Studies of amplification in rhabdomyosarcoma

Division Of Basic Sciences - Nci

Investigators

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Abstract

In our early studies of gene ampli?cation in fusion-positive (FP) rhabdomyosarcoma (RMS), we identi?ed MIR17HG as a key target of the 13q31 amplicon. MIR17HG is a "host gene" in which six microRNAs (miRNAs) (including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1) are processed from the RNA transcribed from an intronic region. We determined that there is elevated expression of these miRNAs in FP RMS tumors with 13q31 ampli?cation as well as in a subset of FP tumors without this ampli?cation. In our studies of FP RMS cell lines, we found variable expression of these miRNAs consistent with the premise that a subset of our FP RMS lines express high levels of these miRNAs and are similar to FP RMS tumors with 13q31 amplicons. In more recent studies of both FP RMS cell lines and our human myoblast model system, we identi?ed a synergistic effect between P3F and MYCN in the upregulation of these miRNAs. To investigate the transcriptional regulation of MIR17HG by P3F in FP-RMS, we analyzed chromatin immunoprecipitation sequencing datasets from Rh4 FP-RMS cells and myoblasts with an inducible P3F construct. These analyses revealed a prominent P3F-bound DNA element in the Rh4 cells located 1.84 Mb upstream of the MIR17HG locus. This same site also appears 24 hours following induction of P3F expression in myoblasts. There were no other P3F binding sites detected between this element and the MIR17HG gene in both cell lines. To assess the functional significance of this distal binding site, we utilized a CRISPR/Cas9-mediated strategy to delete this site in a myoblast line transduced with inducible P3F and constitutive MYCN constructs. After subcloning the treated myoblasts, this deletion was identified in two subclones and was validated by Sanger sequencing of PCR products. Western blot analysis confirmed that P3F and MYCN protein levels remained unchanged following deletion, indicating that the observed effects were not due to loss of these oncogenic transcription factors. Expression analysis by qPCR demonstrated a significant reduction in MIR17HG transcript levels in the deleted subclones compared to parental and undeleted subclones. These findings suggest that this distal P3F-bound region is critical for transcriptional activation of the MIR17HG locus. At the phenotypic level, the deleted subclones showed a marked reduction in focus-forming ability indicative of a loss of oncogenic transformation. Despite the loss of transformation, live-cell imaging revealed that the deleted subclones maintained elevated proliferation rates, suggesting that this regulatory element is dispensable for basal growth but necessary for transformation-associated phenotypes. To determine whether this P3F-binding site regulates other nearby transcripts, we analyzed RNA-seq data for the expression of surrounding genes, including long intergenic non-coding RNAs, miRNAs and protein-coding genes within a 2 Mb window around this P3F binding site. Surprisingly, none of these genes showed detectable expression in the RNA-seq dataset, except for MIR17HG. The complete absence of expression of neighboring loci supports the specificity of this regulatory element in controlling MIR17HG transcription. These findings provide compelling evidence that the identified distal P3F-binding site serves as a regulatory element critical for MIR17HG expression in FP-RMS.