Interleukin-24 (Il-24) Is Suppressed by Pax3-Foxo1 and Is a Novel

Author Manuscript Published OnlineFirst on September 6, 2018; DOI: 10.1158/1535-7163.MCT-18-0118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

INTERLEUKIN-24 (IL-24) 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

1 Department of Veterinary Physiology and Pharmacology Texas A&M University College Station, TX 77843

2 Department of Molecular and Cellular Medicine Texas A&M Health Science Center College Station, TX 77843

Running Title: PAX3-FOXO1 regulates IL-24 in ARMS cells

Keywords: ARMS, PAX3-FOXO1, IL-24, tumor inhibition

Funding: Funding was provided by the National Institutes of Health (P30-ES023512), the Sid Kyle Endowment, and Texas AgriLife Research.

To whom correspondence should be addressed: Stephen Safe Department of Veterinary Physiology and Pharmacology Texas A&M University 4466 TAMU College Station, TX 77843-4466 Tel: 979-845-5988 / Fax: 979-862-4929 Email: [email protected]

Conflict of Interest: There are no conflicts of interest to declare.

Word Count: 4535 words Figures: 7 figures Table: 0 tables

Downloaded from mct.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 6, 2018; DOI: 10.1158/1535-7163.MCT-18-0118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

Alveolar Rhabdomyosarcoma (ARMS) patients have a poor prognosis and this is primarily due to overexpression of the oncogenic fusion protein PAX3-FOXO1. Results of RNASeq studies show that PAX3-FOXO1 represses expression of interleukin-24 (IL-

24) and these two genes are inversely expressed in patient tumors. PAX3-FOXO1 also regulates histone deacetylase 5 (HDAC5) in ARMS cells and results of RNA interference studies confirmed that PAX3-FOXO1-mediated repression of IL-24 is

HDAC5-dependent. Knockdown of PAX3-FOXO1 decreases ARMS cell proliferation, survival, and migration and we also observed similar responses in cells after overexpression of IL-24 consistent with results reported for this tumor suppressor-like cytokine in other solid tumors. We also observed in double knockdown studies that the inhibition of ARMS cell proliferation, survival, and migration after knockdown of PAX3-

FOXO1 was significantly (> 75%) reversed by knockdown of IL-24. Adenoviral- expressed IL-24 was directly injected into ARMS tumors in athymic nude mice and this resulted in decreased tumor growth and weight. Since adenoviral IL-24 has already successfully undergone Phase I in clinical trials, this represents an alternative approach

(alone and/or combination) for treating ARMS patients who currently undergo cytotoxic drug therapies.

INTRODUCTION

Rhabdomyosarcoma (RMS) is primarily observed in children and adolescents

and accounts for approximately 5% of all pediatric cancers with incidence rates of

4.5/106 (1-3) . Embryonal RMS (ERMS) and alveolar RMS (ARMS) are the two major

classes of RMS in children and adolescents and differ with respect to their histology,

genetics, treatment, and prognosis (3,4). ERMS accounts for over 60% of RMS patients

and is associated with loss of heterozygosity at the 11p15 locus (4). ERMS patients

have a favorable initial prognosis; however, the overall survival of patients with

metastatic ERMS is only 40% (5). Cytogenetic analysis of tumors from ARMS patients

have identified that 2;13 and 1;13 chromosomal translocations generating PAX3-

FOXO1 and PAX7-FOXO1 fusion genes, respectively, are highly prevalent (55 and

22%, respectively) (6). The PAX3-FOXO1 fusion gene is the critical prognostic marker

for ARMS patients with metastatic disease, with an estimated overall 4 year survival of

8% compared to 75% survival rate of patients with PAX7-FOXO1-expressing tumors (7-

9).

Results of PAX3-FOXO1 knockdown or overexpression studies in RMS and

other cell lines demonstrate the functional importance of this fusion gene in maintaining

the aggressive cancer cell phenotype and this is due, in part, to the pro-oncogenic

PAX3-FOXO1-regulated genes (10-12). Treatments for RMS patients include radiotherapy, surgery, and chemotherapy with cytotoxic drugs and/or drug combinations of vincristine, dactinomycin, cyclophosphamide, irinotecan, fosfamide, etoposide, doxorubicin, and others. A serious problem exists for RMS patients that survive current

cytotoxic drug therapies, since these individuals as adults have an increased risk for

several diseases (13).

The orphan nuclear receptor NR4A1 is overexpressed in colon, pancreatic,

breast (estrogen receptor positive and negative), and lung tumors. In breast, colon, and

lung tumor patients, high expression of NR4A1 predicts decreased survival (14-19).

Knockdown or overexpression of NR4A1 in cancer cells show that this receptor regulates multiple cell-specific responses including cell proliferation, survival, cell cycle progression, migration, and invasion in lung, melanoma, lymphoma, pancreatic, colon, cervical, 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 to normal muscle tissue (27) and NR4A1 regulated many of the

same genes/pathways observed in other solid tumors (16,19,20,27-32). The NR4A1

ligand 1,1-bis(3-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) which acts as a

receptor antagonist inhibits growth, survival, and migration of RMS cells and also

inhibits the NR4A1-regulated PAX3-FOXO1 oncogene and RMS tumor growth in a

xenograft mouse model (27,32). The present study was initiated after analysis of

RNASeq data showed that knockdown of NR4A1 or PAX3-FOXO1 or treatment with the

NR4A1 antagonist DIM-C-pPHOH resulted in a 2- to 27.9-fold induction of the tumor

suppressor-like factor interleukin-24 (IL-24). In this study, we show that the oncogenic

activity of PAX3-FOXO1 is due, in part to suppression of IL-24 and the anticancer

activities observed after PAX3-FOXO1 knockdown/suppression are due primarily to

induction of IL-24. Overexpression of IL-24 inhibited ARMS cell growth, survival, and migration, and IL-24 (adenoviral) dramatically inhibited tumor growth in vivo suggesting

that the clinically approved IL-24 adenoviral expression vector potentially (33)

represents a promising new approach for ARMS therapy.

MATERIALS AND METHODS

Cell lines, antibodies, chemicals, and other materials. Rh30 human RMS cancer

cells were obtained from the American Type Culture Collection and authenticated in

2014 (Manassas, VA) and were maintained at 37C in the presence of 5% CO2 in

RPMI-1640 Medium and supplemented with 10% fetal bovine serum and 5% antibiotic.

Rh18 and Rh41 cells were received from Texas Tech University Health Sciences

Center-Children’s Oncology Group in 2015. Rh41 and Rh18 cell lines were maintained

in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 20% fetal bovine

serum, 1X ITS (5 g/mL insulin, 5 g/mL transferrin, 5 ng/mL selenous acid), and 5% antibiotic. IMDM was purchased from ThermoFisher Scientific (Waltham, MA). RPMI-

1640 was purchased from Sigma-Aldrich (St. Louis, MO), and Lipofectamine 2000 for siRNA transfection was purchased from Invitrogen (Grand Island, NY). Apoptotic,

Necrotic, and Healthy Cells Quantification Kit was purchased from Biotium (Hayward,

CA). Cells were subsequently viewed using a filter set for FITC, rhodamine, and DAPI on an Advanced Microscopy EVOS fl, fluorescence microscope. RGB-4103 GelRed nucleic acid stain was used in place of Ethedium Bromide from Phenix Research

Products (Candler, NC). SB203580 was purchased from Cell Signaling Technology

(Danvers, MA). The human IL-24 cDNA clone in a pCMV-6 vector was purchased from

Origene (Rockville, MD); the HDAC5-flag expression plasmid was obtained from

Addgene (Cambridge, MA). The C-DIM compounds were prepared as previously described (19) and a summary of the antibodies are provided in Supplemental Table 1.

Total RNA expression analysis, RNASeq analysis, and IL-24 overexpression.

Patient sample data of total RNA for sample sets was acquired from NCBI geo database. Array samples were previously analyzed for quality control, quantile normalized, and multiple probes for each gene were averaged by the submitter. Each sample represented a normalized signal intensity and are plotted as relative expression.

Additional hybridization and sample processing information can be found at https://www.ncbi.nlm.nih.gov/geo/ for NCBI GSE datasets and https://www.ebi/ac/uk/arrayexpress/ for E-TABM-1202 dataset. Relative expression values were listed in JMP Statistical Software and a box plot was generated, from which a t-test was performed; significance was determined as a p-value less than 0.01 or 0.5, shown by an asterick. RMS cells were treated with DIM-C-pPhOH for 48 hr or transfected with siPAX3-FOXO1 or siNR4A1 for 72 hr after which RNA was extracted and sent to the Texas A&M AgriLife Sequencing Core for preparation, sequencing, and analysis. IL-24 cDNA was transfected into RMS cells using lipofectamine 2000 delivery at a concentration of 50 M before endpoint analysis using western blot, PCR, or

Annexin V staining. IL-24 and GFP adenoviral vectors were purchased from Vector

Biolabs (Malvern, PA) with a human adenovirus Type 5 (dE1/E3) viral backbone under the CMV promoter.

Cell proliferation assay. Rh30, Rh18, and Rh41 cells were plated in 12-well plates at 1.0 x 105 and allowed to attach for 24 hr before treatment with DIM-C-pPhOH,

DIM-C-pPhCO2Me, or transfected with siNR4A1, siPAX3-FOXO1, or siIL-24 with DMSO

(dimethyl sulfoxide) as empty vehicle or siCtl siRNA (with lipofectamine vehicle) as

controls, respectively. Cells were then trypsinized and counted at 24 hr using a Coulter

Z1 cell counter

Annexin V and immunostaining. RMS cells were seeded at 1.0 x 105 in 2-well

Lab-Tek chambered B#1.0 Borosilicate coverglass slides from Thermo Scientific and

were allowed to attach for 24 hr transfection with IL-24 overexpression vector, siPAX3-

FOXO1 (100 M), or siIL-24 (100 M) with a control of siCtl (with lipofectamine vehicle)

for 72 hr, and Annexin V staining was determined as described (28). Hoechst staining

from the apoptotic and necrotic cells assay (Biotium, Hayward, CA) was used to

visualize apoptotic cells. Images were taken using an EVOS fluorescence microscopy

from Advance Microscopy. Rh30 cells were immunostained for IL-24 essentially as

described (31).

Boyden Chamber assay. RMS cells were seeded for 24 hr in a 24-well plate,

allowed to attach, and subsequently transfected with IL-24 overexpression vector,

siPAX3-FOXO1 (100 M), or siIL-24 (100 M) with a control of siCtl (non-specific

oligonucleotide). The cells were trypsinized, counted, placed in 24-well 8.0-m-pore

ThinCerts from BD Biosciences (Bedford, MA), allowed to migrate for 24 hr, fixed with

formaldehyde, and then stained with hematoxylin. Cells that migrated through the pores

were then counted.

Western blots. Rh30, Rh18, and Rh41 cells were seeded in 6-well plates at 1.0 x

105 and allowed to attached for 24 hr before treatment with DIM-C-pPhOH, DIM-C- pPhCO2Me, or transfected with siNR4A1, siPAX3-FOXO1, or siIL-24 with DMSO as

empty vehicle or siCtl siRNA (non-specific scrambled oligonucleotide) (with

lipofectamine vehicle) as controls, respectively. Cells were treated with C-DIMs or

DMSO for 48 hr or transfected with siNR4A1, siPAX3-FOXO1, or siIL-24 (all at 100 M) or siCtl for 72 hr, and Western blots of whole cell lysates were determined as previously described (28).

Real-time PCR, and chromatin immunoprecipitation (ChIP) assays. Real time

PCR assays using RMS cell lines transfected with oligonucleotides or treated with C-

DIMs were carried out using the SYBR Green RT-PCR Kit (Bio-rad Laboratories,

Hercules, CA) according to the manufacturer's protocol and the ChIP assay for

determining changes in pol II was carried out as described (28). Oligonucleotides and

primers used are summarized in Supplemental Table S1.

In vivo RMS Xenograft. Female athymic nude mice (6-8 weeks old) were

obtained (Charles River Laboratory, Wilmington, MA) and maintained under specific

pathogen-free conditions, housed in isolated vented cages, and allowed to acclimate for

one week with standard chow diet. The animals were housed at Texas A&M University

in accordance with the standards of the Guide for the Care and Use of Laboratory

Animals and the Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC). The protocol of the animal study was approved by the Institutional

Animal Care and Use Committee (IACUC), Texas A&M University. Rh30 cell lines

(4x106 cells) grown in RPMI media containing 10% FBS were detached, resuspended in

100 l of phosphate-buffered saline with matrigel (BD Bioscience, Bedford, MA) (75:25),

and implanted subcutaneously in the mice. When tumors reached about 200 mm3 size,

the mice were randomized into control and treatment groups (4 animals each).

Intratumoral injections of either in vivo grade Ad-IL24 or Ad-GFP (control) were

administered every third day for and a daily dose of 2x108 PFU suspended in 50 l of

PBS for a total of 5 days.

Tumor volumes and weights, and body weight were determined; the tumor size was measured using Vernier calipers, and the tumor volume was estimated by the formula: tumor volume (mm3) = (L x W2) x ½, where L is the length and W is the width of the tumor.

Statistics. Results for each treatment group were replicated (at least 3X) and expressed a means  SE. Statistical comparisons of the treated groups vs. a control for each treatment were determined using Student's t-test.

RESULTS

Changes in gene expression in Rh30 cells after knockdown of NR4A1 and PAX3-

FOXO1 or treatment with DIM-CpPhOH were determined by RNASeq and comparisons

with controls. Figure 1A illustrates the changes in gene expression in the three

treatment groups and the overlap of common genes that were induced (6) or repressed

(7). The most highly induced gene, IL-24, was induced 27.9-fold after PAX3-FOXO1 knockdown and therefore, we examined the relative expression of IL-24 in ARMS tumors vs. normal muscle (Fig. 1B) using publically available databases (34). IL-24 levels were significantly higher in normal muscle vs. ARMS (Fig. 1B) and also higher in

PAX3-FOXO1 negative vs. PAX3-FOXO1 positive tumors (Fig. 1B). Furthermore, the

expression of IL-24 and NR4A1 are inversely related in ARMS and normal muscle

tissue samples (Fig. 1C). IPA analysis of the changes in gene expression after PAX3-

FOXO1 knockdown involved changes in multiple pathways as illustrated in

Supplemental Figure 1A.

RNASeq analysis shows that both NR4A1 and its ligand (DIM-C-pPhOH), which

acts as an NR4A1 antagonist, induce IL-24 mRNA in Rh30 ARMS cells. Transfection of siNR4A1 or treatment with DIM-C-pPhOH induced IL-24 protein levels in this cell line

(Fig. 2A) and induction was also observed after treatment with DIM-C-pPhCO2Me, another NR4A1 ligand that acts as a receptor antagonist (28) (Fig. 2A). A similar

approach was used in Rh18 (Fig. 2B) and Rh41 (Fig. 2C and Suppl. Fig. 1B) ARMS

cells, and both siNR4A1 and NR4A1 ligands induced IL-24 expression. We also

observed that knockdown of PAX3-FOXO1 (siPF) induced IL-24 protein expression

(Fig. 2D), and these results are consistent with the RNASeq data (Fig. 1A) and confirm

that PAX3-FOXO1 suppresses IL-24 expression in ARMS cells.

Figure 3A shows that NR4A1 knockdown or treatment with NR4A1 antagonists

DIM-C-pPhOH and DIM-C-pPhCO2Me induced IL-24 gene expression in Rh30, Rh18,

and Rh41 ARMS cells and similar results were observed after knockdown of PAX3-

FOXO1 (Fig. 3B). Using primers for 2 proximal regions of the IL-24 promoter,

knockdown of PAX3-FOXO1 resulted in recruitment of pol II (Fig. 3C) and this was

consistent with induction of this gene. ChiPseq results suggest that PAX3-FOXO1 does

not directly bind promoters (35) and induction of IL-24 may be indirect and related to

downregulation of a PAX3-FOXO1-regulated gene such as histone deacetylase 5

(HDAC5) (36,37) which is known to suppress muscle-specific genes (38). Figure 3D

shows that knockdown of PAX3-FOXO1 decreased HDAC5 and this was also observed

in the RNASeq results. Knockdown of HDAC5 by RNAi resulted in induction of IL-24

(Fig. 3E) and overexpression of HDAC5 partially reversed induction of IL-24 after PAX3-

FOXO1 knockdown (Fig. 3F), suggesting that PAX3-FOXO1-mediated repression of IL-

24 is indirect and due to HDAC5.

The anticarcinogenic effect of IL-24 in ARMS cells was investigated by

overexpression studies, and Figure 4A shows that overexpression of IL-24 inhibited

Rh30, Rh18, and Rh41 cell proliferation. IL-24 overexpression induced activation of caspase-3, caspase-9 and PARP cleavage in Rh30 (Fig. 4B), Rh18 (Fig. 4C), and Rh41

(Fig. 4D) cells and also induced Annexin V staining in the 3 ARMS cell lines (Fig. 4E).

IL-24 also inhibited migration of ARMS cells (Fig. 4F) and these results were similar to those previously observed in ARMS cells transfected siPAX3-FOXO1 (27,32).

Supplemental Figure 2 shows by immunostaining of Rh30 cells after transfection with

pCMV-6-IL-24 that a large percentage of the cells expressed IL-24.

Previous studies have characterized several other IL-24 induced responses in

diverse cancer cell lines, including increased phosphorylation of STAT1 and STAT3,

activation or induction of stress/survival genes including Bax, PERK, p38, GADD45, and

GADD34, and downregulation of survival genes Bcl-2 and Bcl-XL (39-44). These

responses were investigated by overexpression of IL-24 in Rh30 (Fig. 5A), Rh18 (Fig.

5B), and Rh41 (Fig. 5C) cells, and all of the IL-24 mediated effects previously reported

in other cancer cell lines were also observed in ARMS cell lines. It has also been

reported that IL-24 expression downregulates Bcl-2 and Bcl-XL and induces Bax,

GADD45, and GADD34 and these effects are p-38 dependent. Figure 5D shows that

the p38 inhibitor SB203580 also inhibited these same IL-24-induced gene products in

Rh30 cells, whereas SB203580 alone did not alter expression of these genes (Fig. 5E).

Previous studies show that knockdown of PAX3-FOXO1 by RNAi inhibits ARMS

cell growth, survival and migration (32), and the role of IL-24 in mediating these

responses was investigated by transfecting cells with siPAX3-FOXO1 (which induces

IL-24) alone and in combination with IL-24 knockdown by RNAi (siIL-24). IL-24

knockdown alone had minimal effects on RMS cell growth, apoptosis and invasion

(Suppl. Figs. 3A-3C). Results summarized in Figures 6A and 6B show that transfection

of ARMS cells with siPAX3-FOXO1 inhibited cell growth and migration, whereas in

ARMS cells cotransfected with siPAX3-FOXO1 plus siIL-24, these inhibitory responses were significantly attenuated. Figure 6C confirms that siPAX3-FOXO1 resulted in

knockdown of PAX3-FOXO1 and induction of IL-24 in Rh30, Rh18, and Rh41 cells and

cotransfection with siIL-24 reversed these responses. Using the same treatment

protocol, we observed that transfection of siPAX3-FOXO1 induced caspase-3, PARP

cleavage (Fig. 6D), and Annexin V staining (Fig. 6E), and in cells cotreated with

siPAX3-FOXO1 plus siIL-24 these same markers of apoptosis were significantly

attenuated. The effects of siIL-24 alone on ARMS cell proliferation, invasion and

induction of Annexin V staining were minimal, and we observed similar effects of siPF

alone and in combination with siIL-24 on caspase 3/PARP cleavage using a second set

(2) of oligonucleotides (Suppl. Fig. 3).

We also investigated the effects of direct injection of adenoviral IL-24 into ARMS

tumors (from Rh30 cells) in athymic nude mice and there was a dramatic decrease in

tumor growth and weight (Figs. 7A and 7B), and IL-24 mRNA and protein were highly

expressed in tumors receiving the adenoviral IL-24 (Fig.7C). Thus, our functional

studies on the in vivo and in vitro effects of IL-24 were complementary. In summary

results of this study show that inactivation of PAX3-FOXO1 results in downregulation of

HDAC5, resulting in depression of IL-24 expression which in turn exhibits tumor

suppressor-like activity (Fig. 7D) and clinical potential for treating RMS patients.

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. RNASeq 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 IL-24 mRNA, which was repressed in ARMS tumors

(compared to normal muscle) and inversely expressed with PAX3-FOXO1 in ARMS

tumors (Figs. 1B and 1C), 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 IL-24 protein (Figs. 1 and 2).

IL-24, which is also known as melanoma differentiation associated gene 7, is a

unique tumor suppressor cytokine that is a member of the IL-24 subfamily. IL-24 activates signaling through the type 1 and type 2 IL-24 receptors (IL-20R), which consists of the IL20RA:IL-20RB and IL-22RA1:IL20RB heterodimeric receptors,

respectively (45). The tumor suppressor-like activities of IL-24 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 [rev. in (39-44,46)].

PAX3-FOXO1 does not bind directly to gene promoters (35), suggesting a

possible indirect mechanism for suppressing IL-24. Results of our RNASeq 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 IL-24

induction was confirmed by the effects of HDAC5 knockdown which resulted in the

induction of IL-24 (Fig. 3). These results are consistent with the scheme illustrated in

Figure 7D. Examination of patient array data (Fig. 1B) coupled with the 27.9-fold

induction of IL-24 (RNASeq) observed after knockdown of PAX3-FOXO1 suggested that

the oncogenic activity of PAX3-FOXO1 may be due, in part, to suppression of IL-24.

Therefore we examined the effects of IL-24 overexpression in ARMS cells (Fig. 4) and

also determined the relative contributions of IL-24 to the tumor suppressor like activity

observed after knockdown of PAX3-FOXO1 (Fig. 6). Overexpression of IL-24 in ARMS

cells inhibited cell growth and migration, activated caspase-dependent PARP cleavage

and Annexin V staining, confirming that the anticarcinogenic activities of IL-24 observed

in ARMS cells are similar to those reported in other solid tumors (39-44,46).

Overexpression of IL-24 activates or suppresses multiple genes and pathways in cancer

cells that contribute to its tumor suppressor like activity. For example, IL-24 induces

activation (phosphorylation) of p38 (MAPK) and induces the growth arrest and DNA

damage (GADD)-inducible genes GADD45 and GADD34, activates STAT1 and STAT3,

increases the Bax/Bcl-2 ratio, and activates (phosphorylation) the stress gene protein

kinase R-like endoplasmic reticulum kinase (PERK) (39-48). We also observed these

same IL-24-dependent responses in ARMS cells and their inhibition by SB203580

confirmed that p38 activation also plays an important role in mediating the effects of IL-

24 in ARMS cells (Fig. 5).

Thus, the direct effects of IL-24 in ARMS cells are similar to those observed in other cancer cell lines and the contributions of IL-24 to the tumor suppressor-like activities observed after PAX3-FOXO1 downregulation were further investigated by

RNAi (Fig. 6). Knockdown of PAX3-FOXO1 by RNA interference inhibited growth, migration, and induced apoptosis in ARMS cells as previously reported (32); however,

cotransfection with siIL-24 significantly attenuated the siPAX3-FOXO1-mediated

anticarcinogenic activity. Moreover, the effectiveness of IL-24 against ARMS tumors

was confirmed in in vivo studies where adenoviral IL-24 administration potently

decreased ARMS tumor growth and weight in an athymic nude mouse model (Fig. 7).

In summary, the results of both in vitro and in vivo studies demonstrate that

PAX3-FOXO1 suppresses expression IL-24 via HDAC5, and knockdown of PAX3-

FOXO1 or overexpression of IL-24 demonstrates that this cytokine exhibits a broad spectrum of anticancer activities in ARMS that are similar to results observed in other solid tumors. The safety of adenoviral-delivered IL-24 has been reported in a Phase I clinical trial in patients with advanced tumors, and our results in ARMS cells and tumors suggest that IL-24 therapy represents a novel treatment modality for ARMS patients.

This approach may be particularly efficacious for this disease in light of the toxic-side effects associated with current therapies for childhood cancers (13).

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FIGURE CAPTIONS

Figure 1. PAX3-FOXO1 and NR4A1 regulate IL-24. (A) RNASeq analysis. Rh30 cells were transfected with siCtl or siNR4A1, siPAX3-FOXO1 or treated with DIM-CpPhOH and induced or repressed mRNAs (compared to siCtl) were determined by RNASeq and common up- or downregulated genes were determined as outlined in the Material and

Methods. Analysis of IL-24 gene expression (B) and IL-24/NR4A1 inverse gene expression (C). Patient-derived mRNA was acquired from the NCBI GSE2851 dataset and comparison of IL-24 mRNA expression with other genes in normal muscle and

ARMS tumors was determined as outlined in the Materials and Methods.

Figure 2. NR4A1 antagonism or inactivation upregulates IL-24. 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 hr 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 IL-24 was determined by western blot analysis of whole cell lysates.

Figure 3. Role of PAX3-FOXO1 in regulation of IL-24 in ARMS cells. (A) Rh30 cells were treated with DIM-C-pPhCO2Me or DIM-C-pPhOH for 24 hr and (B) transfected with siPAX3-FOXO1 for 72 hr, and RNA extracts were examined by real time PCR for expression of IL-24 mRNA. (C) ChIP analysis of the proximal region of the IL-24 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 < .05) differences from the control group are indicated (*).

Figure 4. Anticarcinogenic effect of IL-24 overexpression. ARMS cells were transfected with an empty vector control (pCMV-6) or an IL-24 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 < .05) differences from the control group are indicated (*).

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.

Figure 5. IL-24 dependent responses. Rh30 (A), Rh18 (B), and Rh41 (C) cells were transfected with IL-24 expression plasmid and whole cell lysates were analyzed for expression of survival/apoptotic and stress genes by western blots as outlined in the

Materials and Methods. Effects of SB203580 plus IL-24 (D) and alone (E) on selected

IL-24-regulated proteins. Cells were treated as described in (A)-(C)  addition of

SB203580, and whole cell lysates were analyzed by western blots.

Figure 6. Role of IL-24 in mediating PAX3-FOXO1 activity. Cells were transfected with PAX3-FOXO1 alone or in combination with siIL-24 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. siIL-

24 significantly (p<0.05) reverse the effects of siPF. Results are expressed as means 

SE for at least 3 separate treatments for each group and significant (p < .05) differences

from the control group are indicated (*). In (A) and (B), the control (Ctl) oligonucleotide

treatment was set at 100%.

Figure 7. Tumor growth inhibition after intratumoral injection of adenoviral IL-24.

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 IL-24 daily for 13 days, and effects on tumor volume (A), tumor weight (B) and expression of IL-24 mRNA and protein (C) in tumor lysates were determined as

outlined in the Materials and Methods. (D) Proposed model for regulation of IL-24 by

PAX3-FOXO1.

Interleukin-24 (IL-24) Is Suppressed By PAX3-FOXO1 and Is A Novel Therapy For Rhabdomyosarcoma

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

Mol Cancer Ther Published OnlineFirst September 6, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-0118

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2018/09/06/1535-7163.MCT-18-0118.DC1

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