Abstract
Patients with translocation-positive alveolar rhabdomyosarcoma (ARMS), an aggressive childhood tumor primarily characterized by the PAX3-FOXO1 oncogenic fusion protein, have a poor prognosis because of lack of therapies that specifically target ARMS tumors. This fact highlights the need for novel pharmaceutical interventions. Posttranslational modifications such as phosphorylation are becoming attractive biological targets for the development of such interventions. Along these lines, we demonstrated that PAX3-FOXO1 is phosphorylated at three specific sites and that its pattern of phosphorylation is altered relative to wild-type Pax3 throughout early myogenesis and in ARMS tumor cells. However, little work has been performed examining the effect of directly inhibiting phosphorylation at these sites on ARMS development. To address this gap in knowledge, we used small molecule inhibitors or mutational analysis to specifically inhibit phosphorylation of PAX3-FOXO1 to investigate how altering phosphorylation of the oncogenic fusion protein affects ARMS phenotypes. We found that inhibiting the phosphorylation of PAX3-FOXO1 at Ser201 significantly reduced migration, invasion and proliferation in two independent ARMS tumor cell lines. Further, we found that inhibition of phosphorylation at Ser205 also decreased proliferation and anchorage-independent growth. Consistent with these in vitro results, we demonstrate for the first time that PAX3-FOXO1 is phosphorylated at Ser201 and Ser205 in a primary tumor sample and in tumor cells actively invading the surrounding normal tissue. This report is the first to demonstrate that the direct inhibition of PAX3-FOXO1 phosphorylation reduces ARMS tumor phenotypes in vitro and that these phosphorylation events are present in primary human ARMS tumors and invading tumor cells. These results identify phosphorylation of PAX3-FOXO1, especially at Ser201, as a novel biological target that can be explored as a promising avenue for ARMS therapies.
Highlights
Rhabdomyosarcoma (RMS), one of the most common solid tumors in children,[1] is comprised of two main histological subtypes: embryonal and alveolar (ARMS)
Phosphorylation at Ser[205] is rapidly lost with a concomitant increase in phosphorylation on Ser[209], again mediated by CK2.16,17 In contrast, we found that PAX3-FOXO1 is phosphorylated on Ser[201] and Ser[205] during proliferation; this status remains unaltered throughout myogenesis with no increase in phosphorylation at Ser209.15,16 the aberrant phosphorylation of PAX3-FOXO1 may affect normal myogenesis to contribute the development of alveolar rhabdomyosarcoma (ARMS)
Others demonstrated that small molecule inhibitors of GSK3β affect the proliferation and viability of ARMS tumor cells.[19]
Summary
Rhabdomyosarcoma (RMS), one of the most common solid tumors in children,[1] is comprised of two main histological subtypes: embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily defined by the t(2;13)(q35; q14) translocation, which fuses the amino-terminal region of Pax[3] to the carboxylterminal sequences of FOXO1.2–4 The resulting PAX3-FOXO1 oncogenic fusion protein has altered molecular activities relative to wild-type Pax[3,5,6,7,8,9,10] which are believed to contribute to ARMS tumor phenotypes.[11] Patients diagnosed with PAX3-FOXO1-positive ARMS have a 4-year survival rate of 8%;12 which stems from the chemoresistance of metastatic tumors combined with a current lack of effective therapies specific for targeting ARMS This information highlights the necessity of understanding the underlying biological and biochemical processes that contribute to the genesis of ARMS to develop much needed therapeutic alternatives. Phosphorylation at Ser[205] is rapidly lost with a concomitant increase in phosphorylation on Ser[209], again mediated by CK2.16,17 In contrast, we found that PAX3-FOXO1 is phosphorylated on Ser[201] and Ser[205] during proliferation; this status remains unaltered throughout myogenesis with no increase in phosphorylation at Ser209.15,16 the aberrant phosphorylation of PAX3-FOXO1 may affect normal myogenesis to contribute the development of ARMS
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