PAX3-FOXO1 Controls Expression of the P57kip2 Cell-Cycle Regulator Through Degradation of EGR1

PAX3-FOXO1 controls expression of the p57Kip2 cell-cycle regulator through degradation of EGR1

Wendy Roeb*†, Antonia Boyer*, Webster K. Cavenee*†‡§¶, and Karen C. Arden*‡ʈ

*Ludwig Institute for Cancer Research, †Biomedical Sciences Graduate Program, ‡Department of Medicine, and §Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660

Contributed by Webster K. Cavenee, September 24, 2007 (sent for review August 8, 2007) The chimeric protein PAX3-FOXO1, resulting from a translocation p57KIP2, located at 11p15, is a paternally imprinted gene whose between chromosomes 2 and 13, is the most common genetic decreased expression is a feature common to a variety of human aberration in the alveolar subtype of the human skeletal muscle tumors (12–17). There are several mechanisms through which tumor, rhabdomyosarcoma. To understand how PAX3-FOXO1 con- this occurs, commonly involving changes in genomic imprinting tributes to tumor development, we isolated and characterized or loss of the active maternal allele (18–20). It is interesting to muscle cells from transgenic mice expressing PAX3-FOXO1 under note that the embryonal subtype of rhabdomyosarcoma is char- control of the PAX3 promoter. We demonstrate that these myo- acterized by loss of the maternal 11p15 chromosomal region blasts are unable to complete myogenic differentiation because of (21). Loss of p57KIP2 function is also implicated in Beckwith– an inability to up-regulate p57Kip2 transcription. This defect is Wiedemann syndrome, a complex overgrowth condition associated caused by reduced levels of the EGR1 transcriptional activator with an increased risk for developing rhabdomyosarcomas (22). resulting from a direct, destabilizing interaction with PAX3-FOXO1. Whereas some potential PAX3-FOXO1 downstream targets Neither PAX3 nor FOXO1 share the ability to regulate p57Kip2 have been identified, the mechanism by which PAX3-FOXO1 transcription. Thus, the breakage and fusion of the genes encoding contributes to tumor pathogenesis is unknown. The FOXO1 these transcription factors creates a unique chimeric protein that transactivation domain has been shown to be more robust than controls a key cell-cycle and -differentiation regulator. that of PAX3, enabling PAX3-FOXO1 to more strongly activate PAX3 consensus sequence reporter constructs (23–25). These Fkhr ͉ Foxo ͉ p57 ͉ Pax3 ͉ rhabdomyosarcoma reports have led to the hypothesis that PAX3-FOXO1 drives oncogenesis by overactivating PAX3 transcriptional targets (24, habdomyosarcoma constitutes a group of soft tissue sarco- 26–29). A recent study found that PAX3-FOXO1 bypasses Rmas of childhood and adolescence that are thought to arise cellular senescence by reducing p16INK4A levels (30), whereas from undifferentiated mesenchyme resembling various stages of others have suggested that expression of PAX3-FOXO1 results early embryonic skeletal muscle development. The most aggres- in aberrant regulation of genes involved in myogenic differen- sive pediatric subtype, alveolar rhabdomyosarcoma (ARMS), is tiation (reviewed in ref. 31). The present studies were under- composed of dense aggregates of poorly differentiated cells taken to identify PAX3-FOXO1 target genes in transgenic separated by a framework of fibrous septa forming ‘‘alveolar’’ myoblasts, to test whether these affect myogenic differentiation, spaces. Typical features of ARMS include: physical location of and to determine how PAX3-FOXO1 expression leads to their the tumor, alveolar appearance, the presence of the character- misregulation. istic translocation between chromosomes 2 and 13, and immu- Results nohistochemical reactivity for the myogenic markers desmin, MYOD1, and myosin heavy chain (1). This latter feature might PAX3-FOXO1 Expression in Myoblasts Inhibits Differentiation and suggest that ARMS arises as a consequence of incomplete Decreases p57Kip2. To study the role of PAX3-FOXO1 on myo- myogenic differentiation and abnormal proliferation coupled to genic differentiation, we isolated myoblasts from mice harboring transforming mutations. the PAX3-FOXO1 fusion gene under the control of the PAX3 The PAX3-FOXO1 fusion protein, created by the t(2;13) promoter (32). PAX3-FOXO1 transgenic myoblasts are pheno- chromosomal translocation, is present in most cases of alveolar typically indistinguishable from their wild-type counterparts and rhabdomyosarcoma (2). The translocation severs the transcrip- express normal levels of the myoblast marker, desmin (33). tional transactivation domain of PAX3 but preserves both of its Rhabdomyosarcomas are characterized by deficiencies in two DNA-binding domains. The FOXO1 gene is disrupted in a myogenic differentiation and an inability to exit the cell cycle large intron that bisects its DNA-binding domain. The chromo- (34). Cells derived from these tumors express MYOD1 and somal rearrangement creates a chimeric protein containing the myogenin but do not differentiate into myotubes (34). To transcriptional activation domain of FOXO1 and the DNA- determine if myoblasts derived from PAX3-FOXO1 transgenic binding elements of PAX3, under the control of the PAX3 animals have similar defects, wild-type and transgenic myoblasts promoter (3). were cultured in media that induce myogenic differentiation. As PAX3 is an essential myogenic regulator. Mice lacking Pax3 have multiple skeletal muscle defects, most notably delays in Author contributions: W.R., W.K.C., and K.C.A. designed research; W.R. and A.B. performed muscle differentiation as well as an overall decrease in muscle research; W.R. contributed new reagents/analytic tools; W.R., A.B., W.K.C., and K.C.A. mass (4). In myogenesis, PAX3 functions to induce the expres- analyzed data; and W.R., W.K.C., and K.C.A. wrote the paper. sion of SIX1 and EYA2 (5), transcription factors that up-regulate The authors declare no conﬂict of interest. expression of MYF5, advancing the myogenic differentiation Abbreviation: ARMS, alveolar rhabdomyosarcoma. program. FOXO1 also plays a pivotal role in mediating myogenic ¶To whom correspondence may be addressed. E-mail: [email protected]. differentiation (6, 7). ʈTo whom correspondence may be addressed at: Ludwig Institute for Cancer Research, In myoblasts, terminal differentiation and proliferation are University of California San Diego, 9500 Gilman Drive, CMM-E, Room 3080, La Jolla, mutually exclusive processes. p57KIP2 promotes differentiation CA 92093-0660. E-mail: [email protected]. by stabilizing MYOD1, inhibiting cyclin E-CDK2 activity and This article contains supporting information online at www.pnas.org/cgi/content/full/

proliferating cell nuclear antigen (PCNA) function while main- 0708910104/DC1. CELL BIOLOGY taining RB1 in an active hypophosphorylated state (8–11). © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708910104 PNAS ͉ November 13, 2007 ͉ vol. 104 ͉ no. 46 ͉ 18085–18090 Downloaded by guest on October 1, 2021 Fig. 2. Restoration of p57Kip2 levels in PAX3-FOXO1 transgenic myoblasts promotes their differentiation. (A)(Upper) Proliferating (prolif.) myoblasts, seeded at constant density. (Lower) Myoblasts plated at a constant density and maintained in differentiation (diff.) medium for 24 h. Number of myotubes per high-power ﬁeld is given below the representative photograph (320ϫ). A minimum of four ﬁelds were counted. (B) Immunoblot analysis of myosin heavy chain (MHC) in control and p57Kip2-transduced transgenic myoblasts maintained in either proliferation or differentiation medium. (C) Quantitative PCR analysis of p57Kip2 mRNA from proliferating control and p57Kip2 transduced transgenic myoblasts.

Fig. 1. PAX3-FOXO1 transgenic myoblasts contain low levels of p57KIP2 and are unable to differentiate. (A)(Upper) Proliferating (prolif.) myoblasts, myoblasts were infected with either empty vector or p57Kip2 seeded at constant density. (Lower) Myoblasts plated at a constant density and retroviruses. p57Kip2 transcript levels were increased to slightly maintained in differentiation (diff.) medium for 24 h. Number of myotubes less than the levels seen in wild-type myoblasts, Ϸ4-fold. As per high-power ﬁeld (320ϫ) is given below the representative photograph. A shown in Fig. 2, restoration of p57Kip2 expression profoundly minimum of four ﬁelds were counted. (B) Immunoblot analysis of the myo- improves the ability of PAX3-FOXO1 transgenic myoblasts to genic markers myosin heavy chain (MHC) and desmin in wild-type and trans- ϭ genic myoblasts maintained in either proliferation or differentiation medium. differentiate (P 0.0008). This result demonstrates that (C) Quantitative PCR analysis of p57Kip2 mRNA from early passage myoblasts. p57KIP2 is a major effector of PAX3-FOXO1 in inhibiting P values of the differences between wild-type and transgenic (tg.) myoblasts myogenic differentiation. were calculated by using two-tailed Student’s t test. Error bars represent SDs. (D) Immunoblot analysis of p57KIP2 protein levels in wild-type or transgenic The p57Kip2 Promoter Is Responsive to PAX3-FOXO1. To determine myoblasts maintained in either proliferation or differentiation medium. (E) whether the p57Kip2 promoter is PAX3-FOXO1 responsive and Quantitative PCR analysis of p57Kip2 mRNA accumulation during differenti- which of its elements are responsible for the transcriptional ation. Values shown are the mean fold up-regulation, Ϯ SD. repression, a series of deletions of the full-length mouse promoter linked to a luciferase transcriptional reporter were con- shown in Fig. 1 A and B, PAX3-FOXO1 transgenic myoblasts structed (Fig. 3A). Sequence analysis showed that there are two differentiate poorly, they fail to form myotubes, and they do not putative FOXO1-binding sites and one putative PAX3 binding up-regulate expression of the terminal differentiation marker, site in the full-length promoter, at positions Ϫ2,130, Ϫ2,650, and myosin heavy chain. Ϫ2,900, and all were contained in the PAX3-FOXO1-responsive The inability of transgenic myoblasts to differentiate led us to full-length p57Kip2 promoter construct. However, the deletion hypothesize that the PAX3-FOXO1 fusion gene might play a role of a segment of the p57Kip2 promoter containing all of these sites in suppressing differentiation and preventing cell-cycle exit. To (Ϫ3000 to Ϫ1800 from the transcriptional start site) did not address the nature of the differentiation defect, expression cause the p57Kip2 promoter to become unresponsive to PAX3- profiling with Genechip microarrays was performed on two sets FOXO1 inhibition. On the contrary, PAX3-FOXO1-responsive each of passage-matched primary myoblasts from transgenic and sites were scattered throughout the promoter, with the magni- wild-type animals. For both data sets, among many genes with tude of the repression diminishing with decreasing promoter altered expression, the most profound effect was decreased length. To further define the minimal sequence required for expression of the CDK inhibitor, p57Kip2. This result was PAX3-FOXO1-dependent repression, additional deletion con- validated by using quantitative PCR on independent primary structs were created. As shown in Fig. 3B, deletion of 100 myoblasts from PAX3-FOXO1 transgenic animals, as shown in nucleotides from Ϫ400 to Ϫ300, with respect to the transcrip- Fig. 1C. Wild-type myoblasts showed an Ϸ150-fold increase of tional start site of the minimal p57Kip2 promoter, renders it p57Kip2 expression upon differentiation induction, whereas the PAX3-FOXO1 insensitive. These 100 nucleotides of sequence transgenic myoblasts had Ͻ15% of that response (Fig. 1E), and are also sufficient to mediate repression by PAX3-FOXO1 when this corresponded to the levels of p57KIP2 protein in the cells placed upstream of a synthetic minimal promoter (Fig. 3B). (Fig. 1D). These results suggest that PAX3-FOXO1 might Moreover, this ability is specific to the PAX3-FOXO1 fusion influence the balance between myogenic differentiation and protein and is not evidenced by wild-type PAX3 or FOXO1 proliferation by decreasing the levels of the proliferation inhib- alone (Fig. 3C). These results indicate that PAX3-FOXO1 itor, p57KIP2. represses p57Kip2 transcription through sequences in the Ϫ400 To confirm that diminished quantities of p57KIP2 are suffi- to Ϫ300 region of the promoter that are distinct from PAX3 or cient to inhibit differentiation, we restored p57Kip2 expression in FOXO1 binding sequences and that are unresponsive to PAX3 PAX3-FOXO1 transgenic myoblasts. Early passage transgenic or FOXO1.

18086 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708910104 Roeb et al. Downloaded by guest on October 1, 2021 Fig. 4. PAX3-FOXO1 interferes with Egr1-dependent transcription. (A) Lu- ciferase reporter assays of EGR1, SP1, and SP3 on the full-length p57Kip2 promoter. Values are expressed as fold activation relative to promoter cotransfected with empty vector. (B) EMSA of in vitro transcribed/translated EGR1 on Ϫ400 to Ϫ350 bp p57Kip2 promoter. Competing DNA was either wild-type sequence from Ϫ400 to Ϫ350 bp of the p57Kip2 promoter (Left), or mutated by disruption of EGR1-binding sites (Right). (C) Luciferase reporter assay of the full-length p57Kip2 promoter cotransfected with an increasing quantity of EGR1 (E), with or without PAX3-FOXO1. (D) Luciferase reporter assay of an EGR1 synthetic reporter cotransfected with an increasing quantity of EGR1, with or without PAX3-FOXO1. For all luciferase reporter assays, results are the mean values Ϯ SD of triplicates. Results are representative of at Fig. 3. PAX3-FOXO1 represses p57Kip2 transcription through multiple GC- least three independent experiments. rich sequence elements. (A) Luciferase reporter assays of sequential deletions of the p57Kip2 promoter, cotransfected with increasing quantities of PAX3- FOXO1. The relative positions of the PAX3 (P)- and FOXO1 (F)-binding sites are To establish whether EGR1 acts directly on the p57Kip2 indicated on the schematic. Values are expressed as mean ﬁreﬂy luciferase promoter, EMSA was performed. We used in vitro-translated activity normalized to control Renilla luciferase activity. (B) Luciferase reporter EGR1 and a radiolabeled oligonucleotide representing Ϫ400 to assays of p57Kip2 sequential deletions, cotransfected with increasing quan- Ϫ tities of PAX3-FOXO1. The Ϫ400 to Ϫ300 bp p57Kip2 promoter sequence is 350 bp of the p57Kip2 promoter sequence, because this se- upstream of a minimal tk promoter. Values are expressed as mean fold quence contained the majority of the EGR1-binding sites in this activation relative to promoter cotransfected with empty vector. (C) Lucif- region. Whereas EGR1 could clearly shift the mobility of the erase reporter activity of PAX3, FOXO1, and PAX3-FOXO1 on the p57Kip2 oligonucleotide, disruption of the two EGR1-binding sites in the promoter, Ϫ400 to ϩ30 bp, normalized to background activity on Ϫ300 to ϩ30 unlabeled, competing DNA abrogates its ability to efficiently bp. Values are expressed as mean fold activation relative to promoter cotrans- compete for EGR1 binding (Fig. 4B). Thus, EGR1 can specif- Ϯ fected with empty vector. Results of all experiments are the mean values SD ically bind to and activate p57Kip2 transcription through the of triplicates and representative of at least three independent experiments. minimal promoter sequence important for PAX3-FOXO1 repression. PAX3-FOXO1 Repression of the p57Kip2 Promoter Is Mediated by To determine whether PAX3-FOXO1 could interfere with EGR1-Binding Sequences. The minimum sequence of the p57Kip2 activation of the p57Kip2 promoter by EGR1, luciferase reporter promoter required for PAX3-FOXO1 repression, Ϫ400 to Ϫ300 assays were performed by using the full-length p57Kip2 promoter bp, is rich in GC nucleotides, unlike the PAX3- or FOXO1- in the presence of increasing quantities of Egr1. PAX3-FOXO1 Ϸ binding sites that were the anticipated PAX3-FOXO1 targets. represses EGR1 activation of the p57Kip2 promoter 4-fold We performed chromatin immunoprecipitation several times by (Fig. 4C). Because PAX3-FOXO1 has the ability to interfere using endogenous PAX3-FOXO1 from transgenic myoblasts but with EGR1-dependent activation of the p57Kip2 promoter, we were unable to amplify p57Kip2 promoter sequences (data not tested whether it was able to more generally suppress EGR1- shown). When combined with the absence of a PAX3-binding mediated transcription. Luciferase assays were performed with site in this region, these data suggest that PAX3-FOXO1 con- a synthetic reporter containing three tandem copies of the trols p57Kip2 expression indirectly. An alternative mechanism by EGR1 consensus sequence. Consistent with the effects observed which PAX3-FOXO1 might repress p57Kip2 transcription with- for the p57Kip2 promoter, PAX3-FOXO1 was able to suppress out directly binding to the p57Kip2 promoter could be by EGR1 activity by Ϸ4-fold (Fig. 4D), suggesting that this is one interfering with an activator of p57Kip2 transcription. Because of its general activities. the region of the p57Kip2 promoter from Ϫ400 to Ϫ300 bp is extremely GC-rich, we assessed three of the most-common GC PAX3-FOXO1 Destabilizes EGR1. In the course of performing these box-binding transcription factors, EGR1, SP1, and SP3, for their experiments, we observed that cotransfection of PAX3-FOXO1 ability to activate p57Kip2 transcription. As shown in Fig. 4A, and Egr1 resulted in significantly reduced levels of EGR1. One only EGR1 was able to activate p57Kip2 transcription in a mechanism by which PAX3-FOXO1 might interfere with dose-dependent fashion. This result is consistent with microarray EGR1-dependent transcription is by destabilizing EGR1. Co- experiments that show that p57Kip2 is up-regulated by EGR1 transfection of increasing quantities of PAX3-FOXO1-HA in the CELL BIOLOGY (35, 36). presence of a fixed amount of Egr1-V5 showed that PAX3-

Roeb et al. PNAS ͉ November 13, 2007 ͉ vol. 104 ͉ no. 46 ͉ 18087 Downloaded by guest on October 1, 2021 much of its repressive activity. Additional deletions at either the N or the C terminus dramatically reduce most of the PAX3- FOXO1 transcriptional repression activity. This result suggests that the ability of PAX3-FOXO1 to suppress p57Kip2 transcription is not due to one of its particular domains and implies that the overall protein conformation might be responsible for suppression of p57Kip2 transcription. To establish whether the destabilization of EGR1 by PAX3- FOXO1 involves their direct interaction, we performed coim- munoprecipitations of transfected epitope-tagged proteins (Fig. 5D). In the absence of proteasome inhibition, immunoprecipitation of PAX3-FOXO1-HA yields a faint smear when blots are probed for EGR1-V5. In the presence of MG132, EGR1-V5 coimmunoprecipitates with PAX3-FOXO1-HA, demonstrating that PAX3-FOXO1 and EGR1 directly associate in an unstable complex. We also performed the reciprocal experiment by immunoprecipitating EGR1-V5, resulting in a specific signal for PAX3-FOXO1 (data not shown). To demonstrate that the interaction between EGR1 and PAX3-FOXO1 was not simply a result of overexpression, we performed coimmunoprecipitation experiments on the endogenous proteins normally expressed by the ARMS cell line, Rh28, which harbors the t(2;13) translocation (38). Treatment of these cells with MG132 increases the levels of EGR1 protein (data not shown), and PAX3-FOXO1 and EGR1 form a complex in ARMS cells (Fig. 5E). Lastly, we tested whether the observed decreases in p57Kip2 mRNA were correlated with reduced EGR1 protein levels in our Fig. 5. PAX3-FOXO1 interacts with and destabilizes EGR1. (A) Immunoblot transgenic myoblasts. As shown in Fig. 5F, immunoblot analysis analysis of EGR1 V5 protein in the presence of increasing quantities of PAX3- FOXO1 HA, with or without the proteasome inhibitor MG132. (B) Luciferase revealed that PAX3-FOXO1 significantly reduces the levels of reporter assay of the full-length p57Kip2 promoter, cotransfected with PAX3- EGR1 protein in myoblasts. FOXO1, in the presence of MG132. (C) Deletion mutants of PAX3-FOXO1, included residues given at left (A.A.), with their activity by luciferase reporter Discussion assay, as a mean percentage of wild type, shown at right, Ϯ SD. The relative Here, we show that the accumulation of p57Kip2 mRNA nor- positions of the paired domain (P), homeodomain (H), and partial forkhead mally induced by differentiation signals is suppressed by PAX3- domain (F) are indicated on the schematic. (D) Coimmunoprecipitation anal- FOXO1, thus rendering primary PAX3-FOXO1 myoblasts re- ysis of epitope-tagged proteins, with or without MG132. (E) Coimmunopre- fractory to such stimuli. Taken together with other findings (21, cipitation of endogenous EGR1 and PAX3-FOXO1 from the cell line Rh28, in 39, 40), our results suggest that loss of functional p57KIP2 is a the presence of MG132. (F) Immunoblot analysis of Egr1 in wild-type (W.T.) and PAX3-FOXO1 transgenic (Tg.) myoblasts maintained in proliferation common feature of both subtypes of rhabdomyosarcoma. medium. P57Kip2-null mice display a variety of developmental defects resulting from an inability of cells to differentiate, but they do not exhibit an increased susceptibility to tumors (41). Analysis of FOXO1-HA reduced EGR1-V5 protein levels in a dose- the four PAX3-FOXO1 mouse models described to date (32, dependent manner (Fig. 5A). Inhibition of the proteasome by the 42–44) also suggests that PAX3-FOXO1 expression alone is addition of MG132 abolished this effect, suggesting that the insufficient to produce a malignant phenotype. However, dis- observed reduction of EGR1 protein is due to proteasomal ruption of the Ink4a/ARF or Tp53 pathways, targets of inacti- degradation (Fig. 5A). Consistent with this, levels of Egr-V5 vation in human rhabdomyosarcoma, in PAX3-FOXO1 mice transcript were unaffected by PAX3-FOXO1 (data not shown). substantially increases the frequency of tumor formation (42). To further demonstrate that destabilization of EGR1 is specific These mouse models and our present data suggest that loss of for PAX3-FOXO1, we also performed this assay with PAX3 and function of p57KIP2 is not a dominantly acting transforming FOXO1. As shown in supporting information (SI) Fig. 7, neither event. Rather, our data suggest that reduction of p57KIP2 levels of these transcription factors alone has an effect on EGR1 by PAX3-FOXO1 is more likely to predispose cells to transfor- stability. mation by a secondary genetic event. PAX3-FOXO1 enables cells to bypass cellular senescence checkpoints through loss of Because proteasome inhibition prevents PAX3-FOXO1 from p16INK4a (30). This function, in concert with the failure of destabilizing EGR1, inhibition of proteasome function would be PAX3-FOXO1-expressing cells to differentiate, would create a expected to prevent the repression of p57Kip2 transcription by large pool of proliferating cells primed for transformation. This PAX3-FOXO1. To test this, reporter assays were performed in mechanism of action is different from those described for any the presence of MG132 for 16 h; such proteasome inhibition other cancer-related fusion gene (45). largely reversed the repressive effect of PAX3-FOXO1 on It is interesting that, in this context, PAX3-FOXO1 controls p57Kip2 transcription (Fig. 5B). The inclusion of MG132 for the p57Kip2 transcription not by acting as a transcription factor but, entire course of the assay, 48 h, might have completely restored rather, by interfering with the activation elicited by the inter- p57Kip2 transcription, however this experiment could not be mediary transcription factor, EGR1. We had expected, based on carried out because of the toxicity of the drug (37). the current model of PAX3-FOXO1 function, that previously We next sought to determine which domains of PAX3- identified PAX3 transcription targets, some of which also appear FOXO1 are required for suppression of p57Kip2 transcription by to be PAX3-FOXO1 targets, such as MITF (46), RET (47), using a series of PAX3-FOXO1 deletions (Fig. 5C). Interest- TYRP1 (48), MET (49), NCAM (50), and BCL-xL (28), would ingly, PAX3-FOXO1, despite the loss of its primary DNA- be up-regulated in PAX3-FOXO1 transgenic myoblasts. How- binding domain (see construct 193-836, Fig. 5C), still retains ever, transcription of these genes was unchanged in PAX3-

18088 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708910104 Roeb et al. Downloaded by guest on October 1, 2021 Mouse p57kip2 promoter deletions were constructed by using the firefly luciferase vector, pGL3 basic (Promega, Madison, WI). The Egr1 reporter, containing three tandem EGR1 consensus sites (GCGGGGGCG; see ref. 52) separated by spacers, was derived from the pLucMCS vector (Stratagene, La Jolla, CA).

Cell Culture. All transfections were performed with human 293T cells, in DMEM with 10% FBS. Cells were transfected by using 1 ␮g of DNA and 8 ␮l of Lipofectamine 2000 (Invitrogen) per 3 ϫ 106 cells. For the reporter assays, 1 ϫ 106 cells were transfected with 0.5 ␮g of DNA and 1 ␮l of Lipofectamine 2000.

Western Blots. Ten micrograms of protein were harvested in RIPA buffer [150 mM NaCl/1.0% Triton X-100/0.5% sodium deoxycholate/0.1% SDS/50 mM Tris, pH 8.0/complete protease inhibitor (Roche, Indianapolis, IN)], added to lithium dodecyl- sulfate (LDS) sample buffer (Invitrogen) separated on either Fig. 6. Model for PAX3-FOXO1-driven suppression of myogenic differenti- 4–12% or 3–8% NuPAGE gels (Invitrogen), transferred to ation (adapted from ref. 54). nitrocellulose, blocked (PBS containing 0.1% Tween 20 and 5% nonfat milk), and incubated for1hatroom temperature with FOXO1 transgenic myoblasts, likely reflecting the somewhat primary antibodies. Antibodies and sources are in SI Table 1. lower level of PAX3-FOXO1 expression in our transgenic myoblasts as compared with human ARMS tumor cell lines, Isolation of Mouse Myoblasts. Isolation and culture of mouse either of which is much less than obtained by ectopic overex- myoblasts was performed as described in ref. 53. Briefly, hind- pression. In our system, PAX3-FOXO1 expression is driven by limb muscle tissue was dissected from 3-day-old mice, minced, the endogenous PAX3 promoter, as occurs in vivo. Therefore, and digested with Liberase Blendzyme 3 (Roche) followed by the expression levels in PAX3-FOXO1 transgenic myoblasts trypsin. After washing, this mixture was plated and cultured in should be equivalent to those that would be seen in affected myoblast growth media [20% FBS in a 50/50 mix of DMEM/ myoblasts during the early stages of ARMS. Thus, this system Ham’s F10 (Invitrogen) and 25 ng/ml bFGF (Dako, Glostrup, should accurately model disease initiation. However, in trans- Denmark)]. Fibroblasts were removed by selective plating at fection experiments using other cells that are wild type for Tp53, each subsequent passage. For expression analysis, RNA was we have routinely had difficultly in achieving even modest levels harvested from early passage cells (before or at passage 7) from of PAX3-FOXO1 expression. These findings, taken together cultures grown in triplicate with the RNeasy Plus Kit (Qiagen, Valencia, CA). with our PAX3-FOXO1 mapping data, suggest that PAX3- For differentiation experiments, cells were cultured in DMEM FOXO1 may function as a misfolded protein. If misfolded with 5% horse serum (Invitrogen). After 24 h (48 h for p57KIP2) proteins accumulate to the extent that they overwhelm the in differentiation medium, protein was harvested in RIPA buffer chaperone system, then cells will undergo programmed cell [150 mM NaCl/1.0% Triton X-100/0.5% sodium deoxycholate/ death. Apoptosis occurs through JNK, primarily through stabi- 0.1% SDS/50 mM Tris, pH 8.0/complete protease inhibitor lization of p53 (51). Thus, overexpression of PAX3-FOXO1 (Roche, Indianapolis, IN)], and RNA was extracted as above. would be expected to be selected against in otherwise wild-type For viral transduction, viruses in the murine stem cell virus cells. (MSCV) vector were obtained by cotransfecting 293T cells with Our findings lead to the hypothesis that PAX3-FOXO1 con- an equal mass of MSCV plasmid and the envelope encoding tributes to rhabdomyosarcoma development by repressing the vector, pCL-Eco (Imgenex, San Diego, CA). Viral supernatant transcription of p57Kip2. Reduction of EGR1 protein levels by was prepared 48 h posttransfection by passing the media through PAX3-FOXO1 results in inadequate quantities of p57KIP2 0.45-␮m filters. Myoblasts (3 ϫ 106 cells) were infected with protein, preventing PAX3-FOXO1 transgenic myoblasts from fresh virus diluted 1:2 with myoblast growth media and 3.2 ␮g/ml completing the differentiation program (Fig. 6). Thus, translo- fresh polybrene (Sigma, St. Louis, MO) for 8 h. Forty-eight hours cation-positive myoblasts that are unable to exit the cell cycle, postinfection, transduction efficiency was measured by main- could establish a pool of proliferating precursor cells susceptible taining a separate pool of myoblasts infected with GFP. The to a secondary transforming event, thus illustrating that tumor- entire procedure was repeated four times to obtain a cell specific genetic alterations could be central to the interplay population Ͼ90% GFP-positive. between development and cancer. Finally, these results may have therapeutic applications, because they suggest that ARMS is, at Quantitative Real-Time PCR. Total RNA was harvested as above least in part, a protein degradation-driven disease. Proteasome and reverse transcribed (SuperScript III First Strand Kit; In- inhibitors, such as bortezomib, might be a new and effective vitrogen). Quantitative PCR was performed on the iCycler IQ approach for treating ARMS. using IQ Syber Green (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions and default machine settings with an Materials and Methods annealing temperature of 60°C (primer sequences can be found Plasmids. Mammalian expression vectors encoding EGR1, in SI Table 2). PAX3-FOXO1, PAX3, FOXO1, and PAX3-FOXO1 mutants, both wild-type and epitope-tagged, were derived from EMSA. EGR1 was transcribed and translated in vitro (TNT T7 pcDNA3.1 (Invitrogen, Carlsbad, CA). SP1 and SP3 plasmids Quick; Promega) in a reaction with 10 ␮M ZnSO4 (Sigma). The were derived from pCMV. Epitope tags are as follows: the V5 tag EMSA was performed with 0.2 pmol of 32P- labeled p57Kip2 (Invitrogen) was fused to the N terminus of EGR1 and PAX3- promoter sequence (Ϫ400 to Ϫ350 bp) at 30°C for 30 min in a FOXO1; the 3xHA tag was fused the C terminus of PAX3, reaction buffer containing 20% glycerol, 5 mM MgCl2, 2.5 mM

FOXO1, PAX3-FOXO1, and all of the PAX3-FOXO1 deletion EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris⅐HCl (pH 7.5), CELL BIOLOGY mutants. 0.25 mg/ml poly(dI-dC).poly(dI-dC), and 10 ␮M ZnSO4. Reac-

Roeb et al. PNAS ͉ November 13, 2007 ͉ vol. 104 ͉ no. 46 ͉ 18089 Downloaded by guest on October 1, 2021 tions were separated on 6% DNA retardation gels (Invitrogen) of lysate was used with 2 ␮g of an anti-HA antibody in the that were dried and subjected to autoradiography. presence of Dynabeads Protein G (Invitrogen). Immune complexes were washed [150 mM NaCl/50 mM Tris⅐HCl, pH 8/1% Reporter Assays. 293T cells were cotransfected with the firefly Nonidet P-40/0.5% sodium deoxycholate/0.05% SDS/complete luciferase reporter plasmid and the indicated combinations of protease inhibitor (Roche)]. Complexes were reduced, dena- expression plasmid. The Renilla luciferase plasmid pRL (Pro- tured, and eluted in LDS sample buffer. mega), driven by a minimal tk promoter, was included as an For the coimmunoprecipitation of endogenously expressed internal control. Forty-eight hours posttransfection, the Dual- proteins, the ARMS-derived cell line Rh28 (38) was used. Cells Luciferase Reporter Assay (Promega) was performed according were treated with 20 ␮M MG132 (EMD Biosciences, San Diego, to the manufacturer’s instructions, and values were read on the CA) for 2 h before harvesting in Nonidet P-40 buffer. For the GENios Pro (Tecan, Durham, NC). EGR1 IP, 10 mg of protein was incubated overnight in the presence of 2 ␮g of the anti-EGR1 antibody (C19) with Dyna- EGR1 Destruction Assay. 293T cells were transfected with a fixed beads Protein A (Invitrogen). mass of epitope-tagged EGR1 plasmid with increasing quantities of epitope-tagged PAX3-FOXO1 plasmid. Total DNA mass was We thank Koichi Okumura useful discussions, Tim Fenton for critical kept constant by the addition of pcDNA3.1 plasmid. Sixteen reading, and the University of California at San Diego Cancer Center hours before harvest, cells were treated with either 5 ␮M MG132 Microarray Facility for processing the Genechips. The desmin (D3) and dissolved in DMSO or DMSO alone. myosin heavy chain (MF20) antibodies, developed by Dr. Donald Fischman, and the ␤-actin (JA20) antibody, developed by Jim Jung- Ching Lin, were obtained from the Developmental Studies Hybridoma Coimmunoprecipitation. 293T cells were transfected with equal Bank, developed under the auspices of the National Institute of Child quantities of epitope-tagged PAX3-FOXO1 and EGR1 by using Health and Human Development, and maintained by The University of Lipofectamine 2000 according the manufacturer’s instructions. Iowa Department of Biological Sciences, Iowa City, IA. W.K.C. was Cells were lysed in Nonidet P-40 buffer and either 1 mg of lysate partially supported by a Fellow Award from the National Foundation for was incubated overnight with 2 ␮g of anti-V5 antibody or 2 mg Cancer Research.

1. Weiss S, Goldblum J (2001) Enzinger and Weiss’s Soft Tissue Tumors (Mosby, 26. Bennicelli JL, Fredericks WJ, Wilson RB, Rauscher FJ, III, Barr FG (1995) St Louis). Oncogene 11:119–130. 2. Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, 27. Ginsberg JP, Davis RJ, Bennicelli JL, Nauta LE, Barr FG (1998) Cancer Res Bridge JA, Crist WM, Triche TJ, Barr FG (2002) J Clin Oncol 20:2672–2679. 58:3542–3546. 3. Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ, III, 28. Margue CM, Bernasconi M, Barr FG, Schafer BW (2000) Oncogene 19:2921–2929. Emanuel BS, Rovera G, Barr FG (1993) Nat Genet 5:230–235. 29. Taulli R, Scuoppo C, Bersani F, Accornero P, Forni PE, Miretti S, Grinza A, 4. Franz T, Kothary R, Surani MA, Halata Z, Grim M (1993) Anat Embryol Allegra P, Schmitt-Ney M, Crepaldi T, Ponzetto C (2006) Cancer Res 66:4742– 187:153–160. 4749. 5. Ridgeway AG, Skerjanc IS (2001) J Biol Chem 276:19033–19039. 30. Linardic CM, Naini S, Herndon JE, II, Kesserwan C, Qualman SJ, Counter CM 6. Bois PR, Grosveld GC (2003) EMBO J 22:1147–1157. (2007) Cancer Res 67:6691–6699. 7. Hribal ML, Nakae J, Kitamura T, Shutter JR, Accili D (2003) J Cell Biol 31. Xia SJ, Barr FG (2005) Eur J Cancer 41:2513–2527. 162:535–541. 32. Anderson MJ, Shelton GD, Cavenee WK, Arden KC (2001) Proc Natl Acad Sci 8. Cam H, Griesmann H, Beitzinger M, Hofmann L, Beinoraviciute-Kellner R, USA 98:1589–1594. Sauer M, Huttinger-Kirchhof N, Oswald C, Friedl P, Gattenlohner S, et al. 33. Kaufman SJ, Foster RF (1988) Proc Natl Acad Sci USA 85:9606–9610. (2006) Cancer Cell 10:281–293. 34. Tapscott SJ, Thayer MJ, Weintraub H (1993) Science 259:1450–1453. 9. Watanabe H, Pan ZQ, Schreiber-Agus N, DePinho RA, Hurwitz J, Xiong Y 35. Fu M, Zhu X, Zhang J, Liang J, Lin Y, Zhao L, Ehrengruber MU, Chen YE (1998) Proc Natl Acad Sci USA 95:1392–1397. (2003) Gene 315:33–41. 10. Reynaud EG, Leibovitch MP, Tintignac LA, Pelpel K, Guillier M, Leibovitch 36. Svaren J, Ehrig T, Abdulkadir SA, Ehrengruber MU, Watson MA, Milbrandt SA (2000) J Biol Chem 275:18767–18776. J (2000) J Biol Chem 275:38524–38531. Mol 11. Reynaud EG, Pelpel K, Guillier M, Leibovitch MP, Leibovitch SA (1999) 37. Mimnaugh EG, Chen HY, Davie JR, Celis JE, Neckers L (1997) Biochemistry Cell Biol 19:7621–7629. 36:14418–14429. 12. Baffa R, Negrini M, Mandes B, Rugge M, Ranzani GN, Hirohashi S, Croce CM 38. Hazelton BJ, Houghton JA, Parham DM, Douglass EC, Torrance PM, Holt H, (1996) Cancer Res 56:268–272. Houghton PJ (1987) Cancer Res 47:4501–4507. 13. Bepler G, Garcia-Blanco MA (1994) Proc Natl Acad Sci USA 91:5513–5517. 39. Matsuoka S, Edwards MC, Bai C, Parker S, Zhang P, Baldini A, Harper JW, 14. Henry I, Grandjouan S, Couillin P, Barichard F, Huerre-Jeanpierre C, Glaser Elledge SJ (1995) Genes Dev 9:650–662. T, Philip T, Lenoir G, Chaussain JL, Junien C (1989) Proc Natl Acad Sci USA 40. Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, Thompson JC, Silverman 86:3247–3251. A, Harper JW, DePinho RA, Elledge SJ (1997) Nature 387:151–158. 15. Bourcigaux N, Gaston V, Logie A, Bertagna X, Le Bouc Y, Gicquel C (2000) 41. Yan Y, Frisen J, Lee MH, Massague J, Barbacid M (1997) Genes Dev J Clin Endocrinol Metab 85:322–330. 16. Li JQ, Wu F, Usuki H, Kubo A, Masaki T, Fujita J, Bandoh S, Saoo K, Takeuchi 11:973–983. H, Kuriyama S, et al. (2003) Int J Oncol 23:1537–1543. 42. Keller C, Arenkiel BR, Coffin CM, El-Bardeesy N, DePinho RA, Capecchi MR 17. Rothschild G, Zhao X, Iavarone A, Lasorella A (2006) Mol Cell Biol 26:4351–4361. (2004) Genes Dev 18:2614–2626. 18. Anderson J, Gordon A, McManus A, Shipley J, Pritchard-Jones K (1999) 43. Lagutina I, Conway SJ, Sublett J, Grosveld GC (2002) Mol Cell Biol 22:7204–7216. Neoplasia 1:340–348. 44. Relaix F, Polimeni M, Rocancourt D, Ponzetto C, Schafer BW, Buckingham 19. Hoffmann MJ, Florl AR, Seifert HH, Schulz WA (2005) Int J Cancer M (2003) Genes Dev 17:2950–2965. 114:406–413. 45. Aplan PD (2006) Trends Genet 22:46–55. 20. Soejima H, Nakagawachi T, Zhao W, Higashimoto K, Urano T, Matsukura S, 46. Watanabe A, Takeda K, Ploplis B, Tachibana M (1998) Nat Genet 18:283–286. Kitajima Y, Takeuchi M, Nakayama M, Oshimura M, et al. (2004) Oncogene 47. Lang D, Chen F, Milewski R, Li J, Lu MM, Epstein JA (2000) J Clin Invest 23:4380–4388. 106:963–971. 21. Scrable H, Cavenee W, Ghavimi F, Lovell M, Morgan K, Sapienza C (1989) 48. Galibert MD, Yavuzer U, Dexter TJ, Goding CR (1999) J Biol Chem Proc Natl Acad Sci USA 86:7480–7484. 274:26894–26900. 22. Hatada I, Ohashi H, Fukushima Y, Kaneko Y, Inoue M, Komoto Y, Okada A, 49. Epstein JA, Shapiro DN, Cheng J, Lam PY, Maas RL (1996) Proc Natl Acad Ohishi S, Nabetani A, Morisaki H, Nakayama M, Niikawa N, Mukai T (1996) Sci USA 93:4213–4218. Nat Genet 14:171–173. 50. Kioussi C, Gross MK, Gruss P (1995) Neuron 15:553–562. 23. Maulbecker CC, Gruss P (1993) EMBO J 12:2361–2367. 51. Fuchs SY, Adler V, Pincus MR, Ronai Z (1998) Proc Natl Acad Sci USA 24. Bennicelli JL, Edwards RH, Barr FG (1996) Proc Natl Acad Sci USA 93:5455– 95:10541–10546. 5459. 52. Swirnoff AH, Milbrandt J (1995) Mol Cell Biol 15:2275–2287. 25. Fredericks WJ, Galili N, Mukhopadhyay S, Rovera G, Bennicelli J, Barr FG, 53. Rando TA, Blau HM (1997) Methods Cell Biol 52:261–272. Rauscher FJ, III (1995) Mol Cell Biol 15:1522–1535. 54. Hawke TJ, Garry DJ (2001) J Appl Physiol 91:534–551.

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