ANTICANCER RESEARCH 33: 2029-2036 (2013)

Regulation of Target of PAX3−FOXO1 in Alveolar

EUN HYUN AHN1,2

1Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.; 2Department of Pathology, School of Medicine, University of Washington, Seattle, WA, U.S.A.

Abstract. Background: The majority of alveolar major subtypes based on their histological appearance: rhabdomyosarcoma (ARMS) are distinguished through the embryonal rhabdomyosarcoma (ERMS) and alveolar paired box 3−forkhead box O1 (PAX3−FOXO1) rhabdomyosarcoma (ARMS) (1). ARMS has a higher fusion oncoprotein, being generated by a 2;13 chromosomal frequency of metastases at the initial diagnosis than ERMS, translocation. This fusion-positive ARMS is the most commonly conferring a poorer prognosis than ERMS (2, 3). clinically difficult type of rhabdomyosarcoma. The present A common characteristic of ERMS is a loss of study characterized four genes [gremlin 1 (GREM1), death- heterozygosity at 11p15, however ERMS has not been associated protein kinase-1 (DAPK1), myogenic reported to exhibit a diagnostic genetic alteration. In contrast, differentiation-1 (MYOD1), and hairy/enhancer-of-split chromosomal translocation is frequently observed for ARMS related with YRPW motif-1 (HEY1)] as targets of (4, 5). The translocation t(2;13)(q35;q14) generating the PAX3−FOXO1. Materials and Methods: The expression of paired box 3− (PAX3−FOXO1) the four genes, PAX3−FOXO1, and v- myelocytomatosis fusion was found to occur in 55% of ARMS cases, while the viral-related oncogene, neuroblastoma-derived (avian) translocation t(1;13)(q36;q14) generating the paired box (MYCN) was determined in various ARMS cell models and 7−forkhead box protein O1 (PAX7−FOXO1) gene fusion primary tumors. The roles of PAX3−FOXO1 and MYCN occurred in 22% of cases, and 23% of ARMS were fusion- expression were examined. Results: Pulse-chase and negative (2). The 2;13 translocation present in ARMS is cycloheximide experiments suggest that GREM1, DAPK1, characterized by an overexpression of PAX3−FOXO1 and MYOD1 are directly regulated by PAX3−FOXO1. relative to wild-type PAX3 (6). PAX3−FOXO1 appears to indirectly down-regulate HEY1 by Although PAX3−FOXO1-positive ARMS is the most up-regulating MYCN. Data reveal that the growth- clinically intractable fusion subtype of pediatric suppressive activity of high PAX3−FOXO1 expression is rhabdomyosarcoma, it can be difficult to histologically closely-associated with up-regulation of the GREM1 and classify rhabdomyosarcoma into ARMS and ERMS in some DAPK1 tumor-suppressor genes. Conclusion: This study cases (7), and no specific drugs are available for treating a characterized four downstream targets of PAX3−FOXO1 that specific fusion or histological subtype (1, 2, 7, 8). Therefore, contribute to the biological activities of growth suppression there is a strong incentive to elucidate target genes of and myogenic differentiation. PAX3−FOXO1, which has the utility of being a therapeutic target and marker for the purpose of diagnosis and treatment Rhabdomyosarcoma is the most prominent type of pediatric of rhabdomyosarcoma. Several recent studies have utilized soft tissue sarcoma, being associated with the skeletal muscle the profiles to classify rhabdomyosarcoma lineage. Among pediatric rhabdomyosarcoma, there are two or identify target genes of only PAX3−FOXO1, or both PAX3−FOXO1 and PAX7−FOXO1, however, more research is needed to validate the biological function of the genes in ARMS development (9-15). Correspondence to: Eun Hyun Ahn, Ph.D., Department of Recently Ahn and co-authors identified 34 potential Pathology, University of Washington School of Medicine, Box target genes and validated the four genes, gremlin-1 357705. 1959 NE Pacific St., Seattle, WA 98195, U.S.A. E-mail: [email protected]/[email protected] (GREM1), death-associated protein kinase-1 (DAPK1), myogenic differentiation (MYOD1), and hairy/enhancer- Key Words: Rabdomyosarcoma, PAX3−FOXO1, gene expression, of-split related with YRPW motif-1 (HEY1), as target growth suppression. genes of PAX3−FOXO1 in ARMS by analyzing gene

0250-7005/2013 $2.00+.40 2029 ANTICANCER RESEARCH 33: 2029-2036 (2013) expression profiles from two independent systems: primary RNA extraction and qRT-PCR analysis. Total RNA was extracted tumors (PAX3−FOXO1-positive ARMS and fusion- using RNA STAT-60 (Tel-Test Inc., Friendswood, TX, USA). The negative ERMS) and a cell culture system expressing the qRT-PCR assay was performed as described previously (14). The expression of test genes was normalized to the expression of 18S inducible PAX3−FOXO1-estrogen (ER) ligand ribosomal RNA (18S rRNA). Taqman gene expression assays used binding domain construct (40). The present study focused (Applied Biosystems, Foster City, CA, USA) were: DAPK1 (assay on these four genes in order to investigate whether these ID# Hs00234489_m1), GREM1 (assay ID# Hs00171951_m1), and target genes are directly or indirectly regulated by MYOD1 (assay ID# Hs00159528_m1) and sequences of these gene PAX3−FOXO1, the nature of interactions between target expression assays are not disclosed by Applied Biosystems. The genes of PAX3−FOXO1, and whether specific targets of sequences of forward and reverse primers and probes are for PAX3−FOXO1 are associated with growth suppressive PAX3−FOXO1 [5’CCT CAA CCM CAT GAA CCC3’ (M: either A or C); 5’ CCT TCA TTC TGC ACA CGA ATG A 3’; 5’ VIC_TGG activity of high PAX3−FOXO1 expression. CAA TGG CCT CTC ACC TCA GAA TT_TAMRA 3’], for HEY1 [5’ TGA CCG TGG ATC ACC TGA AA 3’; 5’ GCG TGC GCG Materials and Methods TCA AAG TAA 3’; 5’ FAM_TGC TGC ATA CGG CAG GAG GGA AAG_TAMRA 3’], and for MYCN [5 ‘ACC ACA AGG CCC Tumor samples. Tumor specimens used for quantitative reverse- TCA GTA CCT 3’; 5’ ACG TCG ATT TCT TCC TCT TCA TCT polymerase chain reaction (qRT-PCR) (13 3’; 5’ FAM_CTC TTC ATC ATC TTC ATC ATC TGA ATC GCT PAX3−FOXO1-positive ARMS) were described previously (14). CA _TAMRA 3’]. The presence of PAX3−FOXO1 in ARMS tumor specimens was confirmed by qRT-PCR (16). Extraction of cellular and immunoblot analysis. The RD cells transduced with pKH or pKH-MYCN were seeded at 106 cells Cell culture and cell growth assay. RD ERMS cell line and RD- per 100 mm dish in DMEM containing 10% FBS, 1% P/S, and 1% derived PAX3−FOXO1 and PAX3−FOXO1-ER-positive cells were AM for 24 hours prior to hygromycin B treatment. Cellular proteins maintained in Dulbecco’s modified Eagle’s medium (DMEM) were extracted and immunoblot analysis was performed as described (Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum previously with modifications (22). MYCN (cat# sc-791) and actin (FBS) (HyClone Thermo Scientific, Logan, UT, USA), 1% (sc-1616) used as primary antibodies were from Santa Cruz penicillin:streptomycin (P/S) (Invitrogen) and 1% antibiotic- Biotechnology (Santa Cruz, CA, USA). antimycotic (AM) (Invitrogen). For cell growth experiments, 2×105 cells constitutively expressing PAX3−FOXO1 in pK1 (pK-P3F) and Statistical analysis. The correlation between MYCN and HEY1 2×104 cells carrying the pK1 vector alone (pK) were plated per well expression levels determined by qRT-PCR in 13 PAX3−FOXO1- in six-well plates, trypsinized at the indicated time points, and positive ARMS tumors was analyzed by Pearson’s correlation viable cells were then counted. coefficient after log-transformation of gene expression values.

DNA constructs. Retroviral constructs with relevant DNA inserts Results [inducible PAX3−FOXO1 in pK1 with puromycin resistance marker (pK-P3F-ER), constitutive PAX3−FOXO1 in pK1 with puromycin PAX3−FOXO1 directly regulates GREM1, DAPK1, and resistance marker (pK-P3F), and v-myc myelocytomatosis viral MYOD1, while HEY1 may be indirectly regulated. The two related oncogene, neuroblastoma derived (avian) (MYCN) in pK1 approaches were followed to explore whether GREM1, with hygromycin resistance marker (pKH-MYCN)] were previously generated by Dr. Frederic G. Barr’s laboratory (17-20). A modified DAPK1, MYOD1, and HEY1 are direct transcriptional ER ligand-binding domain was provided by Dr. G. Evan (21). targets regulated by PAX3−FOXO1. Firstly, pulse-chase time course experiments were conducted to determine how Establishment of ERMS cell culture systems inducibly or quickly the maximum expression change was achieved. RD constitutively expressing PAX3−FOXO1 and ERMS cells ERMS-derived cells expressing an inducible PAX3−FOXO1- overexpressing MYCN. Retroviral transduction was performed as ER in pK1 (pK-P3F-ER) or pK1 vector-alone (pK) were described previously with modifications (19, 20). Cells that were treated without or with 100 nM TMF for one hour. Then transduced with retroviral DNA constructs were selected with TMF was removed and cells were cultured with medium for puromycin (BD Biosciences, San Jose, CA, USA) at: 1 μg/ml for RD-derived cells with PAX3−FOXO1-ER in pK1 and 0.8 μg/ml 3, 6, 12, and 24 h. The expression of GREM1, DAPK1, for RD-derived cells with PAX3−FOXO1 in pK1. Cells carrying MYOD1, and HEY1 was determined by qRT-PCR at each inducible PAX3−FOXO1-ER were treated with the ligand time point (Figure 1). In RD cells expressing pK-P3F-ER, 4-hydroxytamoxifen (TMF) (Sigma-Aldrich, St. Louis, MO, the maximum up-regulation of GREM1 (Figure 1A), DAPK1 USA), which activates and induces transcriptional activity of (Figure 1B), and MYOD1 (Figure 1C) was achieved at 6 h. In PAX3−FOXO1-ER by translocating PAX3−FOXO1-ER to the contrast, the maximum down-regulation of HEY1 was nucleus (18). RD cells were transduced with MYCN in pK1 with observed at 12 hours (Figure 1D). the hygromycin resistance marker (pKH-MYCN) or with pKH vector only (pKH) and then were selected by incubation with In the second approach, RD ERMS cells expressing an hygromycin B (Roche Diagnostics, Indianapolis, IN, USA) at inducible PAX3−FOXO1-ER construct in pK1 (pK-P3F-ER) 250 μg/ml. or pK1 vector-alone (pK) were treated with both

2030 Ahn: Regulation of Targets of PAX3−FOXO1 in ARMS

Figure 1. Time course of target gene expression in RD cells expressing paired box 3−forkhead box protein O1- (PAX3−FOXO1-ER). RD cells transduced with PAX3−FOXO1-ER in pK1 (pK-P3F-ER) or pK1-alone (pK) were treated with 100 nM 4- hydroxytamoxifen (TMF) for one hour. Cells were then washed with phosphate buffered saline (PBS) and incubated in medium without TMF. Total RNA was isolated at the indicated time points and expression of genes was determined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expression levels of the genes were normalized to that of 18S ribosomal RNA (18S rRNA) (mean±SD, n=3). Abbreviations of genes are: Gremlin-1 (GREM1), death-associated Figure 2. Effects of cycloheximide on the induction of paired box protein kinase-1 (DAPK1), myogenic differentiation-1 (MYOD1), and 3−forkhead box protein O1 (PAX3−FOXO1) target genes in RD cells hairy/enhancer-of-split related with YRPW motif-1 (HEY1). expressing PAX3−FOXO1-estrogen receptor (ER). RD cells transduced with PAX3−FOXO1-ER in pK1 (pK-P3F-ER) (A-D) or the pK1 vector- alone (pK) (E) were treated with or without 1 μg/ml cyloheximide (Cyclo). After one hour of incubation, cells were treated without or with 30 nM 4-hydroxytamoxifen (TMF) for 24 h. When present, cycloheximide (a de novo protein synthesis inhibitor) and cycloheximide remained in the medium during this second incubation TMF for 24 h following one hour pre-treatment with step. Total RNA was isolated and expression of genes was determined by quantitative reverse-transcription polymerase chain reaction (qRT- cycloheximide (Figure 2). If the gene is a direct target, PCR). The expression levels of the genes were normalized to that of 18S synthesis of intermediate transcription factors or other ribosomal RNA (18S rRNA) (mean±SD, n=3). Abbreviations of genes proteins will not be required to regulate the gene’s expression. are: Gremlin-1 (GREM1), death-associated protein kinase-1 (DAPK1), Therefore, even in the presence of cycloheximide, the down- myogenic differentiation-1 (MYOD1), and hairy/enhancer-of-split regulation or up-regulation of specific PAX3−FOXO1 target related with YRPW motif-1 (HEY1). genes will still occur. Data indicated that even in the presence of cycloheximide, PAX3−FOXO1 induced by TMF resulted in the up-regulation of GREM1 (Figure 2A), DAPK1 (Figure 2B), and MYOD1 (Figure 2C). and that unstimulated PAX3−FOXO1-ER (control: untreated Although HEY1 was down-regulated in the presence of with TMF) is associated with an activity which reverses this cycloheximide and TMF (Figure 2D), a puzzling result was inhibitory effect of cycloheximide on HEY1 expression. found for the effect of cycloheximide alone on HEY1 Based on these findings, it is unclear whether the effect seen expression (Figure 2E). While cells transduced with the in PAX3−FOXO1-ER-transduced cells treated with PAX3−FOXO1-ER in the pK1 vector showed no change in cycloheximide and TMF represents maintainence of the TMF- HEY1 expression in the presence of cycloheximide alone induced down-regulation of HEY1. Therefore, the results of (Figure 2D), there was a significant decrease in HEY1 this cycloheximide experiment for HEY1 cannot be used to expression in cycloheximide-treated cells transduced with the evaluate whether HEY1 is a direct target of PAX3-FOXO1. pK1 vector only (Figure 2E). These findings suggest that Taken together, both pulse-chase and cycloheximide cycloheximide inhibits the expression of a factor needed for experiments suggest that GREM1, DAPK1, and MYOD1 are HEY1 expression in these ERMS cells transduced with pK1, directly regulated by PAX3−FOXO1. In contrast, after

2031 ANTICANCER RESEARCH 33: 2029-2036 (2013)

Figure 3. Effect of v-myc myelocytomatosis viral related oncogene, neuroblastoma-derived (avian) (MYCN) on hairy/enhancer-of-split related with YRPW motif 1 (HEY1) expression. A: The expression levels of MYCN and HEY1 in paired box 3−forkhead box protein O1 (PAX3−FOXO1)-positive alveolar rhabdomyosarcoma (ARMS) tumors (n=13) were determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), normalized to that of 18S ribosomal RNA (18S rRNA) and log-transformed. B: Total protein was extracted after two weeks of hygromycin B selection from RD cells Figure 4. Phenotypic and target gene expression changes following transduced with MYCN in pK1 with the hygromycin resistance marker transduction of a construct constitutively-expressing paired box (pKH-MYCN) or transduced with the pKH vector alone. The protein 3−forkhead box protein O1 (PAX3−FOXO1) into RD cells. A and B: expression of MYCN and β-actin was determined by immunoblot RD cells were transduced with the pK1 retroviral vector-alone (pK) or analysis. The protein expression of MYCN was normalized by β-actin PAX3−FOXO1 subcloned into pK1 (pK-P3F). After three days selection protein expression for a densitometric graph (mean±SE, n=2). C: Total in puromycin-containing medium, 2×105 cells constitutively-expressing RNA was isolated after two weeks of hygromycin B selection from RD PAX3−FOXO1 and 2×104 cells carrying pK1-alone were plated per cells transduced with pKH-MYCN or pKH vector-alone. HEY1 well in six-well plates and selected with puromycin. Viable cells were expression was determined by qRT-PCR and normalized to that of 18S counted at the indicated time points (mean±SD, n=3). C-G: Total RNA ribosomal RNA (18S rRNA) (mean±SD, n=3). was isolated at the indicated time points and the expression of genes was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expression levels were normalized to that of 18S ribosomal RNA (18S rRNA) (mean±SD, n=3). Abbreviations of genes are: Gremlin-1 (GREM1), death-associated protein kinase-1 excluding the cycloheximide result for HEY1, the longer (DAPK1), myogenic differentiation-1 (MYOD1), and hairy/enhancer-of- time requirement for HEY1 down-regulation (Figure 2D) split related with YRPW motif-1 (HEY1). suggests the possible existence of intermediate (s) that regulate HEY1 and must first be induced by PAX3−FOXO1. expression. We hypothesized that MYCN up-regulation PAX3−FOXO1 indirectly down-regulates HEY1 by up- induced by PAX3−FOXO1 may result in HEY1 down- regulating MYCN. Since results from the pulse-chase time regulation, based on published reports. First, PAX3−FOXO1 course experiment suggests that HEY1 may be indirectly up-regulated MYCN (15) and MYCN synergistically regulated by PAX3−FOXO1, further experiments were cooperated with PAX3−FOXO1 to enhance oncogenic conducted to identify which gene(s) induced by activity (15, 23, 24). Second, expression profiles and RT- PAX3−FOXO1 are also able to down-regulate HEY1 PCR data demonstrated that tumors from transgenic mice

2032 Ahn: Regulation of Targets of PAX3−FOXO1 in ARMS overexpressing c-myc, that was targeted to the mammary targets responsible for growth suppressive effects of high gland, showed down-regulation of (25). PAX3−FOXO1 expression. This hypothesis of HEY1 regulation by MYCN was tested In contrast, the induced high expression of MYOD1 by two experimental approaches: First, it was examined (Figure 4F) was similar during all periods. The expression of whether an inverse correlation exists between MYCN and HEY1 (Figure 4G) stayed consistently low throughout all the HEY1 expression in a series of thirteen PAX3−FOXO1- periods and was the least affected by changes in positive ARMS primary tumors determined using qRT-PCR PAX3−FOXO1 levels. These expression findings imply that (Figure 3A). The Pearson’s correlation coefficient for HEY1 different target genes of PAX3−FOXO1 have different dose- versus MYCN expressions was -0.7032, indicating that the response effects. After the initial PAX3−FOXO1 down- and MYCN expression was inversely correlated with HEY1 up-regulation event, HEY1 and MYOD1 are not sensitive to expression (Figure 3A). Second, a population of RD ERMS further changes in PAX3−FOXO1 expression ranges tested cells overexpressing MYCN (pKH-MYCN) was established in this model, whereas GREM1 and DAPK1 are very (Figure 3B), and the expression of HEY1 in these cells by sensitive to these PAX3−FOXO1 fluctuations. qRT-PCR was determined. These RD cells overexpressing MYCN (pKH-MYCN) showed about two-times lower HEY1 Discussion expression as compared with that of the RD cells carrying the vector pK1-Hyg-alone (pKH) (Figure 3C). These results The current study demonstrates that PAX3−FOXO1 directly are consistent with the hypothesis that PAX3−FOXO1 down- up-regulates GREM1, DAPK1, and MYOD1. This result is regulates HEY1 by up-regulating MYCN, and further supported, in part, by the study of Cao et al., which showed support that HEY1 is an indirect downstream target of that DAPK1 and MYOD1 genes are associated with proximal PAX3−FOXO1. PAX3−FOXO1 binding sites, implying these genes as direct targets of PAX3−FOXO1 (26). Growth suppression caused by high PAX3−FOXO1 To my knowledge, the present study is the first to expression is associated with up-regulation of GREM1 and demonstrate that PAX3−FOXO1 down-regulates HEY1 by up- DAPK1. Xia et al. reported that PAX3−FOXO1, when regulating MYCN. Previous studies revealed a role of HEY1 constitutively expressed at a level comparable to the in muscle differentiation. For example, overexpression of expression levels found in ARMS tumors, is growth HEY1 inhibits MYOD1, an early myogenic differentiation suppressive in immortalized murine NIH3T3 fibroblasts (19, marker, possibly through formation of inactive HEY1/MYOD1 20). I hypothesized that the GREM1 and DAPK1 tumor- heterodimers (27). Notch activation robustly induces the suppressor genes may contribute to this growth-suppressive transcriptional repressors HEY1 and hairy/enhancer-of-split activity of high PAX3−FOXO1 expression. related with YRPW motif-like (HEYL) in myoblasts, but only To test this hypothesis, RD cells were transduced with a the constitutive expression of HEY1 blocks (28). construct in which PAX3−FOXO1 is constitutively expressed As demonstrated in some studies, PAX3−FOXO1 may from the pK1 vector. As a control, RD cells were transduced facilitate early steps in myogenesis and thus repression of with pK1 vector-alone and demonstrated continued growth HEY1 expression is consistent with the PAX3−FOXO1- over time (Figure 4A). In contrast, although RD cells induced myogenic developmental program such as up- transduced with PAX3−FOXO1 (Figure 4B) were seeded at regulation of MYOD1. a 10-times higher number than the control RD cells (Figure Pulse-chase time course and cycloheximide experiments 4A), the growth of PAX3−FOXO1-expressing cells declined indicate that PAX3−FOXO1 directly up-regulates MYOD1. for the first 5 to 7 days (Figure 4B). At the later periods of These findings are consistent with results reported in 10, 14, and 21 days, these PAX3−FOXO1-expressing control previous studies of MYOD1 expression in human ARMS cells (Figure 4B-Inset) recovered and grew at a similar (29, 30), PAX3/PAX7−FOXO1-positive cells (12), proliferation rate as the pK1-transduced cells (Figure 4A- mesenchymal stem cells transfected with PAX3−FOXO1 Inset). This initial period of growth decline (Figure 4B) (31), and NIH3T3 fibroblasts transduced with coincided with the period of high PAX3−FOXO1 expression PAX3−FOXO1 (9, 32). The finding of PAX3−FOXO1 (Figure 4C). The PAX3−FOXO1 expression was higher binding to the MYOD core enhancer in transduced NIH3T3 during the early period, peaking at 5 and 7 days. It then went fibroblasts (32) and rabdomyosarcoma cells (26) signified a down and plateaued at approximately half the peak level direct transactivation of MYOD1 by PAX3−FOXO1. It is (Figure 4C). It is noted that this growth suppressive period hypothesized that PAX3−FOXO1 up-regulates MYOD1 (3, 5, 7 days) with high PAX3−FOXO1 expression coincided which causes a reinforcement of myogenic determination, with the period for higher expression of GREM1 (Figure 4D) while simultaneously suppressing terminal myogenic and DAPK1 (Figure 4E). These findings are consistent with differentiation. Thus, ARMS cells remain in undifferentiated the hypothesis that GREM1 and DAPK1 are downstream malignant states (33, 34). Evidence supporting this

2033 ANTICANCER RESEARCH 33: 2029-2036 (2013) hypothesis includes the finding of PAX3−FOXO1 inhibition References of terminal differentiation in C2C12 myoblasts and MYOD- expressing 10T1/2 fibroblasts (33). Use of a siRNA to 1 Linardic CM: PAX3−FOXO1 fusion gene in decrease for PAX3−FOXO1 expression in an ARMS cell line rhabdomyosarcoma. Lett 270: 10-18, 2008. resulted in up-regulation of genes related to terminal 2 Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, myogenic differentiation (34). Maurer HM, Bridge JA, Crist WM, Triche TJ and Barr FG: The oncogenic activity of PAX3−FOXO1 is PAX3−FKHR and PAX7−FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: A Report from the demonstrated, in part, by its stimulatory effects on cell Children’s Oncology Group. J Clin Oncol 20: 2672-2679, 2002. proliferation (35, 36, 37), cell survival/anti- (38, 3 Stevens MC: Treatment for childhood rhabdomyosarcoma: The 39), as well as its inhibitory role on terminal myogenic cost of cure. Lancet Oncol 6: 77-84, 2005. differentiation (1, 33, 34). However, in addition to these 4 Scrable HJ, Witte DP, Lampkin BC and Cavenee WK: effects, PAX3−FOXO1 can also trigger growth suppression Chromosomal localization of the human rhabdomyosarcoma and cell death in other settings (19, 20, 24). This by mitotic recombination mapping. Nature 329: 645-647, 1987. paradoxical feature of PAX−FOXO1 being both growth- 5 Visser M, Sijmons C, Bras J, Arceci RJ, Godfried M, Valentijn LJ, Voute PA and Baas F: Allelotype of pediatric suppressive and oncogenic was shown in previous studies rhabdomyosarcoma. Oncogene 15: 1309-1314, 1997. with immortalized murine fibroblasts and human myoblasts 6 Davis RJ and Barr FG: Fusion genes resulting from alternative (19, 20, 24). These findings led me to postulate that chromosomal translocations are over-expressed by gene-specific chromosomal translocation and PAX3−FOXO1 fusion mechanisms in alveolar rhabdomyosarcoma. Proc Natl Acad Sci formation may first result in low fusion expression. This USA 94: 8047-8051, 1997. early step would be followed by activation of other 7 Anderson J, Gordon T, McManus A, Mapp T, Gould S, Kelsey transcriptional regulators or genetic alternations that block A, McDowell H, Pinkerton R, Shipley J and Pritchard-Jones K: UK Children’s Cancer Study Group (UKCCSG) and the UK growth suppression pathways and permit later high Cancer Cytogenetics Group. Detection of the PAX3−FKHR PAX3−FOXO1 expression (20), which exerts additional fusion gene in paediatric rhabdomyosarcoma: A reproducible oncogenic and tumorigenic activities (24). predictor of outcome? Br J Cancer 85: 831-835, 2001. The current study presents novel evidence that the up- 8 Kelly KM, Womer RB, Sorensen PHB, Xiong QB and Barr FG: regulation of GREM1 and DAPK1 is associated with the Common and variant gene fusions predict distinct clinical growth suppressive activity of high PAX3−FOXO1 phenotypes in rhabdomyosarcoma. J Clin Oncol 15: 1831-1836, expression. The growth inhibition caused by high 1997. 9 Khan J, Bittner ML, Saal LH, Teichmann U, Azorsa DO, PAX3−FOXO1 expression concurrently occurred with the Gooden GC, Pavan WJ, Trent JM and Meltzer PS: cDNA high expression of GREM1 and DAPK1. When microarrays detect activation of a myogenic transcription PAX3−FOXO1 expression was diminished, the expression of program by the PAX3−FKHR fusion oncogene. Proc Natl Acad GREM1 and DAPK1 was also reduced. These findings are Sci USA 96: 13264-13269, 1999. consistent with the hypothesis that high PAX3−FOXO1 10 Wachtel M, Dettling M, Koscielniak E, Stegmaier S, Treuner J, expression inhibits cellular growth by up-regulating GREM1 Simon-Klingenstein K, Bühlmann P, Niggli FK and Schäfer BW: and DAPK1. Gene expression signatures identify rhabdomyosarcoma subtypes and detect a novel t(2:2)(q35;q23) translocation fusing PAX3 to In conclusion, the present study has established the basis NCOA1. Cancer Res 64: 5539-5545, 2004. to propose the four genes (GREM1, DAPK1, MYOD1, and 11 Wachtel M, Runge T, Leuschner I, Stegmaier S, Koscielniak E, HEY1) as targets that function in growth suppression or Treuner J, Odermatt B, Behnke S, Niggli FK and Schäfer BW: myogenic differentiation downstream of PAX3−FOXO1 in Subtype and prognostic classification of rhabdomyosarcoma by ARMS. These studies to define the biological function of immunohistochemistry. J Clin Oncol 24: 816-822, 2006. PAX3−FOXO1 and its target genes and the associated 12 Davicioni E, Finckenstein FG, Shahbazian V, Buckley JD, Triche phenotypes of ARMS will be useful in developing TJ and Anderson MJ: Identification of a PAX−FKHR signature that defines molecular classes and determines the prognosis of therapeutic targets as well as diagnostic and/or prognostic alveolar rhabdomyosarcoma. Cancer Res 66: 6936-6946, 2006. molecular markers and characterizing biological properties 13 De Pittà C, Tombolan L, Albiero G, Sartori F, Romualdi C, of PAX3−FOXO1-positive ARMS. Jurman G, Carli M, Furlanello C, Lanfranchi G and Rosolen A: Gene expression profiling identifies potential relevant genes in Acknowledgements alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3−FKHR-positive and -negative tumors. Int J Cancer 118: This research was supported by NIH grants CA64202 (FG Barr) and 2772-27781, 2006. CA104896 (FG Barr). The Author would like to thank Dr. Frederic 14 Lae M, Ahn EH, Mercado GE, Chuai S, Edgar M, Pawel BR, G. Barr for his support and advice; Gabriela E. Mercado for Olshen A, Barr FG and Ladanyi M: Global gene expression technical assistance; Michael B. Lee and Juseong Lee for profiling of PAX−FKHR fusion-positive alveolar and editorial/graphic assistance and Dr. Lawrence A. Loeb for PAX−FOXO1 fusion-negative embryonal . encouragement. J Pathol 212: 143-151, 2007.

2034 Ahn: Regulation of Targets of PAX3−FOXO1 in ARMS

15 Mercado GE, Xia SJ, Zhang C, Ahn EH, Strzelecki DM, Laé M, 29 Tonin PN, Scrable H, Shimada H and Cavenee WK: Muscle- Ladanyi M and Barr FG: Identification of PAX3−FKHR- specific gene expression in rhabdomyosarcomas and stages of regulated genes differentially expressed between alveolar and human fetal skeletal muscle development. Cancer Res 51: 5100- embryonal rhabdomyosarcoma: Focus on MYCN as a 5106, 1991. biologically relevant target. Genes Cancer 47: 30 Tapscott SJ, Thayer MJ and Weintraub H: Deficiency in 510-520, 2008. rhabdomyosarcomas of a factor required for MyoD activity and 16 Barr FG, Smith LM, Lynch JC, Strzelecki D, Parham DM, myogenesis. Science 59: 1450-1453, 1993. Qualman SJ and Breitfeld PP: Examination of gene fusion status 31 Ren YX, Finckenstein FG, Abdueva DA, Shahbazian V, Chung in archival samples of alveolar rhabdomyosarcoma entered on B, Weinberg KI, Triche TJ, Shimada H and Anderson MJ: the Intergroup Rhabdomyosarcoma Study-III trial: A report from Mouse mesenchymal stem cells expressing PAX−FKHR form the Children’s Oncology Group. J Mol Diagn 8: 202-208, 2006. alveolar rhabdomyosarcomas by cooperating with secondary 17 Bennicelli JL, Edwards RH and Barr FG: Mechanism for . Cancer Res 68: 6587-6597, 2008. transcriptional gain of function resulting from chromosomal 32 Graf Finckenstein F, Shahbazian V, Davicioni E, Ren YX and translocation in alveolar rhabdomyosarcoma. Proc Natl Acad Sci Anderson MJ: PAX−FKHR function as pangenes by simultaneously USA 93: 5455-5459, 1996. inducing and inhibiting myogenesis. Oncogene 27: 2004-2014, 18 Tomescu O, Xia SJ, Strezlecki D, Bennicelli JL, Ginsberg J, 2008. Pawel B and Barr FG: Inducible short-term and stable long-term 33 Epstein JA, Lam P, Jepeal L, Maas RL and Shapiro DN: Pax3 cell culture systems reveal that the PAX3-FKHR fusion inhibits myogenic differentiation of cultured myoblast cells. J oncoprotein regulates CXCR4, PAX3, and PAX7 expression. Biol Chem 270: 11719–11722, 1995. Lab Invest 84: 1060-1070, 2004. 34 Ebauer M, Wachtel M, Niggli FK and Schafer BW: Comparative 19 Xia SJ and Barr FG: Analysis of the transforming and growth expression profiling identifies an in vivo target gene signature suppressive activities of the PAX3−FKHR oncoprotein. with TFAP2B as a mediator of the survival function of Oncogene 23: 6864-6871, 2004. PAX3/FKHR. Oncogene 26: 7267-7281, 2007. 20 Xia SJ, Rajput P, Strzelecki DM and Barr FG: Analysis of 35 Kikuchi K, Tsuchiya K, Otabe O, Gotoh T, Tamura S, Katsumi genetic events that modulate the oncogenic and growth Y, Yagyu S, Tsubai-Shimizu S, Miyachi M, Iehara T and Hosoi suppressive activities of the PAX3−FKHR fusion oncoprotein. H: Effects of PAX3-FKHR on malignant phenotypes in alveolar Lab Invest 87: 318-325, 2007. rhabdomyosarcoma. Biochem Biophys Res Commun 365: 568- 21 Littlewood TD, Hancock DC, Danielian PS, Parker MG and 574, 2008. Evan GI: A modified oestrogen receptor ligand-binding domain 36 Collins MH, Zhao H, Womer RB and Barr FG: Proliferative and as an improved switch for the regulation of heterologous apoptotic differences between alveolar rhabdomyosarcoma proteins. Nucleic Acids Res 23: 1686-1690, 1995. subtypes: A comparative study of tumors containing 22 Ahn EH and Schroeder JJ: Sphinganine causes early activation PAX3−FKHR or PAX7−FKHR gene fusions. Med Pediatr Oncol of JNK and p38 and inhibition of AKT in HT-29 human colon 37: 83-89, 2001. cancer cells. Anticancer Res 26: 121-128, 2006. 37 Anderson J, Ramsay A, Gould S and Pritchard-Jones K: 23 Naini S, Etheridge KT, Adam SJ, Qualman SJ, Bentley RC, PAX3−FKHR induces morphological change and enhances Counter CM and Linardic CM: Defining the cooperative genetic cellular proliferation and invasion in rhabdomyosarcoma. Am J changes that temporally drive alveolar rhabdomyosarcoma. Pathol 159: 1089-1096, 2001. Cancer Res 68: 9583-9588, 2008. 38 Bernasconi M, Remppis A, Fredericks WJ, Rauscher FJ 3rd and 24 Xia SJ, Holder DD, Pawel BR, Zhang C and Barr FG: High Schäfer BW: Induction of apoptosis in rhabdomyosarcoma cells expression of the PAX3−FKHR oncoprotein is required to through down-regulation of PAX proteins. Proc Natl Acad Sci promote tumorigenesis of human myoblasts. Am J Pathol 175: USA 93: 13164-13169, 1996. 2600-2608, 2009. 39 Ayyanathan K, Fredericks WJ, Berking C, Herlyn M, 25 Desai KV, Xiao N, Wang W, Gangi L, Greene J, Powell JI, Balakrishnan C, Gunther E and Rauscher FJ 3rd: Hormone- Dickson R, Furth P, Hunter K, Kucherlapati R, Simon R, Liu ET dependent tumor regression in vivo by an inducible and Green JE: Initiating oncogenic event determines gene- transcriptional repressor directed at the PAX3−FKHR oncogene. expression patterns of human breast cancer models. Proc Natl Cancer Res 60: 5803-5814, 2000. Acad Sci USA 99: 6967-6972, 2002. 40 Ahn EH, Mercado GE, Laé M and Ladanyi M: Identification of 26 Cao L, Yu Y, Bilke S, Walker RL, Mayeenuddin LH, Azorsa DO, target genes of PAX3-FOXO1 in alveolar rhabdomyosarcoma. Yang F, Pineda M, Helman LJ and Meltzer PS: Genome-wide Oncology Reports. In press 2013. identification of PAX3−FKHR binding sites in rhabdomyo- sarcoma reveals candidate target genes important for development and cancer. Cancer Res 70: 6497-6508, 2010. 27 Sun J, Kamei CN, Layne MD, Jain MK, Liao JK, Lee ME and Chin MT: Regulation of myogenic terminal differentiation by the hairy-related transcription factor CHF2. J Biol Chem 276: 18591-18596, 2001. 28 Buas MF, Kabak S and Kadesch T: Inhibition of myogenesis by Received February 12, 2013 Notch: Evidence for multiple pathways. J Cell Physiol 218: 84- Revised March 26, 2013 93, 2009. Accepted March 28, 2013

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