Cell-Based Small-Molecule Compound Screen Identifies Fenretinide as Potential Therapeutic for Translocation-Positive Rhabdomyosarcoma

Abstract

A subset of paediatric sarcomas are characterized by chromosomal translocations encoding specific oncogenic transcription factors. Such fusion proteins represent tumor specific therapeutic targets although so far it has not been possible to directly inhibit their activity by small-molecule compounds. In this study, we hypothesized that screening a small-molecule library might identify already existing drugs that are able to modulate the transcriptional activity of PAX3/FOXO1, the fusion protein specifically found in the pediatric tumor alveolar rhabdomyosarcoma (aRMS). Towards this end, we established a reporter cell line based on the well characterized PAX3/FOXO1 target gene AP2ß. A library enriched in mostly FDA approved drugs was screened using specific luciferase activity as read-out and normalized for cell viability. The most effective inhibitor identified from this screen was Fenretinide. Treatment with this compound resulted in down-regulation of PAX3/FOXO1 mRNA and protein levels as well as in reduced expression of several of its direct target genes, but not of wild-type FOXO1, in a dose- and time-dependent manner. Moreover, fenretinide induced reactive oxygen species and apoptosis as shown by caspase 9 and PARP cleavage and upregulated miR-9. Importantly, it demonstrated a significant anti-tumor effect in vivo. These results are similar to earlier reports for two other pediatric tumors, namely neuroblastoma and Ewing sarcoma, where fenretinide is under clinical development. Our results suggest that fenretinide might represent a novel treatment option also for translocation-positive rhabdomyosarcoma.

Molecular Mechanisms of Apoptosis and Roles in Cancer Development and Treatment

“…In addition, there is evidence that pretreatment of PC-3 cells with the pan-caspase inhibitor Z-VAD-FMK protects cells from troglitazone-and Δ2-TG-activated apoptosis, which verifies the involvement of caspase activation in apoptotic death (Shiau et al, 2005). Furthermore, histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A ) (Duan et al, 2005), synthetic cytotoxic retinoids (e.g., Fenretinide) in a ROS-independent manner (Tiberio et al, 2010;Herrero Martin et al, 2013), cyclin-dependent kinase inhibitors (e.g., Flavopiridol ) (Kitada et al, 2000;Pei et al, 2004;Bose and Grant, 2013), Deubiquitinase inhibitors (e.g., WP1130) are several other molecules that affect gene or protein expression by down-regulating RNA expression in various cancer cells.…”

Pediatric Rhabdomyosarcoma

“…98 Similar efforts have been completed in PAX3-FOXO1 -driven RMS, with interesting results. Using a luciferase reporter placed downstream of the promoter of a PAX3-FOXO1 -induced gene ( TFAP2B ), Martin and colleagues 99 screened a small-molecule library and identified the synthetic retinoid fenretinide as a specific repressor of PAX3-FOXO1 at both the messenger RNA and protein levels, which induced apoptosis of RMS cell lines both in vitro and in vivo. A second drug screen using a similar approach identified that fascaplysin, an inhibitor of CDK4 , worked by abrogating the phosphorylation and subsequent subcellular localization of PAX3-FOXO1 .…”

“…A second drug screen using a similar approach identified that fascaplysin, an inhibitor of CDK4 , worked by abrogating the phosphorylation and subsequent subcellular localization of PAX3-FOXO1 . 100 …”

The Importance of Being Dead: Cell Death Mechanisms Assessment in Anti-Sarcoma Therapy

“…Betulinic acid is able to target the mitochondria in ES, promoting the permeabilization of the outer membrane resulting in the release, from the mitochondria to the cytosol, of soluble factors such as AIF and cytochrome c , who ultimately leads to caspase activation ( 53 ). Direct targeting of mitochondrial physiology was also explored in RMS with photodynamic therapy ( 54 ) and ROS-generation agents ( 55 ). Proteasome inhibitors as Bortezomib generate a major stress in the cell machinery, triggering a number of different reactions, many of them aimed to induce apoptosis.…”