RNA helicase DDX3 has oncogenic activity in breast and
lung cancers and is required for translation of complex
mRNA transcripts, including those encoding key
cell-cycle regulatory proteins. We sought to determine the expression and function of DDX3 in
sarcoma cells, and to investigate the antitumor activity of a novel small molecule DDX3 inhibitor, RK-33. Utilizing various
sarcoma cell lines, xenografts and human tissue microarrays, we measured DDX3 expression at the
mRNA and
protein levels, and evaluated cytotoxicity of RK-33 in
sarcoma cell lines. To study the role of DDX3 in
Ewing sarcoma, we generated stable DDX3-knockdown
Ewing sarcoma cell lines using DDX3-specific
small hairpin RNA (
shRNA), and assessed oncogenic activity. DDX3-knockdown and RK-33-treated
Ewing sarcoma cells were compared with wild-type cells using an isobaric mass-tag quantitative proteomics approach to identify target
proteins impacted by DDX3 inhibition. Overall, we found high expression of DDX3 in numerous human
sarcoma subtypes compared with non-malignant mesenchymal cells, and knockdown of DDX3 by RNA interference inhibited oncogenic activity in
Ewing sarcoma cells. Treatment with RK-33 was preferentially cytotoxic to
sarcoma cells, including
chemotherapy-resistant
Ewing sarcoma stem cells, while sparing non-malignant cells. Sensitivity to RK-33 correlated with DDX3
protein expression. Growth of human
Ewing sarcoma xenografts expressing high DDX3 was inhibited by RK-33 treatment in mice, without overt toxicity. DDX3 inhibition altered the
Ewing sarcoma cellular
proteome, especially
proteins involved in DNA replication, mRNA translation and
proteasome function. These data support further investigation of the role of DDX3 in
sarcomas, advancement of RK-33 to
Ewing sarcoma clinical trials and development of
RNA helicase inhibition as a novel anti-neoplastic strategy.