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Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Effect of RK-33 on Wnt signaling via DDX3A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F–I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT–PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
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fig08: Effect of RK-33 on Wnt signaling via DDX3A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F–I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT–PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.

Mentions: As DDX3 is implicated in Wnt signaling (Cruciat et al, 2013) and Wnt signaling is an important regulator of proliferation and can cause radiation resistance (Woodward et al, 2007; Zhang et al, 2010), we evaluated the relation between DDX3/RK-33 and the Wnt pathway. To determine the spatial pattern of DDX3 and β-catenin expression, we carried out immunofluorescence staining for DDX3 and β-catenin in wild-type H1299 cells, as well as in β-catenin-overexpressing H1299 cells. In wild-type H1299 cells, we could detect DDX3 expression in the cytoplasm, but very little nuclear β-catenin staining (Fig8A, top panel). However, forced expression of β-catenin resulted in a relocalization of DDX3 from the cytoplasm to the nucleus (Fig8A, bottom panel). This indicates that DDX3 may act as a transporter protein to shuttle β-catenin in and out of the nucleus. To establish whether there is a physical interaction between DDX3 and β-catenin, we carried out co-immunoprecipitation with DDX3 and β-catenin. As shown in Fig8B and C, DDX3 binds to β-catenin but not to H3K4Me3 (control), which was confirmed by immunoprecipitation using antibodies against both DDX3 and β-catenin.


Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Effect of RK-33 on Wnt signaling via DDX3A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F–I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT–PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
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Related In: Results  -  Collection

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fig08: Effect of RK-33 on Wnt signaling via DDX3A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F–I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT–PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.Data information: Significance was assessed by two-sided, paired t-test. Error bars represent SD.Source data are available online for this figure.
Mentions: As DDX3 is implicated in Wnt signaling (Cruciat et al, 2013) and Wnt signaling is an important regulator of proliferation and can cause radiation resistance (Woodward et al, 2007; Zhang et al, 2010), we evaluated the relation between DDX3/RK-33 and the Wnt pathway. To determine the spatial pattern of DDX3 and β-catenin expression, we carried out immunofluorescence staining for DDX3 and β-catenin in wild-type H1299 cells, as well as in β-catenin-overexpressing H1299 cells. In wild-type H1299 cells, we could detect DDX3 expression in the cytoplasm, but very little nuclear β-catenin staining (Fig8A, top panel). However, forced expression of β-catenin resulted in a relocalization of DDX3 from the cytoplasm to the nucleus (Fig8A, bottom panel). This indicates that DDX3 may act as a transporter protein to shuttle β-catenin in and out of the nucleus. To establish whether there is a physical interaction between DDX3 and β-catenin, we carried out co-immunoprecipitation with DDX3 and β-catenin. As shown in Fig8B and C, DDX3 binds to β-catenin but not to H3K4Me3 (control), which was confirmed by immunoprecipitation using antibodies against both DDX3 and β-catenin.

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus