Nephroblastoma overexpressed (NOV/CCN3) gene: a paired-domain-specific PAX3-FKHR transcription target that promotes survival and motility in alveolar rhabdomyosarcoma cells

Abstract

The CCN (Cy61, CTGF and NOV) family of proteins is a group of matricellular biomolecules involved in both physiological and pathological processes. Elevated expression of the CCN3 (also known as NOV, Nephroblastoma overexpressed) gene has been detected in clinical samples of the skeletal muscle cancer rhabdomyosarcoma, with the highest expression found in the alveolar subtype (aRMS). Over 80% of aRMSs are characterized by a chromosomal translocation-derived fusion transcription factor PAX3-FKHR. In this study, we linked elevated CCN3 levels in aRMS cells to PAX3-FKHR expression. We found reduced CCN3 levels in aRMS cells following small interfering RNA knockdown of PAX3-FKHR, and increased CCN3 levels in C2 myoblasts following ectopic expression of PAX3-FKHR. Promoter, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses confirmed that the CCN3 gene was a direct target for PAX3-FKHR transcriptional activation through a paired-domain DNA sequence in the first intron of the CCN3 gene. To determine the function of CCN3, we showed that knockdown and ectopic expression of CCN3 decreased survival and increased differentiation in aRMS cells, respectively. In addition, we found that exogenously supplied CCN3 protein promoted aRMS cell adhesion, migration and Matrigel invasion. Taken together, data from this study have (1) provided a mechanistic basis for the CCN3 overexpression in aRMS cells, and (2) identified CCN3 as an autocrine/paracrine factor that contributes to the aggressive behavior of aRMS cells, perhaps through a positive feedback loop. Thus, CCN3 may be an attractive target for therapeutic intervention in aRMS.

Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

“…Mutating the PD-binding site in PRO1 and PRO3 not only abolished the reporters’ transactivation by PAX3-FKHR but also decreased their basal activities (65% decrease for PRO1 and 69% decrease for PRO3), possibly because the basal activation of PRO1 and PRO3 is primarily mediated by mechanism independent of PAX3-FKHR. The requirement of an intact PD binding site for PAX3-FKHR–mediated transactivation is consistent with previous reports [5,21,23]. Our data suggest that the promoter region of CPT1A contains the binding sites for PAX3-FKHR, which are required for PAX3-FKHR-mediated transactivation.…”

PAX3-FKHR Regulates the Expression of Pleiotrophin to Mediate Motility in Alveolar Rhabdomyosarcoma Cells

“…2, mutating the PD-binding site of the PTN promoter abolished its transactivation by PAX3-FKHR. The requirement of an intact PD binding site for PAX3-FKHR–mediated transactivation was consistent with previous reports [10–12]. Our data suggest that the promoter of PTN contains a binding site for PAX3-FKHR, which is required for PAX3-FKHR–mediated transactivation.…”

The CCN family acting throughout the body: recent research developments

“…Activation of CCN2 by MMP-3 was reported to occur in two different types of the cells by independent research groups (56,57). For the expression of CCN3 in rhabdomyosarcomas, PAK3-FKHR is the only indicated transcription factor so far (71). Further research on the transcriptional regulation of other CCN members is to be desired.…”