenotype (−/+), minor extra wing vein (+), minor blistering (++), and severely blistered and deformed wings (+++). Table outlining the genetic interaction between PUM complex members and dREAM components. Download figure Download PowerPoint We next wanted to determine whether the activity of the Pum complex that is required to maintain tissue homeostasis in RBF1/E2F2/dREAM compromised cells is conserved in mammalian cells. To do this, we assayed the effect of depleting Pum1, Pum2, and Nanos1 using shRNA from 3T3 lines derived from mouse embryonic fibroblasts (MEFs) containing mutations in the pocket proteins (Rb1, p107, and p130) (Classon et al, 2000). We did not examine the effects of Nanos2 and Nanos3 knockdown because we were unable to find shRNAs that gave efficient depletion of these targets. As shown in Fig 3A and Supplementary Fig S9A–E, depletion of Nanos1 reduced the number of cells in Rb1 null and triple-negative (Rb1, p107, and p130−/−) 3T3s, suggesting that Nanos1 is a critical component in Rb1-deficient cells. Knockdown of Nanos1 did not affect 3T3s solely lacking p107 or p130. Depletion of either Pum1 or Pum2 did not affect the viability of any of the 3T3s. To understand why Nanos1 depletion reduced the number of only Rb1 null 3T3s, we measured the relative expression levels of p16 and the Pum complex components in each of the 3T3 genotypes. All of the 3T3s except the p107 nulls expressed p16 (Supplementary Fig S10B). The levels of the Pum genes varied little between genotypes and remained high compared to the non-E2F-regulated E2F3 gene; however, Nanos1 and Nanos3 levels were strongly elevated in the 3T3s lacking Rb1 alone or the triple-negative cells (Supplementary Fig S10A). These results show that Nanos1 and Nanos3 are specifically up-regulated following the inactivation of pRb and that depletion of Nanos1 levels reduces the numbers of cells. Figure 3. NANOS1 is required for the maintenance of pRb-deficient cells Crystal violet staining of wild-type (wt), Rb1 null (Rb1−/−), p107 null (p107−/−), and p130 null (p130−/−) 3T3 cells infected with shRNAs targeting Nanos1 (Nanos1-3, Nanos1-4), Pum1 (mP1-2, mP1-3), Pum2 (mP2-1, mP2-5), or scrambled sequence (Scr). Quantification of Alamar blue staining of human Y79 retinoblastoma cells after puromycin selection of uninfected (mock) and cells infected with shRNAs targeting NANOS1 or scrambled sequences (Scr) (mean ± SD, n = 3). Crystal violet stain of BJ cells infect with shRNAs targeting scrambled (Scr), pRb (Rb1 E3, Rb1 D4, Rb1 D5), and NANOS1 (hNOS1-1 and hNOS1-3). Quantification of crystal violet staining of BJ cells infected with shRNAs targeting Rb1, NANOS1, and scrambled sequences, including analysis of cell transfected with pCMV-Rb1 post-infection (mean ± SD, n = 3). Download figure Download PowerPoint To test the idea that NANOS1 contributes to the growth of human pRb-deficient cells, we examined the effects of NANOS1 depletion on (Rb1 null) human retinoblastoma cancer cell lines. Knockdown of NANOS1 using shRNA significantly reduced the number (Alamar Blue) of Y79 retinoblastoma cells compared to scrambled controls (SCR) (Fig 3B). To examine the hypothesis that co-depletion of pRb and NANOS1 may reduce cell number, we treated human cell lines, BJ (Fibroblasts) and Calu-1 cells (non-small cell lung carcinoma cells (NSCLC)), with shRNAs targeting pRb and NANOS1, and assayed cell number using crystal violet staining. Excitingly, lowering the levels of pRb and NANOS1 reduced the number of cells in both fibroblasts (Fig 3C, Supplementary Fig S10C) and NSCLC cells (Supplementary Fig S10D). This reduction in cell number could be rescued by the transfection of plasmid containing a shRNA insensitive pRb (Fig 3C and D). To examine the role of NANOS1 in pRb-deficient cells, we depleted NANOS1 or scrambled control sequences using shRNAs and assayed cell number in 18 cancer cell lines of diverse tissue origins and mutational profiles. The depletion of NANOS1 reduced cell number in a subset of lines (for quantification of cell staining, Supplementary Fig S11A), and we noticed that these lines have the shared property and that they are compromised for pRb function (mutant for either Rb1 or p16INK4a) and retain an intact p53 (Fig 4A and B, Supplementary Fig S11B). To determine the consequence of NANOS1 loss from pRb-deficient cells, we depleted NANOS1 using siRNA from Y79 retinoblastoma and NCI-H1666 NSCLC cell lines and counted cells over time. As shown in Supplementary Fig S12A–C, depletion of NANOS1 resulted in reduced cell number from both cell lines after 5 days, suggesting NANOS1 functions to inhibit cellular expansion of pRb-deficient cells. To evaluate the contribution of p53 to this interaction, we examined HCT116 cells, a p16INK4a mutant cell line that is sensitive to NANOS1 depletion, and compared the effects of NANOS1 depletion in isogenic lines that either lack or retain p53. HCT116 cells retaining p53 activity display a strong reduction in cell number upon NANOS1 depletion; however, HCT116 p53 null cells were unaffected (Fig 4C, Supplementary Fig S11C (quantification of staining)). These results suggest that NANOS1 prevents p53-mediated inhibition of cellular growth in cells that are deficient for normal pRb activity. Figure 4. p53 activity is necessary for the reduction in cell number upon silencing of Rb1 and NANOS1 Crystal violet staining of cancer cell lines from diverse tissue types (fibroblast, retina, head and neck, kidney, bone, bladder, breast, non-small cell lung carcinoma (NSCLC), and colorectal) infected with shRNAs targeting NANOS1 (hNOS1-1 and hNOS1-3) or scrambled sequences. Summary table of the pRb and p53 status of the cells tested in the panel above and a description of the consequence of NANOS1 and scrambled sequence depletion. Crystal violet staining of isogenic HCT116 cells with (p53 wt) and without (p53 mut) p53 infected with shRNAs targeting NANOS1. Download figure Download PowerPoint To investigate how NANOS1-mediated post-translational regulation contributes to the growth of pRb-deficient cells, we examined gene expression profiles from normal retina tissue and retinoblastoma tumors (Ganguly & Shields, 2010). We observed a striking percentage of PUM substrates among the transcripts that were up-regulated (19%, 208/1,083) and down-regulated (22%, 171/770) in retinoblastomas (Fig 5A). To determine how dys-regulation of the E2F transcription factors would affect these putative PUM substrates, we analyzed the promoters of these genes for E2F binding motifs. E2F motifs were identified in 51% of the up-regulated and 7% of the down-regulated PUM substrates in retinoblastomas (Fig 5A). This suggested that the up-regulation of the E2F transcriptional program may be sufficient to counterbalance and override the post-transcriptional regulation of the PUM complex. We therefore focused on the genes that are down-regulated in retinoblastomas and that did not contain E2F motifs. Interestingly, gene ontology classification of the putative PUM targets among these down-regulated genes showed a strong enrichment for kinases and regulators of apoptosis, an enrichment that was not evident when the overall group of down-regulated transcripts was examined (Fig 5B). To determine whether this reduction in the mRNA levels