Dachshund inhibits oncogene-induced breast cancer cellular migration and invasion through suppression of interleukin-8

1. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 1.

   DACH1 inhibits oncogene-induced migration of breast cancer cells. (A) MCF10A or MCF10A-Ras cells were transduced with retroviral expression vectors encoding GFP or DACH1-IRES-GFP and subjected to GFP-FACS sorting with subsequent analysis of cell migration by transwell assay. The data are shown as mean ± SEM of the number of cells migrated in n > 5 separate experiments. Crystal violet dye staining of MCF10A-Ras cells that migrated in the transwell assays is shown. (B) Videomicroscopy of individual cells were quantitated to determine the distance and direction of cellular migration of MCF10A-Ras cells transfected with either control (GFP) or DACH1 expression vector. (C) MCF10A-ErbB2 (NeuT) transformed cells were analyzed for migration. The mean data for the number of cells migrated per field is shown for MCF-10A oncogene-transformed cells transduced with retroviral vector encoding GFP or DACH1-IRES-GFP. (D) Western blot analysis of MCF10A-ErbB2 cells shown in C with antibodies as indicated. (E) MCF10A-c-Myc cells transduced with DACH1 or control vector analyzed for migration as shown in A. *, P < 0.01.

   
   
2. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 2.

   DACH1 inhibits wound healing and persistence of migratory directionality. (A) Migration assays of MCF10A-Raf transformed cells. Cells were transduced with either retroviral vectors encoding GFP or DACH1. Data are mean ± SEM of five separate experiments for either transwell migration (A), wound closure assays (B) or phase contrast video microscopy (C). The velocity and distance migrated was determined for individual MCF10A-Raf cells transduced with either control vector or DACH1. Data mean ± SEM of n > 5 separate events. (D) Migration assays of MCF10A-Ras/ErbB2 cells transformed with either control (GFP) or DACH1. Data mean ± SEM of n > 5 separate experiments (Left) or transwell assays were conducted either in the presence of supernatant derived from cells expressing GFP, DACH1 or a mutant of the DACH1 DS domain (DACH1 ΔDS) (Right). Migration is reduced in media derived from DACH1 tranduced cells. Schematic representation of DACH1 and DACH1 ΔDS domain. *, P < 0.01.

   
   
3. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 3.

   DACH1 inhibits migration and invasion of highly metastatic MDA-MB-231 cells. (A) Phase contrast microscopy of MDA-MB-231 cells induced to express DACH1 (Lower) versus vehicle-treated cells (Upper). (Magnification: Left, 200×; Right, 400×.) (B–D) MDA-MB-231 cells stably expressing ponasterone A inducible DACH1 were assessed for either transwell migration (B), matrigel invasion (C), wound closure (D Left) or velocity-analyzed by videomicroscopy at a single-cell level (D Right), with mean data shown at 360 min. *, P < 0.01.

   
   
4. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 4.

   Proteomic analysis of DACH1-regulated chemokines. (A) Transwell migration assay of MDA-MB-231 cells treated with conditioned media derived from either vehicle control or ponasterone A-treated cells. (B) Cytokine array analysis of supernatant derived from MDA-MB-231 cells treated with either vehicle or ponasterone A to induce DACH1 identified a subset of cytokines differentially regulated by DACH1. Relative abundance is shown, comparing DACH1 expressing cells (PonA) with control. (C) Transwell migration assays of MDA-MB-231 cells treated with cytokines as indicated, including interleukin-8 or control IgG at 5 ng/ml. Migration of cells is shown as cell number per field with data as mean ± SEM for n > 5 separate experiments. (D) 3D collagen invasion assay of MCF10A-Ras cells expressing control (GFP) or DACH1 in the presence of IL-8 or vehicle. The mean depth of migration is shown quantified as mean ± SEM. (E) Cellular migration of MEFs derived from Dach1 WT or knockout mice, using time lapse video microscopy. Dach1 ^−/− (KO) MEFs display enhanced cellular migratory velocity and distance (P < 0.01). (F) The number of lung metastasis identified in nude mice injected with Met-1 cells transduced with pLRT-DACH1, treated with vehicle or Doxycycline DACH1 or immunoneutralizing antibody to CRCL1/KC. *, P < 0.01.

   
   
5. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Fig. 5.

   The IL-8 promoter is repressed by DACH1 requiring the DS domain. (A) IL-8 mRNA abundance was determined by QT-PCR in MCF10A-Ras or MDA-MB-231 cells expressing DACH1. In MDA-MB-231 cells, TNF-α was used to induce IL-8 production. (B and C) Schematic representation of DACH1 expression vectors and mutants, IL-8 promoter and point mutant luciferase reporter gene contractions. The effect of DACH1 on IL-8 promoter activity is shown normalized to Renilla Luciferase activity as an internal control. Data are mean ± SEM of n > 5 separate experiments. (D) ChIP analysis of DACH1 recruitment to either the endogenous IL-8 promoter in MDA-MB-231 cells (above) or to transfected IL-8 promoter constructions encoding either the WT, AP-1, or NFκB point mutants of the IL-8 promoter in HEK293 cells. Each experiment was conducted on at least two separate occasions with the FLAG antibody and was also conducted by using an antibody directed to DACH1 with similar results. Quantitative data of relative abundance of DACH1 recruitment to the IL-8 promoter is shown as mean data. *, P < 0.01.