Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 1. DP103 levels correlate with invasiveness and malignancy. DP103 staining of (A) normal ductal tissue and (B) an IDC. (C) Gene expression value of DP103 (y axis) plotted for each breast cancer subtype, namely basal, claudin-low, luminal-A, luminal-B, ERBB2 (HER2+), and normal-like. (D) Kaplan-Meier curves showing DP103 expression in relation to patients’ OS. Cases that have not experienced a positive event are censored at the date of last follow-up (small vertical lines on the line plots). (E) Kaplan-Meier curves showing DP103 expression in relation to SAR. Cases that have not experienced a positive event are censored at the date of last follow-up (small vertical lines on the line plots). (F) Breast cancer progression model showing isogenic cell lines with increasing invasive potential. (G) Western blotting with DP103 antibody in lysates from the isogenic cell lines (F). (H) qPCR with DP103 mRNA expression in RNA from the isogenic cell lines (F). (I) Gene expression of DP103 correlates with breast metastasis activity by Spearman correlation (64). Red dotted line is curve fitted by linear regression. (J) Primary breast tissues from patients with benign disease, no lymph node metastases (Non-Met), and lymph node metastases (Met) collected and analyzed for DP103 mRNA expression **P < 0.01; ***P < 0.001. (K) RNA from breast cell lines and qPCR performed with DP103 primers. (L) Protein from breast cell lines extracted and levels of DP103 protein evaluated. Fold difference in protein expression indicated in G and L.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: Staining, Gene Expression, Expressing, Western Blot, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 3. DP103 regulates MMP9 expression and function. MMP9 staining of (A) normal ductal tissue and (B) IDC. (C) Kaplan-Meier curves of MMP9 expression in relation to OS. Cases that have not experienced a positive event are censored at the date of last follow-up (small vertical lines on the line plots). (D) Kaplan-Meier curves of MMP9 expression in relation to SAR. Cases that have not experienced a positive event are censored at the date of last follow-up (small vertical lines on the line plots). (E) Primary breast tissues from patients with benign diseases, no metastases, and displaying metastases analyzed for MMP9 mRNA expression. **P < 0.01; ***P < 0.001. (F) Total RNA of different breast cell lines extracted and qPCR performed with MMP9 primers. (G) MDA-MB-231 and BT549 cells were transfected with control siRNA (ctsi) and siRNA against DP103. Total RNA analyzed for mRNA expression of DP103 and MMP9. (H) Expression of DP103 and MMP9 in cells used in G, evaluated using DP103 and MMP9 antibodies. (I) Suppression of DP103 in BT549 cells decreases MMP9 activity. Enzymatic activity of MMP-9 was determined by gelatin zymography.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: Expressing, Staining, Transfection, Control, Activity Assay, Zymography

Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 4. DP103 is an essential regulator of NF-κB signaling. (A) Control siRNA–treated (ctsi) and siDP103-treated MDA-MB-231 cells transfected with Renilla and luciferase reporter plasmid containing NF-κB or AP-1. Luciferase activity normalized and quantified. Results are average of 3 separate experiments. *P < 0.05. Control siRNA–treated (ctsi) and siDP103-treated MDA-MB-231 cells transfected with Renilla and luciferase reporter plasmid driven by NF-κB– binding sites. Cells were stimulated with (B) 10 μM VP16, (C) 25 μM doxorubicin, (D) or 10 μM CPT for 0, 6, and 12 hours and harvested for luciferase assays. Results are the average of 3 separate experiments. *P < 0.05. (E) MDA-MB-231 and BT549 cells transfected with either control siRNA (ctsi) or siDP103. Cells were left either untreated (–) or treated with CPT (10 μM), doxorubicin (10 μM), or VP16 (10 μM) for 2 hours. Protein extracts were analyzed by EMSA (top panel) and Western blotting using DP103 and GAPDH antibodies (bottom panel). (F) MDA-MB-231 cells infected either with lentiviral empty vector (lanes 1–3) or pBO- BI-DP103 (lanes 4–6) were either left untreated or treated with CPT (10 μM) for indicated times. Protein extracts analyzed by EMSA and Western blotting using anti-DP103 and anti–β-actin antibodies (top panel). (G) MDA-MB-231 cells transfected with either control siRNA (ctsi) or siRNA against DP103 and subjected to CPT (10 μM) stimulation at the indicated times. Cells were harvested and lysates evaluated by Western blotting for the indicated proteins. Fold difference in protein DNA binding indicated in E and F for EMSA and for protein expression changes indicated in G for Western blot.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Binding Assay, Western Blot, Infection, Expressing

Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 6. DP103 regulates NF-κB in response to multiple stimuli. (A) MDA-MB-231 cells transfected with either control siRNA or siDP103. Cells untreated (UT) or treated as indicated. Extracts analyzed with EMSA (top) and Western blotting (bottom). White separating lines between different stimuli. (B and C) MDA-MB-231 cells transfected with control siRNA or siDP103. Lysates of cells untreated or treated with TNF-α (B) or CPT (C), tested by EMSA. (D) BT549 cells transfected as in C and stimulated with TNF-α. RNA analyzed for mRNA expression of NF-κB target genes. *P < 0.05; **P < 0.01; ***P < 0.001. (E) MCF10A cells treated with TNF-α or LPS. (F) MCF10A cells either untreated or treated with inhibitors for IKK2, MEK1/2, JNK, p38, or PI3K, then stimulated with LPS. (G) White arrowheads indicate putative NF-κB–binding sites on DP103 promoter. (H) Nuclear proteins from MDA-MB-231 cells either untreated or treated with CPT tested by EMSA. Unlabeled DP103 probe as cold or IgG, p65, or p50 was used. (I) Nuclear proteins from MDA-MB-231 cells untreated or treated with TNF-α tested by EMSA using NF-κB consensus (lanes 1–3) or DP103 probe (lanes 4–6). Unlabeled NF-κB consensus cold probe added to lanes 3 and 6. (J) Cells transfected either with siRNA control or siDP103. Lysates immunoprecipitated with NEMO antibody and kinase assay performed as previously described (36). (K) BT549 cells transfected either with control siRNA or siDP103 as indicated and immunoblotted. Fold difference in protein DNA binding indicated in EMSA for A–C and for protein expression changes in E, F, J, and K.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: Transfection, Control, Western Blot, Expressing, Binding Assay, Immunoprecipitation, Kinase Assay

Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 7. DP103 is a positive cofactor of TAK1-mediated IKK2 activation in MDA-MB-231 cells. (A) Nuclear and cytoplasmic fractions immunoprecipitated with TAK1 and IgG control antibodies and immunoprecipitate material and lysates analyzed by immunoblotting with the indicated antibodies. (B) GST-TAK1 and His-DP103 incubated either separately or together. Immunoprecipitates analyzed by immunoblotting with the indicated antibodies. (C) Kinase assay performed using GST-TAK1 either with GST-IKK2-WT or GST-IKK2-Mut substrates with increasing input of His-DP103 protein. (D) Cells transfected either with control siRNA or siDP103 and stimulated with TNF-α. Lysates were immunoprecipitated using anti-TAK1 antibody, and kinase assay was performed using GST–IKK2 (amino acid residues 152–204) as substrate (top panel). (E) Cells transfected with DP103, siDP103, and respective control vectors. IKK complex immu- noprecipitated with anti-NEMO antibody. IKK activity was determined using phosphorylation of IκBα. (F) Cells transfected either with control siRNA or siTAK1 and stimulated with TNF-α. Total RNA analyzed for mRNA expression of NF-κB target genes. *P < 0.05; ***P < 0.001. (G) Cells treated with IKK2 inhibitor IV or TAK1 inhibitor (5Z)-7-oxozeaenol and stimulated with TNF-α. MMP9 activity evaluated with zymography. (H) Cells treated with control siRNA (ctsi) or siRNAs against TAK1 (siTAK1) and stimulated with TNF-α. MMP9 activity evaluated with zymography. (I) Cells transfected either with control siRNA or siRNAs against TAK1. Invaded cells through Matrigel detached and lysed in assay buffer were presented as relative fluorescence units (RFU). ***P < 0.001. Fold differ- ences in protein expression are indicated in C–E.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: Activation Assay, Immunoprecipitation, Control, Western Blot, Incubation, Kinase Assay, Transfection, Activity Assay, Phospho-proteomics, Expressing, Zymography, Fluorescence

Journal: Journal of Clinical Investigation

Article Title: DEAD-box helicase DP103 defines metastatic potential of human breast cancers

doi: 10.1172/jci73451

Figure Lengend Snippet: Figure 8. Schematic model based on our study showing a role for the RNA helicase DP103 in its ability to bind and stabilize TAK1 and thus activate NF-κB signaling in cancers.

Article Snippet: The DEAD-box protein DP103 (Ddx20 or Gemin-3) represses orphan nuclear receptor activity via SUMO modification.

Techniques: