JMJD2A Promotes Cellular Transformation by Blocking Cellular ...

Cell Reports

Article JMJD2A Promotes Cellular Transformation by Blocking Cellular Senescence through Transcriptional Repression of the Tumor Suppressor CHD5 Fre´de´rick A. Mallette1,2,5 and Ste´phane Richard1,2,3,4,* 1Terry Fox Molecular Oncology Group and the Bloomfield Center for Research on Aging, Sir Mortimer B Davis Jewish General Hospital, Lady Davis Institute for Medical Research, Montreál, Que´bec H3T 1E2, Canada 2Department of Medicine, McGill University, 687 Pine Avenue West, Room A3.09, Montre ´ al, Que´bec H3A 1A1, Canada 3Department of Oncology, McGill University, 546 Pine Avenue West, Room 211, Montre ´ al, Que´bec H2W 1S6, Canada 4Segal Cancer Centre, Lady Davis Institute for Medical Research, 3755 Co ˆ te Ste-Catherine Road, Montreál, Que´bec H3T 1E2, Canada 5Present address: Maisonneuve-Rosemont Hospital Research Center, Department of Medicine, Universite ´ de Montreál, 5415 L’Assomption, Montreál, Que´bec H1T 2M4, Canada *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2012.09.033

SUMMARY

Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5, a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Rasmediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated KRas allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors. INTRODUCTION The Ras GTPases are signaling molecules coupling receptor activation to cellular processes including cell survival, differentiation, and proliferation (Colicelli, 2004). The three cellular Ras genes encode highly homologous 21 kDa proteins (H-Ras, N-Ras, and K-Ras). Ras stimulates a complex signaling network through the activation of a phosphorylation cascade involving Raf/MEK/ERK, RAL, and PI3K pathways. The small GTP-binding proteins Ras are frequently mutated in human cancers, particularly in tumors of the lung, pancreas, and colon (Schubbert et al., 2007). In the majority of cases, missense Ras mutations found in cancer cells create amino acid substitutions at positions 12, 13, and 61 that impair the Ras intrinsic and GAP-mediated ability to

hydrolyze GTP, rendering Ras constitutively activated (Kiaris and Spandidos, 1995). Activation of Ras is not sufficient per se to transform primary cells, and most of the cancers display mutations in more than one gene. The most frequently mutated pathways are the receptor tyrosine kinase/Ras/B-Raf signaling cascade, and the INK4A/CyclinD/Rb and ARF/MDM2/p53 tumor-suppressive pathways (Yeang et al., 2008). Mutations in genes involved in different pathways can occur in the same cancer, whereas mutations of genes implicated in the same pathway are less frequent in the same patient. Normal cells are protected against transformation mediated by the activation of oncogenes, such as Ras, by undergoing a permanent cell-cycle arrest, known as oncogene-induced senescence (OIS) (Serrano et al., 1997). Whereas murine cells rely mostly on p53, human cells entering OIS trigger the activation of the p53 and Rb pathways. Inhibition of these two tumor suppressors blocks the ability of the cell to trigger OIS following Ras activation (Ferbeyre et al., 2000; Mallette et al., 2007; Serrano et al., 1997). OIS constitutes an important tumorsuppressive mechanism in vivo and can be observed in different premalignant tumors such as lung adenomas, neurofibromas, and naevi (Collado et al., 2005; Courtois-Cox et al., 2006; Michaloglou et al., 2005). The senescence program also contributes to the response to cancer therapy in mouse model of lymphomas (Schmitt et al., 2002). Furthermore, restoration of p53 into murine liver carcinomas triggers senescence and tumor regression in vivo (Xue et al., 2007). The p53 tumor suppressor pathway is central to the establishment of cellular senescence. This transcription factor regulates the expression of multiple genes involved in the senescence response such as the regulator of the cell-cycle p21CIP1 (el-Deiry et al., 1993), the nuclear body component promyelocytic leukemia protein (PML) (de Stanchina et al., 2004), and the extracellular matrix component PAI-1 (Kortlever et al., 2006). The activation of p53 during OIS seems to involve multiple mechanisms, for instance, the DNA damage response and the protein ARF. The DNA damage response kinase ATM activates p53 during OIS (Bartkova et al., 2006; Di Micco et al., 2006; Mallette et al., 2007). The phosphorylation of p53 on serine 15 by ATM

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prevents its interaction with the E3 ubiquitin ligase MDM2, thus avoiding the degradation of p53 (Shieh et al., 1997). The tumor suppressor ARF is the product of an alternative reading frame of the INK4A/ARF locus. It is known to stabilize p53 by interacting and sequestering MDM2 to the nucleolus (Weber et al., 1999). ARF is induced by the activation of Ras and required for OIS (Ferbeyre et al., 2002). The regulation of the ARF locus by the Polycomb group (PcG) protein Bmi-1 allows bypass of senescence by downregulating the expression of INK4A and ARF (Jacobs et al., 1999). The T-box member Tbx2 also repressed the ARF locus and was identified for its capacity to inhibit senescence (Jacobs et al., 2000). More recently, the chromodomain helicase DNA-binding domain 5 (CHD5) tumor suppressor has been shown to be required for Ras-induced senescence by regulating the INK4A/ARF locus (Bagchi et al., 2007). CHD5 is a member of the chromatin organizing modulator domain superfamily harboring two Zinc-binding plant homeodomain (PHD) fingers, chromo motifs, and a helicase domain (Marfella and Imbalzano, 2007). CHD5 is a tumor suppressor mapping to 1p36, a site frequently deleted or inactivated in human cancers (Bello et al., 1995; Bie`che et al., 1993; White et al., 1995). Histone posttranslational modifications, such as phosphorylation, ubiquitination, acetylation, and methylation, can modify the structure of chromatin and also contribute to the formation of protein complexes controlling gene expression. Among these modifications, methylation of H3K4 and H3K36 is generally linked to transcriptionally active genes, whereas H3K9 and H3K27 are associated with repressed genes. Multiple lysine methyltransferases are responsible for the methylation of histones and exert control over gene expression (Kouzarides, 2007). However, the discovery of lysine demethylases showed that lysine methylation is a reversible and dynamic process. The Jumonji lysine demethylases are oxygenases catalyzing the demethylation of mono-, di-, or trimethylated lysines. The JMJD2 family of lysine demethylases comprises four members (JMJD2A, JMJD2B, JMJD2C, and JMJD2D) and are Fe(II)- and a-ketoglutarate-dependent enzymes harboring a JmjC catalytic domain (Tsukada et al., 2006; Whetstine et al., 2006). JMJD2A, JMJD2B, and JMJD2C harbor a similar protein structure including a JmjN domain, a tandem PHD, and a tandem tudor domain, whereas JMJD2D lacks the PHD and tandem tudor domains. JMJD2A catalyzes the demethylation of di- and trimethylated H3K9 and H3K36 and is considered as a transcriptional repressor due to its ability to inhibit expression of its only known target gene ASCL2 (Klose et al., 2006; Zhang et al., 2005). JMJD2A also possesses a tandem tudor domain binding to H3K4(me2/3) and H4K20(me2/3) (Huang et al., 2006; Lee et al., 2008). Gene amplification of JMJD2C lysine demethylase has been associated with prostate, breast, esophagus, and brain cancers (Cloos et al., 2006; Ehrbrecht et al., 2006; Liu et al., 2009; Yang et al., 2000); however, little is known about the expression and the role of JMJD2A in cancer. Here, we demonstrate the important role of JMJD2A in the inhibition of RasV12-induced senescence by inhibiting the p53 pathway. Depletion of JMJD2A in normal human diploid fibroblasts induced a premature cellular senescence phenotype requiring the p53 tumor suppressor. In order to identify gene

promoters regulated by JMJD2A, we performed chromatin immunoprecipitation (ChIP)-on-chip and discovered the tumor suppressor CHD5 as a target of JMJD2A. Furthermore, JMJD2A cooperates with RasV12 and E1A to transform primary cells, and we report that human lung carcinomas harbor high expression levels of JMJD2A. We propose that JMJD2A is an oncogene cooperating with RasV12 to promote tumorigenesis through the transcriptional repression of CHD5. RESULTS Depletion of JMJD2A Induces a p53-Dependent Senescence Program and JMJD2A Controls the p53 Pathway during OIS Recently, we showed that JMJD2A is degraded in response to DNA damage (Mallette et al., 2012) and it was shown that in Caenorhabditis elegans JMJD2A depletion increases DNA damage (Whetstine et al., 2006). As DNA damage is a prerequisite for cellular senescence (Mallette et al., 2007), we asked whether depletion of JMJD2A triggered premature cellular senescence. We initially examined the role of JMJD2A in cellular proliferation by depleting JMJD2A in U2OS osteosarcoma cells. U2OS depleted of JMJD2A by RNA interference displayed a G0/G1 growth arrest as evidenced by fluorescence-activated cell sorting (FACS) (Figure S1A). To verify if the observed growth arrest could be linked to senescence, we then depleted JMJD2A in the normal human fibroblasts, IMR90. To do this, we transfected IMR90 cells with small interfering RNAs (siRNAs) targeting JMJD2A (siJMJD2Asp) that efficiently depleted JMJD2A protein expression (Figure 1A). Compared to cells transfected with siGFP, cells depleted of JMJD2A exhibited a flattened vacuolated morphology accompanied by a positive staining to the senescence-associated b-galactosidase assay (SA-b-gal) (Figure 1B). The use of two additional lentiviral small hairpin RNAs (shRNAs) targeting JMJD2A also led to the induction of the senescence phenotype (Figure S1B). A similar phenotype was observed in mouse embryonic fibroblasts (MEFs) depleted of JMJD2A using siRNAs (Figures S1C and S1D). An important hallmark displayed by oncogene-induced senescent cells is the accumulation of tightly packed chromatin structures named senescence-associated heterochromatin foci (SAHF). The SAHF are associated to the accumulation of the heterochromatin associated proteins HP1 and increased trimethylated histone H3 on lysine 9 [H3K9(me3)]. The formation of SAHF seems to be mostly dependent on the p16/Rb pathway and contributes to the stable repression of E2F-dependent genes (Narita et al., 2003). Because JMJD2A catalyzes the demethylation of H3K9(me2/3), we first considered the possibility that JMJD2A-depleted cells could accumulate H3K9 methylated histones and form SAHF. Surprisingly, JMJD2A depletion was insufficient to lead to the formation of SAHF as evidenced by the absence of H3K9(me3) foci (Figure 1C). However, the knockdown of JMJD2A led to the accumulation of PML nuclear bodies (Figure 1C), another wellcharacterized senescence marker (Ferbeyre et al., 2000; Pearson et al., 2000). The senescence response observed in human cells relies mostly on the activation of the p53 and/or Rb pathways (Ferbeyre et al., 2000; Mallette et al., 2007; Serrano et al., 1997). To define the tumor-suppressor requirement of JMJD2A

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JMJD2A levels relies primarily on the p53 pathway. This also suggested that JMJD2A is a potential negative regulator of the p53 pathway. To further investigate this possibility, we examined the capacity of JMJD2A to block the p53 pathway during Rasinduced senescence. In IMR90 cells, Ras-induced senescence depends on either the p53 or the Rb pathway and both pathways need to be inactivated to rescue the senescence phenotype (Mallette et al., 2007; Serrano et al., 1997). Senescence of Ras-transduced cells was severely impaired when sh-p16 and JMJD2A expression were combined (Figures 2 and S2). These findings confirm that JMJD2A negatively controls the p53 pathway. Together, these results indicate that JMJD2A constitutes a negative regulator of p53.

Figure 1. JMJD2A Depletion Causes Cell-Cycle Arrest and p53Dependent Senescence (A) Immunoblot analysis of JMJD2A in IMR90 cells transfected with siGFP or SMARTpool siJMJD2A (siJMJD2Asp) using Lipofectamine RNAiMAX. (B) SA-b-gal staining of IMR90 cells transfected with siGFP or SMARTpool siJMJD2A (siJMJD2Asp) using Lipofectamine RNAiMAX. Cells were transfected on day 0 and day 3 and then fixed on day 6 for SA-b-gal activity. The percentage and SD of SA-b-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells collected 6 days after the initial transfection. (C) Indirect immunofluorescence of PML and H3K9(me3) in IMR90 cells infected with a control shRNA (pLKO.1) or an shRNA targeting JMJD2A (shJMJD2A.3 and sh-JMJD2A.5). Cells were fixed for staining on day 8 postselection. The percentage and SD of the cells displaying more than ten PML nuclear bodies are indicated at the bottom right of each panel. The data represent the average and SD of three independent counts of 100 cells each. (D) IMR90 cells infected with shRNAs targeting p16 (sh-p16) or p53 (sh-p53) were transfected with siGFP or SMARTpool siJMJD2A (siJMJD2Asp) and stained for SA-b-gal activity. The percentage and SD of SA-b-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells that were retransfected every 3 days and fixed for staining 10 days after the initial transfection. See also Figure S1.

depletion-induced senescence, IMR90 cells stably infected with sh-p16 or sh-p53 were generated. Transfection of siRNAs targeting JMJD2A triggered senescence in p16-depleted cells but not in p53-depleted cells (Figures 1D, S1E, and S1F), suggesting that the senescence response triggered by decreased

CHD5 Is a Transcriptional Target of JMJD2A To uncover the molecular mechanism by which JMJD2A regulates the p53 pathway, we took advantage of the p53-proficient U2OS cells, which enter a G0/G1 growth arrest after JMJD2A depletion (Figure S1A), to identify promoters bound by JMJD2A using a ChIP-on-chip approach. Using an array covering more than 10 kb of each gene promoter (from 7,500 to +3,250), we identified multiple potential target gene promoters bound by JMJD2A (Table S1). To date, the only known JMJD2A-regulated gene was the transcription factor ASCL2 (Klose et al., 2006; Zhang et al., 2005). The identification of the ASCL2 promoter by ChIP-on-chip validated our approach to discover additional JMJD2A target genes (Table S1). Among the many JMJD2A target genes, we identified the chromodomain helicase DNAbinding domain 5 (CHD5) tumor suppressor. The CHD5 gene, located at 1p36, is frequently deleted in human cancers and controls cell proliferation and senescence via the p53 pathway (Bagchi et al., 2007). To confirm the binding of JMJD2A to the identified gene promoters, we performed ChIP-qPCR experiments. Again, ASCL2 was enriched by nearly 2-fold in the flag-JMJD2A immunoprecipitated material (Figure 3A). We also validated the presence of JMJD2A at the identified gene promoters including PANX2, RHOQ, PPIC, CDC6, GP5, and CHD5. Among the two putative JMJD2A-binding sites identified by ChIP-on-chip on the CHD5 promoter, only the site located at +741 was enriched by ChIP-qPCR (Figure 3A). We then used RT-qPCR to validate the regulation of the JMJD2A-bound target genes at the transcriptional level following JMJD2A depletion. The transfection of U2OS cells using siJMJD2A SMARTpool led to a dramatic decrease of JMJD2A mRNA but not of the other members of the JMJD2 family, JMJD2B, JMJD2C, and JMJD2D (Figure 3B). Depletion of JMJD2A caused a significant increase in ASCL2 mRNA levels confirming that JMJD2A acts as a transcriptional repressor as previously reported (Klose et al., 2006; Zhang et al., 2005). JMJD2A depletion also led to a concomitant increase of CHD5 mRNA and protein (Figures 3B and 3C). To eliminate the possible contribution of off-target effects using the SMARTpool of siRNAs, we also used two different individual siRNAs deconvoluted from the siJMJD2A SMARTpool to efficiently deplete JMJD2A and observed a concomitant increase in both ASCL2 and CHD5 mRNA levels (Figures S3A and S3B). We then looked at the ability of other members of the JMJD2 family of lysine demethylases to regulate the transcript levels of CHD5. Depletion of JMJD2A, but not of JMJD2B or JMJD2C,


Figure 2. JMJD2A Negatively Controls the p53 Pathway during Oncogene-Induced Senescence SA-b-gal staining of IMR90 cells infected with an empty vector control or RasV12, flag-JMJD2A, and/or an shRNA targeting p16 (sh-p16). The percentage and SD of SA-b-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells fixed for staining on day 8 postselection. See also Figure S2.

led to a significant increase of CHD5 (Figure S3C). Interestingly, PANX2 and RHOQ mRNA levels were also increased following depletion of JMJD2A, whereas CDC6 levels were not significantly regulated (Figure 3B). However, GP5 and PPIC mRNA levels decreased after treatment with siJMJD2A, suggesting that JMJD2A may also act as a transcriptional activator (Figure 3B). To further investigate the role of JMJD2A in the regulation of CHD5, we performed ChIP experiments to assess the changes in histone methylation of the CHD5 promoter. We detected an increase of H3K9(me3) levels at the CHD5 promoter after depletion of JMJD2A (Figure 3D). As a control, we observed an increase of H3K9(me3) at the ASCL2 promoter in siJMJD2Atreated cells as previously reported (Klose et al., 2006; Zhang et al., 2005). Taken together, these results reveal additional target gene promoters regulated by JMJD2A and identify CHD5 as a key target of JMJD2A. To reinforce the link between CHD5 and JMJD2A, we investigated the role of CHD5 during JMJD2A depletion-induced senescence. To do this, we cotransfected IMR90 cells with siRNAs targeting JMJD2A and/or CHD5. While JMJD2Adepleted cells enter senescence, we observed a significant reduction in the SA-b-Gal staining of cells with the combined depletion of CHD5 and JMJD2A in the human fibroblasts IMR90 (Figures 3E and S3D). Similar results were observed in MEFs (Figures S3E–S3G). JMJD2A depletion caused a significant increase of p19ARF protein levels concomitant with the increase of CHD5 (Figure 4A). Although depletion of JMJD2A leads to a modest increase in total p53 protein levels, it causes an important increase in p53 nuclear localization (Figure 4B). The senescence induced following depletion of JMJD2A not only required p53, but was also p19ARF dependent as confirmed by the use of knockout MEFs (Figures 4C and 4D). This suggests that the p53-dependent senescence program triggered by decreased JMJD2A levels is mediated by CHD5 via p19ARF. JMJD2A Blocks OIS-Mediated Activation of p53 To determine the role of JMJD2A in OIS, we examined the protein levels of JMJD2A during RasV12-induced senescence. Following introduction of activated Ras in IMR90 cells, JMJD2A protein levels are significantly decreased, whereas CHD5 and p21 proteins are increased (Figure 5A). To investigate whether CHD5 is the target gene by which JMJD2A regulates the p53 pathway during Ras-induced senescence, we looked at the ability of JMJD2A to repress CHD5 expression in normal human diploid fibroblasts. According to what we observed in U2OS cells, JMJD2A depletion in IMR90 cells infected using two different shRNAs also led to a significant increase of CHD5 (Figure S4). We then looked at the mRNA and protein levels of the JMJD2A target genes ASCL2 and CHD5 during OIS. Concomitant with the reduction in JMJD2A previously observed in 1236 Cell Reports 2, 1233–1243, November 29, 2012 ª2012 The Authors

Figure 3. JMJD2A Binds to the CHD5 Gene and Controls Its Transcriptional Expression (A) Validation by ChIP-qPCR of selected genes identified by ChIP-on-chip. U2OS cells were transfected with an empty control vector or flag-JMJD2A and ChIP was performed using Flag M2 antibody 48 hr after transfection. (B) Expression levels of mRNA of genes regulated by JMJD2A. U2OS cells were transfected with siGFP or a SMARTpool targeting JMJD2A (siJMJD2Asp) using Lipofectamine RNAiMAX and mRNA extracted 72 hr after transfection. Relative mRNA levels were normalized to GAPDH mRNA. (C) Immunoblot analysis of CHD5 protein levels in U2OS cells transfected with siGFP or a SMARTpool targeting JMJD2A (siJMJD2Asp) using lipofectamine RNAiMAX. (D) Chromatin immunoprecipitation of H3K9(me3) at the promoters of ASCL2 and CHD5 genes in U2OS transfected with siGFP or siJMJD2Asp. (E) SA-b-gal staining of IMR90 transfected with siGFP, siJMJD2A-02, siJMJD2A-03, and siCHD5. The percentage and SD of SA-b-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells that were retransfected every 3 days and fixed for staining 10 days after the initial transfection. See also Figure S3.

Ras-expressing cells, ASCL2 and CHD5 mRNA and protein were strikingly upregulated during RasV12-induced senescence (Figures 5B and 5C). Ectopic expression of JMJD2A inhibited the RasV12-induced increase of ASCL2 and CHD5 levels (Figures 5B and 5C) and led to an associated decrease of the levels of the p53 target genes p21 and NOXA (Figure 5C). In some experimental settings, the control of p21 expression by Ras may be p53-independent (Delgado et al., 2000). To further support the p53-dependent control of p21 expression exerted by JMJD2A during OIS, we generated different IMR90 cell lines expressing an shRNA against p53 (sh-p53) in combination with RasV12 and JMJD2A. The accumulation of p21 during Rasinduced senescence in normal human fibroblasts is dependent

Figure 4. JMJD2A Depletion-Induced Senescence Requires p19ARF (A) Immunoblot of p19ARF in MEFs transfected with siGFP or SMARTpool siJMJD2A (siJMJD2Asp) using Lipofectamine RNAiMAX. (B) Indirect immunofluorescence against p53 in MEF as prepared in (A). (C) SA-b-gal staining of wild-type, p53/, and p19ARF/ MEF transfected with siGFP or siJMJD2Asp. The percentage and SD of SA-b-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments performed with cells that were retransfected every three days and fixed for staining 10 days after the initial transfection. (D) Indirect immunofluorescence against PML of the cells prepared in (C). The percentage and SD of the cells displaying more than ten PML nuclear bodies are indicated at the bottom left of each panel. The data represent the average and SD of three independent counts of 100 cells each.

on the presence of p53 (Figure 5D). Ectopic expression of JMJD2A caused a significant decrease of both p53 and p21 in Ras-expressing cells (Figure 5D). These observations suggest that JMJD2A negatively controls the Ras-induced activation of p53 by mediating the repression of CHD5. JMJD2A Cooperate with Ras Activation in Promoting Tumorigenesis To investigate the effect of JMJD2A expression on RasV12driven transformation, we took advantage of the experimental system where RasV12, E1A, and MDM2 cooperate to transform normal diploid fibroblasts (Seger et al., 2002). We rationalized


Figure 5. JMJD2A Blocks RasV12-Mediated p53 Activation (A) Immunoblot analysis of JMJD2A, CHD5, p21, and a-tubulin as a loading control in IMR90 cells infected with a control vector or RasV12. (B) mRNA levels of ASCL2 and CHD5 in IMR90 cells infected with an empty vector control, H-RasV12 and flag-JMJD2A. RNA was extracted at day 8 postselection and quantitative RT-PCR performed. Relative mRNA levels were normalized to GAPDH mRNA. (C) Immunoblot analysis of CHD5 and p53 target genes in IMR90 cells prepared as in (B). (D) Immunoblot analysis of p53 and p21 in IMR90 cells expressing different combinations of H-RasV12, flag-JMJD2A, and sh-p53. See also Figure S4.

that, according to our genetic experiment showing that JMJD2A negatively controls the p53 pathway during Ras-induced senescence (Figure 2), JMJD2A could replace MDM2 to promote Rasmediated transformation. We first infected IMR90 cells with retroviral vectors encoding RasV12 and E1A. Then, we introduced MDM2, JMJD2A, JMJD2AH188A, or an empty control vector and injected the selected cells into the flanks of immunocompromised mice. As expected, the E1A/RasV12/MDM2 cells formed tumors at 100% of the injection sites (8/8) (Figure 6A). The replacement of MDM2 by JMJD2A in combination with RasV12 and E1A, also significantly promoted the Ras-mediated formation of cells leading to the formation of tumors at most of the injection sites (14/22), although less efficiently than the combination with MDM2 (Figure 6A). Mutation of the catalytic domain of JMJD2A (JMJD2AH188A) completely abolished the ability of JMJD2A to promote Ras-mediated transformation since no tumors (0/6) were observed following injection of the E1A/ RasV12/JMJD2AH188A cells (Figure 6A). The catalytic activity of JMJD2A is then required to cooperate with Ras in cellular transformation. The tumors formed following injection of E1A/ RasV12/JMJD2A displayed high levels of flag-tagged JMJD2A, confirming that these tumors arose from the initial JMJD2Aexpressing fibroblasts (Figure 6B). These results strongly

Figure 6. JMJD2A Contributes to the Transformation of Primary Cells and Is Overexpressed in Mouse Model of Lung Tumors (A) Tumorigenicity assay using IMR90 cells infected with combinations of empty vector, E1A, RasV12, JMJD2A, JMJD2AH188A, or MDM2, and injected subcutaneously in immunocompromised mice. (B) Immunoblot analysis of flag-JMJD2A expression in tumors observed following injection of IMR90 cells expressing E1A/RasV12/JMJD2A (E/R/J) or E1A/RasV12/MDM2 (E/R/M). (C) Immunohistochemical analysis of JMJD2A protein expression in lungs from wild-type and K-RasLA2 mice. Scale bar represents 200 mm.

suggest that JMJD2A cooperates with Ras in promoting cell transformation by negatively regulating the p53 pathway. To further investigate the role of JMJD2A in Ras-mediated tumorigenesis, we used the K-RasLA2 mouse model of lung carcinomas (Johnson et al., 2001). K-Ras activation led to the formation of tumors displaying strong JMJD2A-positive staining, whereas lungs of wild-type littermates showed weak staining (Figure 6C). To examine the role of JMJD2A in human cancer, we analyzed several human lung carcinomas in which Ras mutations occur


Figure 7. JMJD2A Is Overexpressed in Human Lung Tumors and Depletion of JMJD2A Triggers Senescence in the Human Lung Cancer Cell Line A549 (A) Immunohistochemical analysis of JMJD2A protein expression in human lung carcinomas and normal lung tissues. Scale bar represents 50 mm. (B) Immunohistochemical score (IHS) of 10 normal lung tissues and 108 lung carcinomas immunostained for JMJD2A as displayed in (A). An IHS score >3 was considered as positive expression. (C) SA-b-gal staining of A549 human lung cancer cells infected with lentiviral vectors pLKO.1 or shRNA against JMJD2A. The percentage and SD of SA-b-galpositive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells collected 8 days postselection. (D) Growth curve of A549 human lung cancer cells infected with lentiviral vectors pLKO.1 or shRNA against JMJD2A as in (C). See also Figure S5.

frequently (Schubbert et al., 2007). We observed high levels of JMJD2A in nearly 17% (18/108) of the human lung carcinomas, whereas none of the normal lung tissue (0/10) displayed JMJD2A expression (Figures 7A and 7B). Furthermore, the JMJD2A staining was intense in lung carcinomas but absent in normal lung tissues (Figure 7B). To study the role of JMJD2A in cancer cell growth, we depleted JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele. Interestingly, depletion of JMJD2A in A549 cells led to the establishment of senescence and growth arrest, suggesting that JMJD2A plays a crucial role in the growth of Ras-expressing cancer cell lines (Figures 7C and 7D). We further test the cellular response to JMJD2A knockdown in different normal fibroblasts (WI-38 and BJ) and cancer cell lines (MCF7, HeLa, and 293T). The depletion of JMJD2A caused defects in proliferation of the p53-proficient WI-38, BJ, and MCF7, but not in the p53-inactivated 293T and HeLa (Figures S5A and S5B). This reduced proliferation observed was not associated with cell death, but displayed features of senescence (Figures S5C and S5D). Taken together, these results reveal that JMJD2A is an oncogene cooperating with RasV12 in promoting cellular transformation by negatively controlling the p53 pathway through the transcriptional repression of the tumor suppressor CHD5.

DISCUSSION Understanding the molecular mechanisms leading to the establishment of tumor suppression such as OIS is crucial for the development of innovative therapeutics for blocking tumor progression. Based on the results presented here, we define a molecular pathway contributing to the activation of p53 during OIS. The lysine demethylase JMJD2A is known to catalyze the removal of di- and trimethyl groups from lysine 9 and 36 of histone H3. Here we describe that JMJD2A depletion is sufficient to trigger a p53-dependent senescence response (Figure 1D) and ectopic expression of JMJD2A is able to negatively regulate p53 during OIS (Figure 5). Taken together, these results strongly suggest that JMJD2A negatively regulates p53. Using ChIP, we identified a myriad of JMJD2A target genes including the tumor suppressor CHD5 (Figure 3). CHD5 plays an important role in the activation of p53 during OIS (Bagchi et al., 2007) and the CHD5 gene is frequently deleted in human cancers (Bello et al., 1995; Bie`che et al., 1993; White et al., 1995). We show that ectopic expression of JMJD2A hinders Ras-induced expression of CHD5 leading to p53 activation defects (Figures 5B–5D). The increased PML nuclear bodies observed following JMJD2A depletion (Figure 1C) also indicates that JMJD2A negatively


controls p53 activation, since PML is a transcriptional target of p53 (de Stanchina et al., 2004). However, ectopic expression of JMJD2A does not completely abrogate the expression of p53 target genes (Figures 5C and 5D) and the residual p53 activation observed in cells expressing both Ras and JMJD2A might explain the less efficient transformation mediated by JMJD2A compared to MDM2 (Figure 6A). This implies that other molecular pathways contribute to p53 activation during OIS. In fact, a DNA damage response caused by replicative stress (Di Micco et al., 2006), as well as reactive oxygen species (Mallette and Ferbeyre, 2007) is initiated by oncogenic activation in normal cells. This DNA damage response triggered by the PIK-related kinase ATM is required to activate p53 during OIS (Bartkova et al., 2006; Di Micco et al., 2006; Mallette et al., 2007). Downregulation of JMJD2A and the DNA damage response could possibly cooperate to achieve full p53 activation during OIS. The CHD5 tumor suppressor is frequently deleted in human cancers (Bello et al., 1995; Bie`che et al., 1993; White et al., 1995). Here, we describe a mechanism of inactivation of CHD5 through the transcriptional repression mediated by JMJD2A. This suggests that the increased JMJD2A expression observed in lung tumors of human and mice could contribute to the control of the p53 pathway through the control of CHD5. Recently, it has been demonstrated that CHD5 protein levels are low or absent in lung cancer and that enforced expression of CHD5 in lung cancer cell lines causes a significant decrease in clonogenicity (Zhao et al., 2012). The elevated JMJD2A expression observed in lung tumors of both human and mouse could contribute to the reduction of CHD5 levels. Furthermore, JMJD2A expression can replace the MDM2 oncogene in Ras-mediated transformation of normal diploid fibroblasts confirming its capacity to negatively regulate the p53 pathway (Figure 6A). Altogether, our results imply that JMJD2A cooperates with Ras during tumorigenesis. The expression of the lysine demethylase JMJD2C is significantly increased in prostate cancer compared to normal tissue (Cloos et al., 2006), but the levels of JMJD2A in cancer tissues remained to be determined. Here we show that JMJD2A levels are also elevated in lung adenocarcinomas and that JMJD2A cooperates with Ras-mediated tumorigenesis by restraining p53 activation. Interestingly, JMJD2A seems to be essential for different cancer cell lines since its depletion triggers either apoptosis (Kim et al., 2012) or senescence (Figure 7C), thus limiting the proliferation potential of the cells. JMJD2A also seems to be required for tumor cell invasion and migration in breast cancer (Li et al., 2011). JMJD2A has been linked to the control of the cell cycle by regulating DNA replication and chromatin accessibility (Black et al., 2010). As demonstrated also by Black et al. (2010), depletion of JMJD2A in 293T cells does not cause proliferation defects or senescence and this is due to the lack of a functional p53 tumor suppressor pathway in 293T cells (Figure S5). Since its discovery, the JMJD2A lysine demethylase has been proposed to act as a transcriptional repressor. JMJD2A has been identified as an interacting protein with the transcriptional repressor complex N-CoR including HDAC1 and HDAC3 (Gray et al., 2005; Zhang et al., 2005). Depletion of JMJD2A has been shown to trigger the accumulation of H3K9(me3) levels

and lead to a concomitant increase in ASCL2 gene expression (Figure 3) (Klose et al., 2006; Zhang et al., 2005). We now extend the list of JMJD2A transcriptionally repressed target genes with the identification of CHD5, PANX2, and RHOQ (Figures 3A and 3B). Although we confirm that JMJD2A can act as a transcriptional repressor, we also identified JMJD2A-activated genes (GP5 and PPIC), suggesting that JMJD2A could also act as a transcriptional activator. Although CDC6 is an interesting candidate, we were unable to validate it as a transcriptional target of JMJD2A (Figure 3B). The choice between activation and repression may be dictated by the chromatin structure, the presence or absence of other transcriptional regulators, and environmental stresses such as hormone stimulation. Senescence represents an excellent cellular model to study putative oncogenes cooperating with Ras. The exploitation of the requirement of the ARF/p53 and INK4A/Rb pathways during Ras-induced senescence allowed the identification of additional regulators of these pathways. Among them, KLF4 caused the bypass of OIS through the transcriptional repression of p53 (Rowland et al., 2005), and hDRIL1 led to activation of E2F1 to inhibit OIS (Peeper et al., 2002). Here, we identify JMJD2A as an oncogene cooperating with Ras through its ability to suppress cellular senescence. Furthermore, JMJD2A depletion triggers a senescence response, suggesting that its inactivation by small molecule inhibitors might trigger senescence in cancer cells. Clinically, some treatments rely on their capacity to force cancer cells into a senescent state (Ewald et al., 2010; Nardella et al., 2011). Recent studies indicate that cellular senescence plays a crucial role in the suppression of oncogene-induced tumorigenesis in vivo (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005; Lin et al., 2010; Michaloglou et al., 2005; Ventura et al., 2007; Xue et al., 2007). EXPERIMENTAL PROCEDURES Cells, Vectors, and Retroviral Infections Human diploid fibroblasts IMR90, WI-38, BJ, MEF, MEF p53/, MEF p19ARF/, MCF7, A549, HeLa, 293T, and osteosarcoma cells U2OS (ATCC) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics. The following retroviral vectors were used: pLPC-puro, pWZL-hygro E1A, pWZL-Blast RasV12, MSCV-sh-p16, pRS-sh-p53, and pWZL-neo MDM2 (Narita et al., 2006). The pLPC-puro flag-JMJD2A was generated by introducing an amino-terminal flag epitope by PCR and cloning into the BamHI site of pLPC-puro. Retroviral infections, cell-cycle analysis, and senescence-associated b-galactosidase assays were carried out as described previously (Mallette et al., 2007). Western Blot Analysis and Immunofluorescence For western blotting, cells were lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and EDTA-free protease inhibitors (Complete, Roche) and sonicated three times for 10 s each. Proteins were separated on SDS-PAGE and transferred to nitrocellulose membranes (BioRad). Primary antibodies used were anti-a-tubulin (B-5-1-2, Sigma, 1:5,000), anti-Flag (M2, Sigma, 1:4,000), anti-JMJD2A (NB110-40585, Novus Biologicals, 1:1,000), anti-CHD5 (ab66516, Abcam, 1:500), anti-p53 HRPconjugated (HAF1355, R&D Systems, 1:5,000), anti-p16 (F-12, Santa Cruz, 1:500), anti-p21 (05-345, Millipore, 1:1,000), anti-p19ARF (Ab80, Abcam, 1:1,000), and anti-NOXA (114C307, EMD, 1:500). For immunofluorescence, IMR90 cells infected with different combinations of retroviral plasmids were grown on coverslips in 6-well plates. Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with PBS, 3%


BSA, 0.2% Triton X-100 for 5 min. Cells were then washed three times with PBS and 3% BSA and stained with anti-Flag (M2, Sigma, 1:400), anti-HP1a (2616, Cell Signaling, 1:400), anti-H3K9(me3) (AR-0170, Lake Placid Biologicals, 1:400), anti-p53 (2524, Cell Signaling, 1:400), or anti-PML (PG-M3, Santa Cruz Biotechnology, 1:400). Alexa Fluor 488- or Alexa Fluor 546-conjugated secondary antibodies (Molecular Probes, 1:1,000) were used. shRNA Lentiviral Infections and siRNA Transfections The pLKO.1 lentiviral vectors containing shRNAs pLKO.1-shJMJD2A.3 (RHS3979-9581182) and pLKO.1-shJMJD2A.5 (RHS3979-9581184) were obtained from OpenBiosystems. To produce lentiviral particles, 293T cells were transfected using 9 mg of pLKO.1 shRNA plasmid, 6.75 mg of psPAX2 packaging plasmid, and 2.25 mg pMD2.G envelope plasmid. Media was changed 24 hr after transfection and lentiviral particles were harvested 48 hr after transfection. Viral supernatant was filtered through 0.45 mm filters and then supplemented with 4 mg/ml polybrene and 10% FBS. The list of siRNAs used is provided in the Extended Experimental Procedures. Subcutaneous Tumorigenicity Assay Cells were assessed for in vivo tumorigenicity as described (Seger et al., 2002). Briefly, IMR90 cells were infected with a combination of pWZL-hygro E1A, pWZL-Blast RasV12, and a control vector pLPC-puro, pLPC-puro flagJMJD2A, pLPC-puro flag-JMJD2AH188A, or pWZL-neo MDM2 and injected in the flank of 8-week-old immunocompromised athymic nude mice. Cells (5 3 106) were resuspended in 100 ml of sterile PBS and injected using a 25 gauge needle into anesthetized mice. The mice were monitored daily and were handled and sacrificed in accordance with a protocol approved by the Animal Care Committee at McGill University. Immunohistochemistry Staining Lungs from wild-type or K-RasLA2 mice and human lung carcinomas and normal tissue array (BC041115, US Biomax) were tested using immunohistochemistry staining according to manufacturer recommendations on an automated immunostainer (Discovery XT system, Ventana Medical Systems, Tucson, AZ, USA). Antigen retrieval was performed with proprietary reagents. JMJD2A (5328, Cell Signaling, 1:20) antibody was applied on every sample for 60 min at room temperature. Sections were then incubated with a specific secondary biotinylated antibody. Streptavidin horseradish peroxidase, and 3,30 -diaminobenzidine were used according to the manufacturer’s instructions (Ventana Medical Systems). Finally, sections were counterstained with hematoxylin. As for quantitative assessment, slides were scanned using the Hamamatsu’s NanoZoomer 2.0-HT Digital Pathology system. Virtual slides were then uploaded on a server dedicated to histology facility users, http:// www.histo.iric.ca, and visualized with the mScope Education Portal 3.6.2 (Aurora, Montreal). The expression level of JMJD2A was evaluated by the pathologist by combining an estimate of the percentage of immunoreactive cells (quantity score) with an estimate of the staining intensity (staining intensity score). For instance, less than 10% labeled cells were scored as 1, 10%–50% as 2, 50%–70% as 3, and 70%–100% as 4. Staining intensity was rated on a scale from 0 to 3 as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The raw data were then converted to the immunohistochemical score (IHS) by multiplying the quantity and the staining intensity scores. Therefore, the score could range from 0 to 12. A IHS score >3 was considered as positive expression. SUPPLEMENTAL INFORMATION Supplemental Information includes five figures, three tables, and Extended Experimental Procedures and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2012.09.033. LICENSING INFORMATION This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which

permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ACKNOWLEDGMENTS We thank Masashi Narita for reagents, and members of the Richard laboratory for helpful comments. We thank Gerardo Ferbeyre for the critical reading of the manuscript and for providing the p19ARF-deficient MEFs. We also thank Gillian Vogel and Chau Tuan-Anh Ngo for technical assistance. We are grateful to Raphae¨lle Lambert, the Genomics core facility of the Institute for Research in Immunology and Cancer (IRIC, Montreal), Dr. Louis Gaboury, Julie Hinsinger, and the Histology core facility of IRIC. F.A.M. received postdoctoral fellowships from the Fonds de Recherche en Sante´ du Que´bec (FRSQ), the Canadian Institutes of Health Research (CIHR), and the Terry Fox Foundation through an award from the National Cancer Institute of Canada (NCIC). This work was funded by CIHR grants MOP-67070 and MOP-93811. Received: March 30, 2012 Revised: August 30, 2012 Accepted: September 27, 2012 Published: November 15, 2012 REFERENCES Bagchi, A., Papazoglu, C., Wu, Y., Capurso, D., Brodt, M., Francis, D., Bredel, M., Vogel, H., and Mills, A.A. (2007). CHD5 is a tumor suppressor at human 1p36. Cell 128, 459–475. Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D., Issaeva, N., Vassiliou, L.V., Kolettas, E., Niforou, K., Zoumpourlis, V.C., et al. (2006). Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444, 633–637. Bello, M.J., Leone, P.E., Vaquero, J., de Campos, J.M., Kusak, M.E., Sarasa, J.L., Pestanã, A., and Rey, J.A. (1995). Allelic loss at 1p and 19q frequently occurs in association and may represent early oncogenic events in oligodendroglial tumors. Int. J. Cancer 64, 207–210. Bie`che, I., Champe`me, M.H., Matifas, F., Cropp, C.S., Callahan, R., and Lidereau, R. (1993). Two distinct regions involved in 1p deletion in human primary breast cancer. Cancer Res. 53, 1990–1994. Black, J.C., Allen, A., Van Rechem, C., Forbes, E., Longworth, M., Tscho¨p, K., Rinehart, C., Quiton, J., Walsh, R., Smallwood, A., et al. (2010). Conserved antagonism between JMJD2A/KDM4A and HP1g during cell cycle progression. Mol. Cell 40, 736–748. Braig, M., Lee, S., Loddenkemper, C., Rudolph, C., Peters, A.H., Schlegelberger, B., Stein, H., Do¨rken, B., Jenuwein, T., and Schmitt, C.A. (2005). Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436, 660–665. Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M., Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W., et al. (2005). Crucial role of p53dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436, 725–730. Cloos, P.A., Christensen, J., Agger, K., Maiolica, A., Rappsilber, J., Antal, T., Hansen, K.H., and Helin, K. (2006). The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature 442, 307–311. Colicelli, J. (2004). Human RAS superfamily proteins and related GTPases. Sci. STKE 2004, RE13. Collado, M., Gil, J., Efeyan, A., Guerra, C., Schuhmacher, A.J., Barradas, M., Bengurıá, A., Zaballos, A., Flores, J.M., Barbacid, M., et al. (2005). Tumour biology: senescence in premalignant tumours. Nature 436, 642. Courtois-Cox, S., Genther Williams, S.M., Reczek, E.E., Johnson, B.W., McGillicuddy, L.T., Johannessen, C.M., Hollstein, P.E., MacCollin, M., and Cichowski, K. (2006). A negative feedback signaling network underlies oncogene-induced senescence. Cancer Cell 10, 459–472.


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