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Link to original content: https://pubmed.ncbi.nlm.nih.gov/30190424/
Interleukin-24 (IL24) Is Suppressed by PAX3-FOXO1 and Is a Novel Therapy for Rhabdomyosarcoma - PubMed Skip to main page content
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. 2018 Dec;17(12):2756-2766.
doi: 10.1158/1535-7163.MCT-18-0118. Epub 2018 Sep 6.

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

Alexandra Lacey  1 , Erik Hedrick  1 , Yating Cheng  1 , Kumaravel Mohankumar  1 , Melanie Warren  2 , Stephen Safe  3
Affiliations

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

Alexandra Lacey et al. Mol Cancer Ther. 2018 Dec.

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 RNA-sequencing studies show that PAX3-FOXO1 represses expression of interleukin-24 (IL24), 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 IL24 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 IL24, 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 IL24. Adenoviral-expressed IL24 was directly injected into ARMS tumors in athymic nude mice, and this resulted in decreased tumor growth and weight. Because adenoviral IL24 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.

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Conflict of interest statement

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

Figures

Figure 1.
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.
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.
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.
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.
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.
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.
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.
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