Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy For

Published OnlineFirst September 6, 2018; DOI: 10.1158/1535-7163.MCT-18-0118

Cancer Biology and Translational Studies Molecular Cancer Therapeutics Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma Alexandra Lacey1, Erik Hedrick1, Yating Cheng1, Kumaravel Mohankumar1, Melanie Warren2, and Stephen Safe1

Abstract

Alveolar rhabdomyosarcoma (ARMS) patients have a poor with results reported for this tumor suppressor–like cytokine prognosis, and this is primarily due to overexpression of the in other solid tumors. We also observed in double knockdown oncogenic fusion protein PAX3-FOXO1. Results of RNA- studies that the inhibition of ARMS cell proliferation, survival, sequencing studies show that PAX3-FOXO1 represses expres- and migration after knockdown of PAX3-FOXO1 was signif- sion of interleukin-24 (IL24), and these two genes are inverse- icantly (>75%) reversed by knockdown of IL24. Adenoviral- ly expressed in patient tumors. PAX3-FOXO1 also regulates expressed IL24 was directly injected into ARMS tumors in histone deacetylase 5 (HDAC5) in ARMS cells, and results of athymic nude mice, and this resulted in decreased tumor RNA interference studies conﬁrmed that PAX3-FOXO1– growth and weight. Because adenoviral IL24 has already suc- mediated repression of IL24 is HDAC5-dependent. Knock- cessfully undergone phase I in clinical trials, this represents an down of PAX3-FOXO1 decreases ARMS cell proliferation, alternative approach (alone and/or combination) for treating survival, and migration, and we also observed similar ARMS patients who currently undergo cytotoxic drug thera- responses in cells after overexpression of IL24, consistent pies. Mol Cancer Ther; 17(12); 2756–66. 2018 AACR.

Introduction Results of PAX3-FOXO1 knockdown or overexpression studies in RMS and other cell lines demonstrate the functional impor- Rhabdomyosarcoma (RMS) is primarily observed in children tance of this fusion gene in maintaining the aggressive cancer cell and adolescents and accounts for approximately 5% of all pedi- phenotype, and this is due, in part, to the pro-oncogenic PAX3- atric cancers, with incidence rates of 4.5/106 (1–3). Embryonal FOXO1–regulated genes (10–12). Treatments for RMS patients RMS (ERMS) and alveolar RMS (ARMS) are the two major classes include radiotherapy, surgery, and chemotherapy with cytotoxic of RMS in children and adolescents and differ with respect to their drugs and/or drug combinations of vincristine, dactinomycin, histology, genetics, treatment, and prognosis (3, 4). ERMS cyclophosphamide, irinotecan, ifosfamide, etoposide, doxorubi- accounts for over 60% of RMS patients and is associated with cin, and others. A serious problem exists for RMS patients who loss of heterozygosity at the 11p15 locus (4). ERMS patients have survive current cytotoxic drug therapies, because these individuals a favorable initial prognosis; however, the overall survival of as adults have an increased risk for several diseases (13). patients with metastatic ERMS is only 40% (5). Cytogenetic The orphan nuclear receptor NR4A1 is overexpressed in colon, analysis of tumors from ARMS patients has identiﬁed that 2;13 pancreatic, breast (estrogen receptor–positive and negative), and and 1;13 chromosomal translocations generating PAX3-FOXO1 lung tumors. In breast, colon, and lung tumor patients, high and PAX7-FOXO1 fusion genes, respectively, are highly prevalent expression of NR4A1 predicts decreased survival (14–19). Knock- (55% and 22%, respectively; ref. 6). The PAX3-FOXO1 fusion gene down or overexpression of NR4A1 in cancer cells shows that this is the critical prognostic marker for ARMS patients with metastatic receptor regulates multiple cell-speciﬁc responses including cell disease, with an estimated overall 4-year survival rate of 8% proliferation, survival, cell-cycle progression, migration, and inva- compared with 75% survival rate of patients with PAX7- sion in lung, melanoma, lymphoma, pancreatic, colon, cervical, FOXO1–expressing tumors (7–9). ovarian, and gastric cancer cell lines (11, 14, 17, 19–27). Studies in this laboratory have reported that NR4A1 is also overexpressed in RMS tumors compared with normal muscle tissue (27), and NR4A1 regulated many of the same genes/ 1Department of Veterinary Physiology and Pharmacology, Texas A&M Univer- pathways observed in other solid tumors (16, 19, 20, 27–32). 2 sity, College Station, Texas. Department of Molecular and Cellular Medicine, The NR4A1 ligand 1,1-bis(3-indolyl)-1-(p-hydroxyphenyl)methane Texas A&M Health Science Center, College Station, Texas. (DIM-C-pPhOH), which acts as a receptor antagonist, inhibits Note: Supplementary data for this article are available at Molecular Cancer growth, survival, and migration of RMS cells and also inhibits the Therapeutics Online (http://mct.aacrjournals.org/). NR4A1-regulated PAX3-FOXO1 oncogene and RMS tumor Corresponding Author: Stephen Safe, Texas A&M University, 4466 TAMU, growth in a xenograft mouse model (27, 32). The present study College Station, TX 77843-4466. Phone: 979-845-5988; Fax: 979-862-4929; was initiated after analysis of RNA-seq data showed that knock- E-mail: [email protected] down of NR4A1 or PAX3-FOXO1 or treatment with the NR4A1 doi: 10.1158/1535-7163.MCT-18-0118 antagonist DIM-C-pPhOH resulted in a 2- to 27.9-fold induction 2018 American Association for Cancer Research. of the tumor suppressor–like factor interleukin-24 (IL24). In this

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PAX3-FOXO1 Regulates IL24 in ARMS Cells

study, we show that the oncogenic activity of PAX3-FOXO1 is due, Cell proliferation assay in part, to suppression of IL24, and the anticancer activities Rh30, Rh18, and Rh41 cells were plated in 12-well plates at observed after PAX3-FOXO1 knockdown/suppression are due 1.0 105 and allowed to attach for 24 hours before treatment primarily to induction of IL24. Overexpression of IL24 inhibited with DIM-C-pPhOH, DIM-C-pPhCO2Me, or transfected with ARMS cell growth, survival, and migration, and IL24 (adenoviral) siNR4A1, siPAX3-FOXO1, or siIL24 with DMSO (dimethyl sulf- dramatically inhibited tumor growth in vivo, suggesting that the oxide) as empty vehicle or siCtl siRNA (with lipofectamine clinically approved IL24 adenoviral expression vector potentially vehicle) as controls, respectively. Cells were then trypsinized and (33) represents a promising new approach for ARMS therapy. counted at 24 hours using a Coulter Z1 cell counter.

Materials and Methods Annexin V and immunostaining RMS cells were seeded at 1.0 105 in 2-well Lab-Tek cham- Cell lines, antibodies, chemicals, and other materials bered B#1.0 Borosilicate coverglass slides from Thermo Scientific Rh30 human RMS cancer cells were obtained from the ATCC and were allowed to attach for 24-hour transfection with IL24 and authenticated in 2014 and were maintained at 37 C in the overexpression vector, siPAX3-FOXO1 (100 mmol/L), or siIL24 presence of 5% CO2 in RPMI-1640 medium and supplemented (100 mmol/L) with a control of siCtl (with lipofectamine vehicle) with 10% fetal bovine serum and 5% antibiotic. Rh18 and Rh41 for 72 hours, and Annexin V staining was determined as described cells were received from Texas Tech University Health Sciences (28). Hoechst staining from the apoptotic and necrotic cells assay Center-Children's Oncology Group in 2015. Rh41 and Rh18 cell (Biotium) was used to visualize apoptotic cells. Images were taken fi lines were maintained in Iscove's Modi ed Dulbecco's Medium using an EVOS fluorescence microscopy from Advance Micros- (IMDM) supplemented with 20% fetal bovine serum, 1 ITS copy. Rh30 cells were immunostained for IL24 essentially as m m (5 g/mL insulin, 5 g/mL transferrin, 5 ng/mL selenous acid), described (31). and 5% antibiotic. IMDM was purchased from Thermo Fisher fi Scienti c. RPMI-1640 was purchased from Sigma-Aldrich, and Boyden Chamber assay Lipofectamine 2000 for siRNA transfection was purchased from RMS cells were seeded for 24 hours in a 24-well plate, allowed fi Invitrogen. Apoptotic, necrotic, and Healthy Cells Quanti cation to attach, and subsequently transfected with IL24 overexpression Kit was purchased from Biotium. Cells were subsequently viewed vector, siPAX3-FOXO1 (100 mmol/L), or siIL24 (100 mmol/L) fi using a lter set for FITC, rhodamine, and DAPI on an Advanced with a control of siCtl (nonspecific oligonucleotide). The cells fl fl Microscopy EVOS , uorescence microscope. RGB-4103 GelRed were trypsinized, counted, placed in 24-well 8.0-mm-pore Thin- nucleic acid stain was used in place of Ethedium Bromide from Certs from BD Biosciences, allowed to migrate for 24 hours, fixed Phenix Research Products. SB203580 was purchased from Cell with formaldehyde, and then stained with hematoxylin. Cells that Signaling Technology. The human IL24 cDNA clone in a pCMV-6 migrated through the pores were then counted. vector was purchased from Origene; the HDAC5-flag expression plasmid was obtained from Addgene. The C-DIM compounds Western blots were prepared as previously described (19), and a summary of the Rh30, Rh18, and Rh41 cells were seeded in 6-well plates at antibodies is provided in Supplementary Table S1. 1.0 105 and allowed to attached for 24 hours before treatment with DIM-C-pPhOH, DIM-C-pPhCO2Me, or transfected with Total RNA expression analysis, RNA-seq analysis, and IL24 siNR4A1, siPAX3-FOXO1, or siIL24 with DMSO as empty vehicle overexpression or siCtl siRNA (nonspecific scrambled oligonucleotide; with Patient sample data of total RNA for sample sets were lipofectamine vehicle) as controls, respectively. Cells were treated acquired from the NCBI geo database. Array samples were with C-DIMs or DMSO for 48 hours or transfected with siNR4A1, previously analyzed for quality control, quantile normalized, siPAX3-FOXO1, or siIL24 (all at 100 mmol/L) or siCtl for 72 hours, and multiple probes for each gene were averaged by the and Western blots of whole-cell lysates were determined as submitter. Each sample represented a normalized signal inten- previously described (28). sity and is plotted as relative expression. Additional hybrid- ization and sample processing information can be found at Real-time PCR and chromatin immunoprecipitation (ChIP) https://www.ncbi.nlm.nih.gov/geo/ for NCBI GSE data sets assays and https://www.ebi.ac.uk/arrayexpress/ for the E-TABM- Real-time PCR assays using RMS cell lines transfected with 1202 data set. Relative expression values were listed in JMP oligonucleotides or treated with C-DIMs were carried out using Statistical Software, and a box plot was generated, from which the SYBR Green RT-PCR Kit (Bio-Rad Laboratories) according a t test was performed; significance was determined as a P value to the manufacturer's protocol, and the ChIP assay for deter- less than 0.01 or 0.5, shown by an asterisk. RMS cells were mining changes in pol II was carried out as described (28). treated with DIM-C-pPhOH for 48 hours or transfected with Oligonucleotides and primers used are summarized in Supple- siPAX3-FOXO1 or siNR4A1 for 72 hours, after which RNA was mentary Table S1. extracted and sent to the Texas A&M AgriLife Sequencing Core for preparation, sequencing, and analysis. IL24 cDNA was In vivo RMS Xenograft transfected into RMS cells using Lipofectamine 2000 delivery Female athymic nude mice (6–8 weeks old) were obtained at a concentration of 50 mmol/L before endpoint analysis using (Charles River Laboratory) and maintained under specific path- Western blot, PCR, or Annexin V staining. IL24 and GFP ogen-free conditions, housed in isolated vented cages, and adenoviral vectors were purchased from Vector Biolabs with allowed to acclimate for 1 week with standard chow diet. The a human adenovirus type 5 (dE1/E3) viral backbone under the animals were housed at Texas A&M University in accordance with CMV promoter. the standards of the Guide for the Care and Use of Laboratory

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Figure 1. PAX3-FOXO1 and NR4A1 regulate IL24. A, RNA-seq analysis. Rh30 cells were transfected with siCtl or siNR4A1, siPAX3-FOXO1, or treated with DIM-CpPhOH and induced or repressed mRNAs (compared with siCtl) were determined by RNA-seq, and common upregulated or downregulated genes were determined as outlined in the Materials and Methods. Analysis of IL24 gene expression (B) and IL24/NR4A1 inverse gene expression (C). Patient-derived mRNA was acquired from the NCBI GSE2851 data set, and comparison of IL24 mRNA expression with other genes in normal muscle and ARMS tumors was determined as outlined in the Materials and Methods.

3 2 1 Animals and the Association for Assessment and Accreditation of (mm ) ¼ (L W ) /2, where L is the length and W is the width Laboratory Animal Care (AAALAC). The protocol of the animal of the tumor. study was approved by the Institutional Animal Care and Use Committee (IACUC), Texas A&M University. Rh30 cell lines (4 Statistical analysis 106 cells) grown in RPMI media containing 10% FBS were Results for each treatment group were replicated (at least 3) detached, resuspended in 100 mL of phosphate-buffered saline and expressed a means SE. Statistical comparisons of the treated with matrigel (BD Bioscience) (75:25), and implanted subcuta- groups versus a control for each treatment were determined using neously in the mice. When tumors reached about 200 mm3 size, the Student t test. the mice were randomized into control and treatment groups (4 in vivo animals each). Intratumoral injections of either grade Ad- Results IL24 or Ad-GFP (control) were administered every third day for and a daily dose of 2 108 PFU suspended in 50 mL of PBS for a Changes in gene expression in Rh30 cells after knockdown of total of 5 days. NR4A1 and PAX3-FOXO1 or treatment with DIM-CpPhOH were Tumor volumes and weights and body weight were deter- determined by RNA-seq and comparisons with controls. Figure 1A mined; the tumor size was measured using Vernier calipers, and illustrates the changes in gene expression in the three treatment the tumor volume was estimated by the formula: tumor volume groups and the overlap of common genes that were induced (6) or

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PAX3-FOXO1 Regulates IL24 in ARMS Cells

Figure 2. NR4A1 antagonism or inactivation upregulates IL24. Rh30 (A), Rh41 (B), and Rh18 (C) cells were transfected with siRNA for NR4A1 and treated with the NR4A1 antagonists DIM-C-pPhOH and DIM-C-pPhCO2Me for 24 hours, and whole-cell lysates were analyzed by Western blots as outlined in the Materials and Methods. D, ARMS cells were transfected with siRNA targeted for PAX3-FOXO1, and IL24 was determined by Western blot analysis of whole-cell lysates.

repressed (7). The most highly induced gene, IL24, was induced induced IL24 gene expression in Rh30, Rh18, and Rh41 ARMS 27.9-fold after PAX3-FOXO1 knockdown, and therefore, we cells, and similar results were observed after knockdown of examined the relative expression of IL24 in ARMS tumors versus PAX3-FOXO1 (Fig. 3B). Using primers for two proximal regions normal muscle (Fig. 1B) using publicly available databases (34). of the IL24 promoter, knockdownofPAX3-FOXO1resultedin IL24 levels were significantly higher in normal muscle versus recruitmentofpolII(Fig.3C),andthiswasconsistentwith ARMS (Fig. 1B) and also higher in PAX3-FOXO1–negative versus induction of this gene. ChiPseq results suggest that PAX3- PAX3-FOXO1–positive tumors (Fig. 1B). Furthermore, the FOXO1 does not directly bind promoters (35), and induction expression of IL24 and NR4A1 is inversely related in ARMS and of IL24 may be indirect and related to downregulation of a normal muscle tissue samples (Fig. 1C). IPA analysis of the PAX3-FOXO1–regulated gene such as histone deacetylase 5 changes in gene expression after PAX3-FOXO1 knockdown (HDAC5) (36, 37), which is known to suppress muscle-specific involved changes in multiple pathways as illustrated in Supple- genes (38). Figure 3D shows that knockdown of PAX3-FOXO1 mentary Fig. S1A. decreased HDAC5, and this was also observed in the RNA-seq RNA-seq analysis shows that both NR4A1 and its ligand (DIM- results. Knockdown of HDAC5 by RNAi resulted in induction C-pPhOH), which acts as an NR4A1 antagonist, induce IL24 of IL24 (Fig. 3E) and overexpression of HDAC5 partially mRNA in Rh30 ARMS cells. Transfection of siNR4A1 or treatment reversed induction of IL24 after PAX3-FOXO1 knockdown with DIM-C-pPhOH induced IL24 protein levels in this cell line (Fig. 3F), suggesting that PAX3-FOXO1–mediated repression (Fig. 2A) and induction was also observed after treatment with of IL24 is indirect and due to HDAC5. DIM-C-pPhCO2Me, another NR4A1 ligand that acts as a receptor The anticarcinogenic effect of IL24 in ARMS cells was inves- antagonist (ref. 28; Fig. 2A). A similar approach was used in Rh18 tigated by overexpression studies, and Fig. 4A shows that over- (Fig. 2B) and Rh41 (Fig. 2C and Supplementary Fig. S1B) ARMS expression of IL24 inhibited Rh30, Rh18, and Rh41 cell pro- cells, and both siNR4A1 and NR4A1 ligands induced IL24 expres- liferation. IL24 overexpression induced activation of caspase-3, sion. We also observed that knockdown of PAX3-FOXO1 (siPF) caspase-9, and PARP cleavage in Rh30 (Fig. 4B), Rh18 (Fig. 4C), induced IL24 protein expression (Fig. 2D), and these results are and Rh41 (Fig. 4D) cells and also induced Annexin V staining consistent with the RNA-seq data (Fig. 1A) and confirm that in the three ARMS cell lines (Fig. 4E). IL24 also inhibited PAX3-FOXO1 suppresses IL24 expression in ARMS cells. migration of ARMS cells (Fig. 4F), and these results were similar Figure 3A shows that NR4A1 knockdown or treatment with to those previously observed in ARMS cells transfected with NR4A1 antagonists DIM-C-pPhOH and DIM-C-pPhCO2Me siPAX3-FOXO1 (27, 32). Supplementary Fig. S2 shows by

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Figure 3.

Role of PAX3-FOXO1 in regulation of IL24 in ARMS cells. A, Rh30 cells were treated with DIM-C-pPhCO2Me or DIM-C-pPhOH for 24 hours and (B) transfected with siPAX3-FOXO1 for 72 hours, and RNA extracts were examined by real-time PCR for expression of IL24 mRNA. C, ChIP analysis of the proximal region of the IL24 promoter after PAX3-FOXO1 knockdown was determined essentially as described. Cells were transfected with siPAX3-FOXO1 (D) or siHDAC5 (E), and siPAX3-FOXO1 HDAC5 expression plasmid (F), and whole-cell lysates were analyzed by Western blots as outlined in the Materials and Methods. Results are expressed as means SE for at least 3 separate treatments for each group, and signiﬁcant (P < 0.05) differences from the control group are indicated by an asterisk.

immunostaining of Rh30 cells after transfection with pCMV-6- are p-38 dependent. Figure 5D shows that the p38 inhibitor IL24 that a large percentage of the cells expressed IL24. SB203580 also inhibited these same IL24-induced gene products Previous studies have characterized several other IL24 induced in Rh30 cells, whereas SB203580 alone did not alter expression of responses in diverse cancer cell lines, including increased phos- these genes (Fig. 5E). phorylation of STAT1 and STAT3, activation or induction of Previous studies show that knockdown of PAX3-FOXO1 by stress/survival genes including Bax, PERK, p38, GADD45, and RNAi inhibits ARMS cell growth, survival, and migration (32), GADD34, and downregulation of the survival genes Bcl-2 and and the role of IL24 in mediating these responses was inves- Bcl-XL (39–44). These responses were investigated by overexpres- tigated by transfecting cells with siPAX3-FOXO1 (which sion of IL24 in Rh30 (Fig. 5A), Rh18 (Fig. 5B), and Rh41 (Fig. 5C) induces IL24) alone and in combination with IL24 knockdown cells, and all of the IL24-mediated effects previously reported in by RNAi (siIL24). IL24 knockdown alone had minimal effects other cancer cell lines were also observed in ARMS cell lines. It has on RMS cell growth, apoptosis, and invasion (Supplementary also been reported that IL24 expression downregulates Bcl-2 and Figs. S3A–S3C). Results summarized in Fig. 6A and B show that Bcl-XL and induces Bax, GADD45, and GADD34, and these effects transfection of ARMS cells with siPAX3-FOXO1 inhibited cell

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Figure 4. Anticarcinogenic effect of IL24 overexpression. ARMS cells were transfected with an empty vector control (pCMV-6) or an IL24 expression plasmid, and effects on cell proliferation (A), apoptosis markers (B–D), Annexin V staining (E), and cell invasion (F) (Boyden chamber) were determined as outlined in the Materials and Methods. Results are expressed as means SE for at least 3 separate treatments for each group, and significant (P < 0.05) differences from the control group are indicated by an asterisk. In A and F, the empty vector or oligonucleotide control value was set at 100%, and the value for untreated cells (Ctl) is also shown. growth and migration, whereas in ARMS cells cotransfected were significantly attenuated. The effects of siIL24 alone on with siPAX3-FOXO1 plus siIL24, these inhibitory responses ARMS cell proliferation, invasion, and induction of Annexin V were significantly attenuated. Figure 6C confirms that staining were minimal, and we observed similar effects of siPF siPAX3-FOXO1 resulted in knockdown of PAX3-FOXO1 and alone and in combination with siIL24 on caspase 3/PARP induction of IL24 in Rh30, Rh18, and Rh41 cells, and cotrans- cleavage using a second set (2) of oligonucleotides (Supple- fection with siIL24 reversed these responses. Using the same mentary Fig. S3). treatment protocol, we observed that transfection of siPAX3- We also investigated the effects of direct injection of ade- FOXO1 induced caspase-3, PARP cleavage (Fig. 6D), and noviral IL24 into ARMS tumors (from Rh30 cells) in athymic Annexin V staining (Fig. 6E),andincellscotreatedwith nude mice, and there was a dramatic decrease in tumor growth siPAX3-FOXO1 plus siIL24 these same markers of apoptosis and weight (Fig. 7A and B), and IL24 mRNA and protein were

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Discussion NR4A1 regulates PAX3-FOXO1 gene expression in ARMS cells, and treatment with the NR4A1 antagonist DIM-C-pPhOH downregulated PAX3-FOXO1 expression, resulting in inhibition of ARMS cell and tumor growth. RNA-seq was used to investigate the common and divergent effects of NR4A1 and PAX3-FOXO1 knockdown and DIM-C-pPhOH treatment on expression of genes in Rh30 cells, and we observed induction of 6 and inhibition of 7 genes in common by all 3 treatments (Fig. 1A). The most dramatic response was observed for IL24 mRNA, which was repressed in ARMS tumors (compared with normal muscle) and inversely expressed with PAX3-FOXO1 in ARMS tumors (Fig. 1B and C), but was induced 2.0-, 2.0-, and 27.9-fold after treatment with DIM- C-pPhOH or knockdown or NR4A1 and PAX3-FOXO1, respectively, and these treatments also induced IL24 protein (Figs. 1 and 2). IL24, which is also known as melanoma differentiation- associated gene 7, is a unique tumor suppressor cytokine that is a member of the IL24 subfamily. IL24 activates signaling through the type1and2IL24receptors(IL20R),whichconsistsoftheIL20RA: IL20RB and IL22RA1:IL20RB heterodimeric receptors, respectively (45). The tumor suppressor–like activities of IL24 include inhibition of cancer cell growth and migration, induction of apoptosis, and inhibition of drug resistance, and these activities are observed in several different cancer cell lines through activation/repression of multiple genes/pathways (reviewed in refs. 39–44, 46). PAX3-FOXO1 does not bind directly to gene promoters (35), suggesting a possible indirect mechanism for suppressing IL24. Results of our RNA-seq studies and previous reports identified HDAC5 as a PAX3-FOXO1–regulated gene (36, 37), and previous studies showed a role for HDAC5 as an inhibitor of muscle gene expression in RMS cells (38). Our results confirm expression of HDAC5 protein in Rh30 cells, and the decrease in HDAC5 expression after PAX3-FOXO1 knockdown and the linkage to IL24 induction was confirmed by the effects of HDAC5 knockdown which resulted in the induction of IL24 (Fig. 3). These results are consistent with the scheme illustrated in Fig. 7D. Examination of patient array data (Fig. 1B) coupled with the 27.9-fold induction of IL24 (RNA-seq) observed after knockdown of PAX3-FOXO1 suggested that the oncogenic activity of PAX3- FOXO1 may be due, in part, to suppression of IL24. Therefore, we examined the effects of IL24 overexpression in ARMS cells (Fig. 4) and also determined the relative contributions of IL24 to the tumor suppressor–like activity observed after knockdown of Figure 5. PAX3-FOXO1 (Fig. 6). Overexpression of IL24 in ARMS cells IL24-dependent responses. Rh30 (A), Rh18 (B), and Rh41 (C) cells were inhibited cell growth and migration, activated caspase-dependent transfected with IL24 expression plasmid, and whole-cell lysates were analyzed PARP cleavage and Annexin V staining, confirming that the for expression of survival/apoptotic and stress genes by Western blots as anticarcinogenic activities of IL24 observed in ARMS cells are outlined in the Materials and Methods. Effects of SB203580 plus IL24 (D)and similar to those reported in other solid tumors (39–44, 46). alone (E) on selected IL24-regulated proteins. Cells were treated as Overexpression of IL24 activates or suppresses multiple genes described in A–C addition of SB203580, and whole-cell lysates were analyzed by Western blots. and pathways in cancer cells that contribute to its tumor suppressor–like activity. For example, IL24 induces activation (phosphorylation) of p38 (MAPK) and induces the growth highly expressed in tumors receiving the adenoviral IL24 arrest and DNA damage (GADD)-inducible genes GADD45 (Fig. 7C). Thus, our functional studies on the in vivo and and GADD34, activates STAT1 and STAT3, increases the Bax/ in vitro effects of IL24 were complementary. In summary, Bcl-2 ratio, and activates (phosphorylation) the stress gene results of this study show that inactivation of PAX3-FOXO1 protein kinase R-like endoplasmic reticulum kinase (PERK; results in downregulation of HDAC5, resulting in depression refs. 39–48). We also observed these same IL24-dependent of IL24 expression, which in turn exhibits tumor suppressor– responses in ARMS cells and their inhibition by SB203580 like activity (Fig. 7D) and clinical potential for treating RMS confirmed that p38 activation also plays an important role in patients. mediating the effects of IL24 in ARMS cells (Fig. 5).

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Figure 6. Role of IL24 in mediating PAX3-FOXO1 activity. Cells were transfected with PAX3-FOXO1 alone or in combination with siIL24, and effects on cell growth (A), cell migration (B), knockdown efficiency (C), caspase-3 and PARP cleavage (D), and Annexin V staining (E) were determined as outlined in the Materials and Methods. siIL24 significantly (P < 0.05) reversed the effects of siPF. Results are expressed as means SE for at least 3 separate treatments for each group, and significant (P < 0.05) differences from the control group are indicated by an asterisk. In A and B, the control (Ctl) oligonucleotide treatment was set at 100%.

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Figure 7. Tumor growth inhibition after intratumoral injection of adenoviral IL24. Athymic nude mice bearing Rh30 cells as xenografts were allowed to grow to reach a volume of approximately 200 mm3 and then injected with commercially available adenoviral IL24 daily for 13 days, and effects on tumor volume (A), tumor weight (B), and expression of IL24 mRNA and protein (C) in tumor lysates were determined as outlined in the Materials and Methods. D, Proposed model for regulation of IL24 by PAX3-FOXO1.

Thus, the direct effects of IL24 in ARMS cells are similar to Disclosure of Potential Conflicts of Interest those observed in other cancer cell lines, and the contributions No potential conflicts of interest were disclosed. of IL24 to the tumor suppressor–like activities observed after PAX3-FOXO1 downregulation were further investigated by Authors' Contributions RNAi (Fig. 6). Knockdown of PAX3-FOXO1 by RNA interfer- Conception and design: A. Lacey, E. Hedrick, S. Safe Development of methodology: A. Lacey, E. Hedrick, K. Mohankumar, ence inhibited growth, migration, and induced apoptosis in M. Warren, S. Safe ARMS cells as previously reported (32); however, cotransfec- Acquisition of data (provided animals, acquired and managed patients, tion with siIL24 significantly attenuated the siPAX3-FOXO1– provided facilities, etc.): A. Lacey, E. Hedrick, K. Mohankumar, M. Warren, mediated anticarcinogenic activity. Moreover, the effectiveness S. Safe of IL24 against ARMS tumors was confirmed in in vivo studies Analysis and interpretation of data (e.g., statistical analysis, biostatistics, where adenoviral IL24 administration potently decreased computational analysis): A. Lacey, E. Hedrick, Y. Cheng, K. Mohankumar Writing, review, and/or revision of the manuscript: A. Lacey, E. Hedrick, ARMS tumor growth and weight in an athymic nude mouse K. Mohankumar, S. Safe model (Fig. 7). Administrative, technical, or material support (i.e., reporting or organizing In summary, the results of both in vitro and in vivo studies data, constructing databases): K. Mohankumar, S. Safe demonstrate that PAX3-FOXO1 suppresses expression of IL24 Study supervision: S. Safe via HDAC5, and knockdown of PAX3-FOXO1 or overexpres- Other (performed experiments): K. Mohankumar sion of IL24 demonstrates that this cytokine exhibits a broad spectrum of anticancer activities in ARMS that are similar to Acknowledgments results observed in other solid tumors. The safety of adenoviral- This study was funded by the NIH (P30-ES023512), the Sid Kyle Endowment, Texas AgriLife Research, and the Robert J. Kleberg, Jr. and Helen C. Kleberg delivered IL24 has been reported in a phase I clinical trial in Foundation. patients with advanced tumors, and our results in ARMS cells and tumors suggest that IL24 therapy represents a novel treat- The costs of publication of this article were defrayed in part by the payment of advertisement ment modality for ARMS patients. This approach may be page charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. particularly efficacious for this disease in light of the toxic side effects associated with current therapies for childhood Received January 29, 2018; revised May 8, 2018; accepted August 29, 2018; cancers (13). published first September 6, 2018.

References 1. Paulino AC, Okcu MF. Rhabdomyosarcoma. Curr Probl Cancer 2008; 2. Parham DM, Ellison DA. Rhabdomyosarcomas in adults and children: an 32:7–34. update. Arch Pathol Lab Med 2006;130:1454–65.

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PAX3-FOXO1 Regulates IL24 in ARMS Cells

3. Sebire NJ, Malone M. Myogenin and MyoD1 expression in paediatric 24. Li QX, Ke N, Sundaram R, Wong-Staal F. NR4A1, 2, 3–an orphan nuclear rhabdomyosarcomas. J Clin Pathol 2003;56:412–6. hormone receptor family involved in cell apoptosis and carcinogenesis. 4. Scrable HJ, Witte DP, Lampkin BC, Cavenee WK. Chromosomal localiza- Histol Histopathol 2006;21:533–40. tion of the human rhabdomyosarcoma locus by mitotic recombination 25. Kolluri SK, Bruey-Sedano N, Cao X, Lin B, Lin F, Han YH, et al. Mitogenic mapping. Nature 1987;329:645–7. effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer 5. Breneman JC, Lyden E, Pappo AS, Link MP, Anderson JR, Parham cells. Mol Cell Biol 2003;23:8651–67. DM, et al. Prognostic factors and clinical outcomes in children and 26. Ke N, Claassen G, Yu DH, Albers A, Fan W, Tan P, et al. Nuclear hormone adolescents with metastatic rhabdomyosarcoma–areportfromthe receptor NR4A2 is involved in cell transformation and apoptosis. Cancer Intergroup Rhabdomyosarcoma Study IV. J Clin Oncol 2003;21: Res 2004;64:8208–12. 78–84. 27. Lacey A, Hedrick E, Li X, Patel K, Doddapaneni R, Singh M, et al. Nuclear 6. Sorensen PH, Lynch JC, Qualman SJ, Tirabosco R, Lim JF, Maurer HM, et al. receptor 4A1 (NR4A1) as a drug target for treating rhabdomyosarcoma PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in (RMS). Oncotarget 2016;7:31257–69. alveolar rhabdomyosarcoma: a report from the children's oncology group. 28. Lee SO, Li X, Hedrick E, Jin UH, Tjalkens RB, Backos DS, et al. Diindo- J Clin Oncol 2002;20:2672–9. lylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon 7. Davicioni E, Anderson MJ, Finckenstein FG, Lynch JC, Qualman SJ, cancer cells. Mol Endocrinol 2014;28:1729–39. Shimada H, et al. Molecular classification of rhabdomyosarcoma– 29. Hedrick E, Lee SO, Doddapaneni R, Singh M, Safe S. Nuclear receptor 4A1 genotypic and phenotypic determinants of diagnosis: a report from the as a drug target for breast cancer chemotherapy. Endocr Relat Cancer Children's Oncology Group. Am J Pathol 2009;174:550–64. 2015;22:831–40. 8. Davicioni E, Anderson JR, Buckley JD, Meyer WH, Triche TJ. Gene 30. Hedrick E, Lee SO, Kim G, Abdelrahim M, Jin UH, Safe S, et al. Nuclear expression profiling for survival prediction in pediatric rhabdomyosar- receptor 4A1 (NR4A1) as a drug target for renal cell adenocarcinoma. PLoS comas: a report from the children's oncology group. J Clin Oncol 2010; One 2015;10:e0128308. 28:1240–6. 31. Hedrick E, Lee SO, Doddapaneni R, Singh M, Safe S. NR4A1 antagonists 9. Missiaglia E, Williamson D, Chisholm J, Wirapati P, Pierron G, Petel F, et al. inhibit b1-integrin-dependent breast cancer cell migration. Mol Cell Biol PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in 2016;36:1383–94. rhabdomyosarcoma and significantly improves current risk stratification. 32. Lacey A, Rodrigues-Hoffman A, Safe S. PAX3-FOXO1A expression in J Clin Oncol 2012;30:1670–7. rhabdomyosarcoma is driven by the targetable nuclear receptor NR4A1. 10. Linardic CM. PAX3-FOXO1 fusion gene in rhabdomyosarcoma. Cancer Cancer Res 2017;77:732–41. Lett 2008;270:10–8. 33. Cunningham CC, Chada S, Merritt JA, Tong A, Senzer N, Zhang Y, et al. 11. Ahn EH, Mercado GE, Lae M, Ladanyi M. Identification of target genes of Clinical and local biological effects of an intratumoral injection of mda-7 PAX3-FOXO1 in alveolar rhabdomyosarcoma. Oncol Rep 2013;30: (IL24; INGN 241) in patients with advanced carcinoma: a phase I study. 968–78. Mol Ther 2005;11:149–59. 12. Ahn EH. Regulation of target genes of PAX3-FOXO1 in alveolar rhabdo- 34. Sarver A. Endothelial and alveolar rhabdomyosarcoma mRNA expression. myosarcoma. Anticancer Res 2013;33:2029–35. NCBI GEO database; 2011. 13. Hudson MM, Ness KK, Gurney JG, Mulrooney DA, Chemaitilly W, Krull 35. Gryder BE, Yohe ME, Chou HC, Zhang X, Marques J, Wachtel M, et al. PAX3- KR, et al. Clinical ascertainment of health outcomes among adults treated FOXO1 establishes myogenic super enhancers and confers BET bromo- for childhood cancer. JAMA 2013;309:2371–81. domain vulnerability. Cancer Discov. 2017;7:884–99. 14. Zhou F, Drabsch Y, Dekker TJ, de Vinuesa AG, Li Y, Hawinkels LJ, 36. Davicioni E, Finckenstein FG, Shahbazian V, Buckley JD, Triche TJ, Ander- et al. Nuclear receptor NR4A1 promotes breast cancer invasion and son MJ. Identification of a PAX-FKHR gene expression signature that metastasis by activating TGF-beta signalling. Nat Commun 2014; defines molecular classes and determines the prognosis of alveolar rhab- 5:3388. domyosarcomas. Cancer Res 2006;66:6936–46. 15. Muscat GE, Eriksson NA, Byth K, Loi S, Graham D, Jindal S, et al. Research 37. Marshall AD, Grosveld GC. Alveolar rhabdomyosarcoma–the molecular resource: nuclear receptors as transcriptome: discriminant and prognostic drivers of PAX3/7-FOXO1-induced tumorigenesis. Skelet Muscle value in breast cancer. Mol Endocrinol 2013;27:350–65. 2012;2:25. 16. Lee SO, Andey T, Jin UH, Kim K, Singh M, Safe S. The nuclear receptor TR3 38. Zhang M, Zhu B, Davie J. Alternative splicing of MEF2C pre-mRNA controls regulates mTORC1 signaling in lung cancer cells expressing wild-type p53. its activity in normal myogenesis and promotes tumorigenicity in rhab- Oncogene 2012;31:3265–76. domyosarcoma cells. J Biol Chem 2015;290:310–24. 17. Cho SD, Yoon K, Chintharlapalli S, Abdelrahim M, Lei P, Hamilton S, et al. 39. Chada S, Bocangel D, Ramesh R, Grimm EA, Mumm JB, Mhashilkar Nur77 agonists induce proapoptotic genes and responses in colon AM, et al. mda-7/IL24 kills pancreatic cancer cells by inhibition of cancer cells through nuclear receptor-dependent and nuclear receptor- the Wnt/PI3K signaling pathways: identification of IL-20 receptor- independent pathways. Cancer Res 2007;67:674–83. mediated bystander activity against pancreatic cancer. Mol Ther 18. Wu H, Lin Y, Li W, Sun Z, Gao W, Zhang H, et al. Regulation of Nur77 2005;11:724–33. expression by beta-catenin and its mitogenic effect in colon cancer cells. 40.WangCJ,XiaoCW,YouTG,ZhengYX,GaoW,ZhouZQ,etal. FASEB J 2011;25:192–205. Interferon-alpha enhances antitumor activities of oncolytic adenovi- 19. Lee SO, Abdelrahim M, Yoon K, Chintharlapalli S, Papineni S, Kim rus-mediated IL-24 expression in hepatocellular carcinoma. Mol Cancer K, et al. Inactivation of the orphan nuclear receptor TR3/Nur77 2012;11:31. inhibits pancreatic cancer cell and tumor growth. Cancer Res 2010; 41. Yacoub A, Park MA, Gupta P, Rahmani M, Zhang G, Hamed H, et al. 70:6824–36. Caspase-, cathepsin-, and PERK-dependent regulation of MDA-7/IL-24- 20.LeeSO,JinUH,KangJH,KimSB,GuthrieAS,SreevalsanS,etal.The induced cell killing in primary human glioma cells. Mol Cancer Ther orphan nuclear receptor NR4A1 (Nur77) regulates oxidative and endo- 2008;7:297–313. plasmic reticulum stress in pancreatic cancer cells. Mol Cancer Res 42. Sarkar D, Su ZZ, Lebedeva IV, Sauane M, Gopalkrishnan RV, Valerie 2014;12:527–38. K, et al. mda-7 (IL-24) mediates selective apoptosis in human 21. Smith AG, Lim W, Pearen M, Muscat GE, Sturm RA. Regulation of NR4A melanoma cells by inducing the coordinated overexpression of the nuclear receptor expression by oncogenic BRAF in melanoma cells. GADD family of genes by means of p38 MAPK. PNAS 2002;99: Pigment Cell Melanoma Res 2011;24:551–63. 10054–9. 22. Zhan YY, He JP, Chen HZ, Wang WJ, Cai JC. Orphan receptor TR3 is 43. Su ZZ, Madireddi MT, Lin JJ, Young CS, Kitada S, Reed JC, et al. The cancer essential for the maintenance of stem-like properties in gastric cancer cells. growth suppressor gene mda-7 selectively induces apoptosis in human Cancer Lett 2013;329:37–44. breast cancer cells and inhibits tumor growth in nude mice. PNAS 23. Bras A, Albar JP, Leonardo E, de Buitrago GG, Martinez AC. Ceramide- 1998;95:14400–5. induced cell death is independent of the Fas/Fas ligand pathway and is 44. Chada S, Sutton RB, Ekmekcioglu S, Ellerhorst J, Mumm JB, Leitner WW, prevented by Nur77 overexpression in A20 B cells. Cell Death Differ et al. MDA-7/IL-24 is a unique cytokine–tumor suppressor in the IL-10 2000;7:262–71. family. Int Immunopharmacol 2004;4:649–67.

www.aacrjournals.org Mol Cancer Ther; 17(12) December 2018 2765

Lacey et al.

45. Menezes ME, Bhatia S, Bhoopathi P, Das SK, Emdad L, Dasgupta S, et al. 47. Rutz S, Wang X, Ouyang W. The IL-20 subfamily of cytokines–from MDA-7/IL-24: multifunctional cancer killing cytokine. Adv Exp Med Biol host defence to tissue homeostasis. Nat Rev Immunol 2014;14: 2014;818:127–53. 783–95. 46. Gupta P, Su ZZ, Lebedeva IV, Sarkar D, Sauane M, Emdad L, et al. mda-7/IL- 48. Dash R, Bhoopathi P, Das SK, Sarkar S, Emdad L, Dasgupta S, et al. Novel 24: multifunctional cancer-speciﬁc apoptosis-inducing cytokine. Pharma- mechanism of MDA-7/IL-24 cancer-speciﬁc apoptosis through SARI induc- col Ther 2006;111:596–628. tion. Cancer Res 2014;74:563–74.

2766 Mol Cancer Ther; 17(12) December 2018 Molecular Cancer Therapeutics

Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma

Alexandra Lacey, Erik Hedrick, Yating Cheng, et al.

Mol Cancer Ther 2018;17:2756-2766. Published OnlineFirst September 6, 2018.

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