Date of Award


Degree Name

Doctor of Philosophy


Molecular Biology, Microbiology and Biochemistry

First Advisor

Davie, Judith


Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.




This dissertation is only available for download to the SIUC community. Current SIUC affiliates may also access this paper off campus by searching Dissertations & Theses @ Southern Illinois University Carbondale from ProQuest. Others should contact the interlibrary loan department of your local library or contact ProQuest's Dissertation Express service.