Regulation of Target Genes of PAX3−FOXO1 in Alveolar Rhabdomyosarcoma

ANTICANCER RESEARCH 33: 2029-2036 (2013) Regulation of Target Genes of PAX3−FOXO1 in Alveolar Rhabdomyosarcoma 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 protein 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−forkhead box protein O1 (PAX3−FOXO1) gene the four genes, PAX3−FOXO1, and v-myc 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 gene expression 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 receptor (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 transcription 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 proteins 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.

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