The PAX3-FKHR fusion oncoprotein inhibits myogenic differentiation and collaborates with MYCN to transform immortalized human myoblasts Free

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PAX3-FKHR is a fusion protein generated by the t(2;13)(q35;q14) chromosomal translocation, which specifically occurs in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Though previous studies of this protein demonstrated oncogenic properties in murine and avian fibroblasts, its properties have never been investigated in human cells, particularly myoblasts, which are postulated to be related to the cell of origin of this myogenic neoplasm. To explore this issue, PAX3-FKHR was introduced into human myoblasts immortalized with BMI1 (which represses p16INK4a and p14ARF expression) and TERT (telomerase catalytic subunit). PAX3-FKHR was expressed in a retroviral vector as a fusion with the estrogen receptor ligand binding domain in which its function is inducible by 4-hydroxytamoxifen (TMF).

To examine myogenic differentiation capability, cells were cultured in differentiation medium. In these conditions, cells with empty vector and cells expressing PAX3-FKHR without TMF transiently upregulated myogenin, an early myogenic marker, and then upregulated myosin heavy chain, a late myogenic marker. However, in cells expressing PAX3-FKHR with TMF, myogenin expression was increased to a higher level and there was little or no induction of myosin heavy chain. At the microscopic level, whereas control cells converted from a small polygonal to a large elongated appearance with occasional cell fusion, cells with active PAX3-FKHR retained a small polygonal phenotype. These findings indicate that PAX3-FKHR enhances initiation of myogenesis, yet blocks later steps.

In cellular transformation assays, PAX3-FKHR alone failed to transform human myoblasts in both soft agar colony and focus formation assays. It is hypothesized that additional genetic alterations collaborate with PAX3-FKHR in transforming human myoblasts. MYCN was selected as a possible collaborating oncogene because MYC genes serve in this collaborating role in other human transformation models, and MYCN is highly expressed and sometimes amplified in ARMS. MYCN was then introduced into human myoblasts with a second retroviral vector. Soft agar colony and focus formation assays of human myoblasts expressing both MYCN and PAX3-FKHR, each gene alone, or empty vector revealed that only cells expressing both PAX3-FKHR and MYCN showed transforming activity whereas the other three groups showed background activity. These findings indicate that MYCN collaborates with PAX3-FKHR in transformation of human myoblasts. It is also noted that, though MYCN is not sufficient for transformation, expression of this protein promotes growth of cells.

In summary, these findings indicate that PAX3-FKHR carries out its oncogenic role in human myoblasts in two ways, inhibiting myogenic differentiation and promoting transformation in collaboration with MYCN.