Sin3b Expression Is Required for Cellular Senescence and Is Up-Regulated Upon Oncogenic Stress

Published OnlineFirst August 4, 2009; DOI: 10.1158/0008-5472.CAN-09-0537

Cell, Tumor, and Stem Cell Biology

Sin3B Expression Is Required for Cellular Senescence and Is Up-regulated upon Oncogenic Stress

Kathryn B. Grandinetti,1 Petar Jelinic,1 Teresa DiMauro,1 Jessica Pellegrino,1 Rube´n Ferna´ndez Rodrı´guez,1 Patricia M. Finnerty,1 Rachel Ruoff,1,2,3 Nabeel Bardeesy,4 Susan K. Logan,1,2,3 and Gregory David1,3

Departments of 1Pharmacology and 2Urology, and 3NYU Cancer Institute, NYU Langone Medical Center, NewYork, NewYork; and 4Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts

Abstract null primary fibroblasts are not immortal and remain susceptible to oncogene-induced senescence (9–11). Disruption of E2F-mediated Serial passage of primary mammalian cells or strong mitogenic transcription using a dominant-negative form of E2F1 prevents signals induce a permanent exit from the cell cycle called p19ARF-, p53-, or activated Ras–mediated senescence (12). Reinforc- senescence. A characteristic of senescent cells is the hetero- ing the importance of the E2F proteins and their target genes in chromatinization of loci encoding pro-proliferative genes, senescence is the established role for Rb and Rb-like proteins in leading to their transcriptional silencing. Senescence is thought silencing pro-proliferative E2F target genes (13, 14). Upon undergo- to represent a defense mechanism against uncontrolled pro- ing senescence, E2F target loci are embedded in nuclear foci, termed liferation and cancer. Consequently, genetic alterations that senescence-associated heterochromatic foci (SAHF; ref. 15), which allow senescence bypass are associated with susceptibility to are characterized by the presence of constitutive heterochromatin- oncogenic transformation. We show that fibroblasts genetically specific marks, including H3K9me3 and HP1 proteins, and by the inactivated for the chromatin-associated Sin3B protein are absence of acetylated histones. The enzymes responsible for the refractory to replicative and oncogene-induced senescence. chromatin modifications observed at E2F target loci during Conversely, overexpression of Sin3B triggers senescence and senescence are not completely understood. the formation of senescence-associated heterochromatic foci. Importantly, loss-of-function experiments have indicated that Although Sin3B is strongly up-regulated upon oncogenic stress, bypass of Ras-induced senescence correlates with oncogenic decrease in expression of Sin3B is associated with tumor transformation. For example, p53- and p19ARF-null fibroblasts are progression in vivo, suggesting that expression of Sin3B may transformed upon activated Ras overexpression (9, 16), leading to the represent a barrier against transformation. Together, these notion that immortalization correlates with susceptibility to cellular results underscore the contribution of senescence in tumor transformation on oncogenic activation. Consistently, activation of a suppression and suggest that expression of chromatin modi- senescence program has been evidenced in a variety of preneoplasic fiers is modulated at specific stages of cellular transformation. lesions in human, including melanocytic nevi and prostatic Consequently, these findings suggest that modulation of Sin3B- adenomas (3, 4), whereas further progression of the tumor requires associated activities may represent new therapeutic opportu- inactivation of the senescence pathway. nities for treatment of cancers. [Cancer Res 2009;69(16):6430–7] To further examine the interplay between chromatin modifications, senescence, and oncogenic transformation, we investigated Introduction the role of one potential effector protein, Sin3B. The Sin3 complex is Various cellular stresses have been shown to trigger senescence in a large multiprotein complex that is recruited by several sequence- cultured cells, including telomere attrition, accumulation of DNA specific transcription factors. It is characterized by the presence of damage, and increased expression of the products of the INK4a/ the Sin3A or the highly related Sin3B protein, which serve as scaffold ARF locus (1). However, the biological significance of cellular proteins between transcription factors and the repressor activities of senescence remains controversial. Recent reports have suggested the complex (17). Among these repressive activities, the histone that it may serve as a barrier against tumorigenesis in vivo (2–4), as deacetylase activity carried out by Sin3-associated HDAC1 and well as a driving force in organismal aging (5–7). In mouse cells, HDAC2 is essential for the transcriptional repression mediated by cellular senescence is driven primarily by p19ARF, which serves as a the Sin3 complex. Several lines of evidence suggest the involvement sensor for oncogenic signals to activate the p53 response (8). of the mammalian Sin3 complex in establishing the senescent Consistent with the notion that p19ARF, and not p16INK4a, mediates phenotype. First, a Sin3-containing complex has been shown to be replicative and oncogene-induced senescence in mouse cells, murine integral in the formation of pericentric heterochromatin (18), fibroblasts genetically inactivated for p19ARF failed to arrest on serial heterochromatic regions molecularly similar to SAHFs. Furthermore, INK4a genetic inactivation studies in the mouse showed that Sin3B, although passaging or forced expression of activated Ras, whereas p16 - dispensable for cellular growth, is necessary to mediate the onset of quiescence by transcriptional repression of E2F-responsive genes Note: Supplementary data for this article are available at Cancer Research Online (19, 20). Therefore, we investigated the ability of Sin3B-null (http://cancerres.aacrjournals.org/). fibroblasts to undergo replicative or oncogene-induced senescence. Current address for K.B. Grandinetti: The Scripps Research Institute, La Jolla, CA 92037. We showhere that Sin3B is required for heterochromatinization and Requests for reprints: Gregory David, Department of Pharmacology, MSB417, silencing of E2F target genes. Although primary cells devoid of Sin3B NYU Langone Medical Center, 550 First Avenue, NewYork, NY 10016. Phone: are resistant to senescence, they are not transformed upon 212-263-2926; Fax: 212-263-7133; E-mail: [email protected]. I2009 American Association for Cancer Research. expression of activated Ras, thus uncoupling immortalization from doi:10.1158/0008-5472.CAN-09-0537 susceptibility to oncogenic transformation. Importantly, expression

Cancer Res 2009; 69: (16). August 15, 2009 6430 www.aacrjournals.org

Sin3B-Induced Senescence and Tumor Suppression of Sin3B increases upon oncogenic stress, both in vitro and in vivo, correlating with the induction of the senescence program, but is strongly decreased as tumors progress, making it a potential therapeutic target in cancer.

Materials and Methods Cellular analysis. IMR-90 human fibroblasts were grown in DMEM plus 10% FCS and penicillin/streptomycin and used before reaching P20. Mouse embryonic fibroblasts (MEF) were generated from E13.5 embryos. Retroviral infection, growth curves, bromodeoxyuridine (BrdUrd) incorporation, senescence-associated h-galactosidase (SA-h-gal) staining, and protein analysis were conducted as described in refs. 16, 19, 21. P values were calculated using Student’s t test. Quantitative reverse transcription-PCR. Reverse transcription was done by using Moloney murine leukemia virus polymerase and oligo(dT) primers. Real-time PCR analyses were done using the SYBR Green method (primer sequences available on request). Results were reported as relative to the abundance of h2-microglobulin transcripts. Chromatin immunoprecipitation. Immunoprecipitations were done essentially as described by Boyd and colleagues (22) using Sin3B (Santa Cruz Biotechnology), H3 (Abcam), H3K9me3 (Abcam), HDAC1 (Millipore), and HP1g (Millipore) antibodies, followed by analysis of recovered chromatin using real-time PCR (primer sequences and detailed protocols available on request). Protein analysis. Paraformaldehyde-fixed cells were incubated with antibodies against H3K9me3 (Abcam), H3K4me3 (Abcam), and PML (Santa Cruz Biotechnology). Secondary antibodies were obtained from Molecular Probes. Nuclei were counterstained using either propidium iodide or 4¶,6- diamidino-2-phenylindole (DAPI) as indicated and analyzed on a Zeiss LSM 510 confocal microscope. For Western blot, antibodies used were Sin3B (Santa Cruz Biotechnology), Ras (Santa Cruz Biotechnology), Sin3A (Santa Cruz Biotechnology), p19 (Santa Cruz Biotechnology), Flag M2, and tubulin (Sigma). For immunohistochemistry, Sin3B (A-20, Santa Cruz Biotechnology) Figure 1. Sin3B is required for senescence in MEFs. A, growth curves using +/+ n À/À w and MCL-1 (S-19, Santa Cruz Biotechnology) antibodies were used. a 3T3 protocol on early-passage Sin3B ( ; n = 3) and Sin3B ( ; n =3) MEFs. Points, mean; bars, SD. B, quantification of SA-h-gal–positive cells in Sin3B+/+ (n =3)orSin3BÀ/À (n = 3) MEFs at passage 6. *, P < 0.05. C, representative morphology of Sin3B+/+ or Sin3BÀ/À MEFs at passage 7. Results Genetic inactivation of Sin3B prevents entry into replicative senescence. To establish immortalized cell lines, Sin3B+/+ and growth and that loss of Sin3B alone is sufficient to immortalize À À Sin3B / MEFs were generated and cultured using a standard 3T3 primary fibroblasts. Altogether, these results strongly argue that the protocol. As expected, Sin3B+/+ MEFs proliferated for 8 to 10 absence of Sin3B delays replicative senescence in the absence of passages before losing proliferative capacity, at which point, alteration of p53 status. À À immortalized cultures ultimately emerged. Surprisingly, Sin3B / We investigated the possibility that Sin3B might be essential not cells proliferated steadily for up to 26 passages (Fig. 1A), suggesting only for the establishment but also for the maintenance of a that Sin3B genetic inactivation promotes spontaneous immortali- senescent phenotype in mouse cells as previously reported for the Rb zation. In mouse cells, accumulation of genotoxic damage resulting protein (25). Primary MEFs harboring one floxed Sin3B allele and from oxidative stress is believed to account for replicative sen- either one wild-type Sin3B allele (Sin3BL/+) or one null Sin3B allele À escence (23). To assess the contribution of oxidative stress to the (Sin3BL/ ) were generated. These MEFs were serially propagated growth differences observed upon Sin3B inactivation, primary MEFs using a 3T3 protocol, and at passage 8, virtually 100% of the cells were h were cultured in low-oxygen conditions (3% O2). By contrast to what senescent as revealed by SA- -gal staining of a subset of the cells we observed in high-oxygen conditions, the growth rates of Sin3B+/+ (Supplementary Fig. S2A). Senescent cells were then infected with À À and Sin3B / primary MEFs were comparable over 18 passages, adenovirus expressing Cre recombinase, resulting in efficient and consistent with a specific function of Sin3B in replicative senescence rapid excision of the floxed allele (Supplementary Fig. S2C). As À À (Supplementary Fig. S1A). The percentage of Sin3B / MEFs that shown in Supplementary Fig. S2B, acute deletion of Sin3B was not were positive for SA-h-gal staining (24) was significantly lower than sufficient to restore cellular proliferation in senescent cells. Together, that of their wild-type counterparts at passage 6 (Fig. 1B). This these results showthat although Sin3B is required for the induction observation corroborates the noticeable differences in cell morphol- of senescence, it is, by contrast to Rb (25), dispensable for the À À ogy after six to eight passages between Sin3B / and Sin3B+/+ MEFs, maintenance of the senescent state. which appeared significantly more flat and enlarged, irrespective of Genetic inactivation of Sin3B prevents oncogene-induced the cell density (Fig. 1C). Of note, p53 levels were greatly increased senescence but is not sufficient for Ras-induced transformation. À À upon UV irradiation in both Sin3B+/+ and Sin3B / fibroblasts at Given the requirement for Sin3B during replicative senescence passage 6 (Supplementary Fig. S1B). This suggested that the p53 (Fig. 1), we investigated whether a Sin3B-dependent pathway is À À pathway remained intact in Sin3B / cells despite their immortal equally engaged in oncogene-induced senescence. 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Cancer Research

Figure 2. Sin3B-null primary MEFs are refractory to oncogene-induced senescence. A, representative Western blot of Sin3B+/+ or Sin3BÀ/À early-passage MEFs whole-cell extracts after infection with RasV12 (R) or vector (V) using the indicated antibodies. B, left, SA-h-gal assay on Sin3B+/+ (n =5)orSin3BÀ/À (n = 5) MEFs after RasV12 infection. Right, percentage of BrdUrd-positive cells 3 d after vector or RasV12 infection of early-passage Sin3B+/+ (n =2)orSin3BÀ/À (n = 2) MEFs. Columns, mean; bars, SE. *, P < 0.05. n.s., nonsignificant. C, representative Western blot of Sin3B+/+ or Sin3BÀ/À passage-2 MEFs after infection with RasV12 or vector using the indicated antibodies. D, left, number of colonies growing in soft agar after infection with RasV12 per 1,000 plated cells from the indicated source or genotype. Right, transformation assay of Sin3B+/+ and Sin3BÀ/À early-passage MEFs by SV40 large Tantigen and RasT24. Left, number of foci from two independent experiments using two cell lines per genotype. **, P < 0.01. Right, representative plates 10 d after replating. retrovirally transduced into early-passage primary MEFs, leading does not account for the changes in onset of senescence between to high-level expression of activated Ras as well as a reproducible Sin3B-wild-type and Sin3B-null cells upon induction of oncogene- up-regulation of endogenous Sin3B (Fig. 2A). As previously reported induced stress, we examined the levels of gH2AX after infection with (16), expression of activated Ras in wild-type MEFs induced activated Ras. As shown in Supplementary Fig. S3A, no difference in senescence, as evidenced by the strong increase in the proportion of the amount of gH2AX-positive cells was observed in Sin3B-wild-type cells staining positive for SA-h-Gal (Fig. 2B, left) and the significant or Sin3B-null cells following RasV12 infection. Along with the normal À À reduction in BrdUrd incorporation (Fig. 2B, right). By contrast, the p53 accumulation upon UV treatment in Sin3B / cells (Supple- proportion of senescent cells was significantly reduced and BrdUrd mentary Fig. S3B), these results suggest that Sin3B acts downstream incorporation was only marginally affected by RasV12 expression in of the DNA damage response pathway to mediate senescence. À À Sin3B / cells (Fig. 2B). Therefore, we investigated the possibility that It has been shown that genetic deletion of p53 or of the Ink4a locus RasV12-dependent activation of the p19ARF/p53 pathway is impaired renders cells both resistant to senescence and susceptible to trans- À À in Sin3B / fibroblasts. As previously reported, expression of p19ARF formation upon forced expression of activated Ras, enforcing the was induced upon transduction of activated Ras into wild-type hypothesis that bypassing senescence is a critical step in transfor- À À cells (Fig. 2C; ref. 26). Surprisingly, in Sin3B / cells overexpressing mation (30). However, by contrast to p53 or p19ARF, deletion of Sin3B activated Ras, the level of p19ARF protein was comparable with that was not sufficient to allow soft-agar growth upon activated Ras over- detected in Sin3B+/+ cells. However, the amount of p19ARF detected in expression (Fig. 2D, left). Yet, forced expression of SV40 large T À À À À the absence of oncogenic stress was significantly higher in Sin3B / antigen and activated Ras in Sin3B / primary MEFs led to the À À cells compared with Sin3B+/+ cells (Fig. 2C). This result is consistent formation of transformed foci, indicating that Sin3B / fibroblasts with the previous observation that p19ARF is an E2F target (27) and are not inherently refractory to cellular transformation (Fig. 2D, that E2F-driven repression is impaired in the absence of Sin3B (19). right). Interestingly, expression of large T antigen and activated Ras À À À À Furthermore, our observation that Sin3B / fibroblasts do not in Sin3B / cells resulted in a significantly higher number of trans- senesce upon RasV12 expression, in spite of high levels of p19ARF, formed colonies than that in Sin3B+/+ cells. The basis for the in- À À strongly suggests that Sin3B functions downstream of p19ARF in creased susceptibility of Sin3B / primary MEFs to transformation oncogene-induced senescence. These results showthat, although by large T antigen/Ras remains elusive, but it is possible that Sin3B participates in the transcriptional repression of p19ARF under disruption of the E2F repression pathway sensitizes primary normal culture conditions, it is required for the induction of fibroblasts to transformation. Altogether, these experiments strongly senescence upon activation of the p19ARF/p53 pathway. suggest that Sin3B is required for oncogene-induced senescence. Recent work has suggested that the DNA damage response However, in the absence of additional genetic lesions, its inactivation pathway may be a driver of oncogene-induced senescence (28, 29). To is not sufficient to promote cellular transformation upon oncogene confirm that differential activation of the DNA damage response activation.

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Sin3B-Induced Senescence and Tumor Suppression

Sin3B is recruited to the promoters of E2F target genes upon To showthe direct role of Sin3B in mediating E2F transcriptional oncogenic stress and its presence correlates with their hetero- control, we performed chromatin immunoprecipitation experiments. chromatinization and silencing. It has been previously observed We found that Sin3B is strongly and specifically enriched at E2F target that transcriptional repression of E2F targets correlates with the onset promoters upon RasV12 overexpression, but not at nonrelevant of either replicative or oncogene-induced senescence (12). Therefore, promoters such as oct4 (Supplementary Fig. S4A, left;Fig.3C). In we investigated whether inactivation of Sin3B impairs the transcrip- control experiments done in Sin3B-null cells, we observed no increase tional repression program triggered by oncogenic stress in primary in the amount of Sin3B-associated chromatin on senescence fibroblasts. We first observed that the amount of transcripts induction. We confirmed that expression of activated Ras induces corresponding to every E2F target gene we looked at was significantly recruitment of heterochromatin marks, including H3K9me3 and HP1g increased upon Sin3B deletion, irrespective of the presence of RasV12, at E2F target promoters (Supplementary Fig. S4A and B, right;Fig.3D; consistent with our previous observations (Fig. 3A and B,compare ref. 15). Importantly, we showed that this enrichment for heterochro- À À Sin3B / to Sin3B+/+; ref. 19). We then confirmed the down-regulation matin marks at E2F target promoters upon oncogenic stress requires of several E2F targets, including cycA2 and ccne1, in wild-type primary the presence of Sin3B (Supplementary Fig. S4; Fig. 3D). Of note, total fibroblasts upon RasV12 overexpression (Supplementary Fig. S4B, left; levels of H3K9me3 were not affected by the absence of Sin3B À À Fig. 3A). By contrast to their wild-type counterparts, Sin3B / MEFs (Supplementary Fig. S5). Consistent with the notion that HP1g is a did not exhibit transcriptional repression of any of these E2F target marker rather than an inducer of senescence (33), we observed an genes upon RasV12 overexpression (Supplementary Fig. S4B, left;Fig.3A increase in global levels of HP1g upon RasV12 overexpression only in and B). A second group of E2F target genes, including cdc2a and mcm5, the presence of Sin3B (Supplementary Fig. S5). Altogether, these were not affected by RasV12 overexpression in wild-type cells but were observations suggest that the recruitment of Sin3B and the subsequent À À significantly up-regulated in Sin3B / cells (Fig. 3B). This observation modifications to the promoters of E2F target genes are required for is in agreement with the recent demonstration of heterogeneous accurate transcriptional repression of these pro-proliferative genes transcriptional responses among E2F target genes (31) and correlates and the resultant onset of oncogene-induced senescence. with the finding that forced expression of oncogenes in primary Sin3B up-regulation is sufficient to induce senescence. We fibroblasts induces senescence through uncontrolled transition from initially observed an increase in the level of Sin3B protein upon Ras G1 to S phase (28, 32). Altogether, these experiments indicate that the induction (Fig. 2A). To document the specificity of this increase, the À À impairment of oncogene-induced senescence in Sin3B / MEFs levels of Sin3B and of the related Sin3A protein were investigated. As closely correlates with altered E2F target transcription. shown in Fig. 4A (left), whereas Sin3B protein levels increased 3- to

Figure 3. Sin3B directly regulates the chromatin modifications and transcription of E2F target genes in primary MEFs infected with RasV12. A and B, relative amount of transcripts as determined by quantitative RT-PCR corresponding to the indicated E2F target genes in early-passage Sin3B+/+ (n =2;black columns) or Sin3BÀ/À (n =2; white columns) MEFs infected with vector or RasV12. Shown is the average of two independent experiments. C, chromatin immunoprecipitations (ChIP) on promoters of the indicated genes with an anti-Sin3B antibody in early-passage Sin3B+/+ (n =2; black columns) or Sin3BÀ/À (n =2;white columns) MEFs infected with vector or RasV12. Shown is the average of two independent experiments. D, chromatin immunoprecipitation of the dihydrofolate reductase (dhfr) promoter with the indicated antibodies in early-passage Sin3B+/+ (n =2; black columns) or Sin3BÀ/À (n =2;white columns) MEFs infected with vector or RasV12. Shown is the average of two independent experiments. Bars, SE. *, P < 0.05.

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Figure 4. Sin3B levels are up-regulated in Ras-infected MEFs and Sin3B overexpression induces senescence in primary fibroblasts. A, left, Western blot of wild-type MEFs infected with RasV12 at P2 using the indicated antibodies; 40, 20, and 10 Ag of protein were used. Right, relative expression of Sin3B was determined using quantitative RT-PCR (n = 2 for each). B, Western blot of proteins overexpressed in wild-type MEFs using the indicated antibodies. *, nonspecific band. C, percentage of SA-h-Gal–positive MEFs 7 d after infection with the indicated retroviruses (>200 cells per point in duplicate). Columns, mean; bars, SE. *, P < 0.05.

4-fold upon expression of activated Ras, the levels of Sin3A remained also noted that the well-characterized subnuclear structures called unchanged, emphasizing the specificity of Sin3B in the cellular re- PML bodies were not disrupted on Sin3B overexpression (Supple- sponse to oncogenic stress. As shown by quantitative reverse mentary Fig. S6B), suggesting that the effect of Sin3B on the transcription-PCR (RT-PCR) analysis, the increase in Sin3B levels formation of SAHF is specific and does not result from an overall upon activated Ras expression occurs at the transcriptional level alteration of nuclear organization. The action of Sin3B downstream (Fig. 4A, right). of Ras in the establishment of senescence was further substantiated Having established that Sin3B is required for cellular senescence by the observation that overexpression of RasV12, but not of Sin3B, and that Sin3B levels are up-regulated upon oncogenic stress, we in- leads to relocalization of the propidium iodide staining within the vestigated whether Sin3B overexpression in primary cells was suf- nucleolus (Supplementary Fig. S6B;Fig.5B). Overall, these results ficient to engage the senescence program. Early-passage wild-type showthat, upon overexpression, Sin3B is able to coordinate the MEFs were infected with retroviruses encoding either full-length histone modifications characteristic of heterochromatin and con- Sin3A or full-length Sin3B (Fig. 4B). Although Sin3B overexpression tributes to the formation of SAHF. was sufficient to significantly increase the proportion of cells positive Sin3B levels are modulated during oncogenic progression for SA-h-gal compared with the control, overexpression of Sin3A had in vivo. Previous reports have shown that the bypass of senescence no effect (Fig. 4C). This result indicated that overexpression of Sin3B, plays an important role in oncogenic progression in animal models but not Sin3A, is sufficient to engage a senescence program. (2, 34) and that reactivation of the senescence program may be Because the abundance of pericentric heterochromatin present in sufficient to lead to tumor clearance (35). Although we have observed mouse cells makes visualization of SAHF structures difficult, we like- that Sin3B expression is induced upon oncogenic stress in vitro wise infected human primary diploid fibroblasts (IMR90) with retro- (Fig. 4A), we sought to investigate the relevance of this result as it viruses encoding full-length mouse Sin3B or activated Ras. As expected, relates to tumor progression in vivo. We used a well-characterized overexpression of activated Ras in IMR90 triggered the onset of mouse model that exhibits multistage pancreatic ductal adenocar- senescence as detected by SA-h-gal staining (Fig. 5A). By contrast, <20% cinoma progression. In this model, pancreas-specific expression of of same-passage IMR90 cells infected with the corresponding empty activated K-Ras, driven by its own promoter, leads to the generation retroviral vector were positive for SA-h-gal staining (Fig. 5A). Strik- of local premalignant lesions, namely, metaplastic acini, which have ingly, upon Sin3B overexpression, f40% of the cells were positive for been shown to stain positive for senescence markers (3). These SA-h-gal staining, a significant increase in the amount of senescent metaplastic acinar structures are believed to be precursors for cells compared with empty vector–infected cells (Fig. 5A). pancreatic intraepithelial neoplasias (PanIN lesions), which subse- Consistent with the ability of Sin3B to induce SA-h-gal positivity in quently progress to pancreatic ductal adenocarcinoma in associa- primary human fibroblasts, Sin3B-overexpressing IMR90 displayed tion with homozygous loss of Ink4a/Arf or p53 (36–38). We nuclear foci that were brightly stained for H3K9me3 and DAPI, investigated here the levels of expression of Sin3B in K-Ras– comparable with those observed on overexpression of activated Ras activated, p53-heterozygous mouse pancreas at different stages of (Supplementary Fig. S6A;Fig.5B). Furthermore, these DNA dense tumor progression. Sin3B expression is strongly up-regulated in foci excluded histone marks that correspond to transcriptionally metaplastic acini and to a lesser extent in PanINs compared with active chromatin, such as H3K4me3 (Supplementary Fig. S6B). We adjacent normal pancreas or pancreas from wild-type animals

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Sin3B-Induced Senescence and Tumor Suppression

Figure 5. Sin3B overexpression induces senescence in primary human fibroblasts. A, percentage of SA-h-Gal–positive, early-passage IMR90 cells 5 d after infection with the indicated retroviruses and puromycin selection (>200 cells per point). Columns, average of two independent experiments done in duplicate; bars, SE. Differences between pBABE and RasV12 and between pBABE and Sin3B are significant (P < 0.05). B, immunofluorescence of IMR90 cells 5 d after infection with the indicated constructs and puromycin selection using antibody to H3K9me3 (green) and counterstained with propidium iodide (PI; red).

(compare b and c to a in Fig. 6A). Quantification of the Sin3B signal showing the presence of markers of senescence in preneoplastic in sections from at least eight independent animals per type of lesion lesions in a Ras-induced mouse model of pancreatic cancer (3), we revealed a significant up-regulation of Sin3B in preneoplastic lesions also observed positivity for markers of senescence in metaplastic compared with normal pancreas (Fig. 6B). This result is consistent lesions (Fig. 6C). Importantly, in pancreatic ductal adenocarcinoma, with our in vitro observations that Sin3B expression is up-regulated Sin3B expression decreases dramatically and is comparable with the upon oncogenic stress (Fig. 4A). Consistent with a previous report levels detected in normal pancreas (Fig. 6Aa–d). Altogether, these

Figure 6. Expression of Sin3B throughout tumorigenesis. A, a and a¶, Sin3B immunostaining on wild-type mouse pancreas. a¶, the inset shown in a (dashed lines). b and b¶, Sin3B immunostaining on mouse pancreas (Pdx-Cre+; Lox-Stop-Lox-K-Ras; p53L/+) with acinar-ductal metaplasia. Note the strong nuclear staining in metaplasia. b¶, the inset shown in b (dashed lines). c and c¶, Sin3B immunostaining on mouse pancreas (Pdx-Cre+; LSL-K-Ras; p53L/+) with acinar-ductal metaplasia (white arrow) and PanIN lesions (black arrow). d ¶, the inset shown in c (dashed lines). d and d¶, Sin3B immunostaining on mouse pancreas (Pdx-Cre+; LSL-K-Ras; p53L/+) with ductal adenocarcinoma. d ¶, the inset shown in d (dashed lines). Pancreas sections from at least eight independent animals were observed for each type of lesion. Shown are representative sections of more than eight independent slides analyzed. B, quantification of the Sin3B signal in normal pancreas (N), metaplastic lesions (M), or pancreatic adenocarcinoma (A). At least eight samples were scored for each type of lesion, and quantification ranges from 0 (no signal) to 4 (strong signal). Columns, mean; bars, SD. C, MCL-1 immunostaining on metaplastic lesions (left) or adenocarcinoma (right) in the same mouse model. Shown are representative sections from multiple independent animals. www.aacrjournals.org 6435 Cancer Res 2009; 69: (16). August 15, 2009

Cancer Research observations suggest that Sin3B is up-regulated in early premalig- Interestingly, genetic ablation of Sin3B uncouples immortalization nant lesions but becomes strongly down-regulated on full oncogenic and sensitivity to oncogenic transformation. Indeed, Sin3B-null transformation. Consistent with the results obtained in the mouse fibroblasts are immortal and do not exit the cell cycle on oncogenic model, the Oncomine online database revealed that Sin3B levels are stress, but expression of activated Ras is not sufficient to lead to a fully À significantly (P <10 5) reduced in multiple cancer types compared transformed state. This is reminiscent of what has been observed on with the corresponding normal tissues (Supplementary Fig. S7). simultaneous genetic ablation of Rb and p107 (40). Loss of Sin3B Along with the results of this study, the demonstration that chromatin expression sensitizes to oncogenic transformation by large T antigen modifications associated with cellular senescence represent a barrier and Ras, and decrease in Sin3B expression is observed in several against tumorigenesis in vivo (2) strongly suggests that the modulation tumors in vivo, suggesting that Sin3B may act as a nonclassic tumor of Sin3B-associated activities acts as a nonclassic tumor suppression suppressor. One hypothesis is that, although Sin3B acts downstream mechanism and could serve as a potential therapeutic approach in of p19ARF to signal senescence, disruption of the senescence pathway preventing oncogenic transformation. is not sufficient to lead to transformation but allows the accumulation of deleterious mutations leading to the oncogenic phenotype. This is Discussion consistent with our observation that Sin3B-null cells infected with RasV12 do not exit the cell cycle, allowing them to accumulate In this study, we investigated the ability of Sin3B-null fibroblasts to additional mutations (Fig. 2B, right). Importantly, loss of Suv39h1/2, a undergo replicative and oncogene-induced senescence. We showed family of chromatin modifiers that contribute to the formation of that Sin3B is present at promoters undergoing heterochromatiniza- SAHF, was recently shown to accelerate tumorigenesis in a mouse tion and is required for the induction of senescence. Notably, model of B-cell lymphoma (2). In this regard, it will be informative to although primary cells devoid of Sin3B were resistant to senescence, investigate the contribution of Sin3B to the prevention of cancer they were not transformed upon expression of activated Ras, thus in vivo. Of note, multiple clinical trials are aimed at investigating the uncoupling immortalization from susceptibility to oncogenic efficacy of inhibitors of histone deacetylases as therapeutic agents in transformation. Finally, we found that expression of Sin3B increased human cancer (41). Based on our findings, inhibiting Sin3B- on oncogenic stress, consistent with the induction of the senescence associated HDAC activity may promote transformation, rather than program, but strongly decreased as tumors progress, suggesting that prevent it. Furthermore, modulation of Sin3B expression via it may represent a barrier to oncogenic transformation. stimulation of Sin3B transcriptional activity may provide a potential Our results suggest that Sin3B coordinates the recruitment of therapeutic approach to restore the senescence program in tumors. chromatin modifiers to the promoters of E2F target genes, leading to Our results suggest that understanding the precise contribution of their heterochromatinization. Although the identity of these mod- discrete chromatin modifiers in oncogenesis may lead to a better ifiers is still under investigation, we hypothesize that the tethering of targeted therapeutic approach modulating the function of specific Sin3B leads to the coordinated removal of active chromatin marks chromatin modifiers at precise loci. and the addition of inactive marks. Supporting this hypothesis, it has recently been shown that Sin3B physically interacts with the H3K4 demethylase RBP2 (39). It is tempting to speculate that at the onset Disclosure of Potential Conflicts of Interest of senescence, Sin3B is recruited to E2F target promoters through No potential conflicts of interest were disclosed. the Rb-related pocket proteins, tethering RBP2, leading to the removal of the H3K4me3 active mark. Similarly, it is likely that, as we Acknowledgments have previously observed for mSds3 at pericentric loci (18), Sin3B is Received 2/12/09; revised 5/22/09; accepted 6/9/09; published OnlineFirst 8/4/09. able to coordinate the recruitment of a H3K9 histone methyltrans- Grant Support: Rett Syndrome Research Foundation (G. David), March of Dimes ferase and HP1g at E2F promoters. One alternative explanation is (G. David), the American Federation for Aging Research (G. David), American Cancer Society (G. David and K.B. Grandinetti), NIH grant CA112226 (S.K. Logan), and NIH that Sin3B recruitment at E2F target promoters upon oncogenic training grant CA009161 (K.B. Grandinetti). stress represents the first step in the silencing of these genes, as The costs of publication of this article were defrayed in part by the payment of page previously shown at the induction of quiescence (19). This initial step charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. would then be necessary for the subsequent recruitment of specific We thank Lawrence Gardner, Eva Hernando, and Iman Osman for helpful discussions, chromatin modifiers, leading to the permanent silencing of these loci. and Chris Van Oevelen and Brian Dynlacht for their help with E2F targets analyses.

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www.aacrjournals.org 6437 Cancer Res 2009; 69: (16). August 15, 2009

Sin3B Expression Is Required for Cellular Senescence and Is Up-regulated upon Oncogenic Stress

Kathryn B. Grandinetti, Petar Jelinic, Teresa DiMauro, et al.

Cancer Res 2009;69:6430-6437. Published OnlineFirst August 4, 2009.

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