1 Oncogene 2008 Vol: 27(28):3912-3922. DOI: 10.1038/onc.2008.33

Oncogenic role of DDX3 in breast cancer biogenesis

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

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Figures
Figure 1: Induction of DDX3 by BPDE in mammary epithelial cells. (a) Effect of BPDE on the growth (left) and viability (right) of MCF 10A cells in a time-dependent manner. (b) Left: immunoblot analyses of the levels of DDX3, at different incubation time points, in MCF 10A and MCF 12A cells following incubation with 0.5 M BPDE. Right: immunoblot analyses of the levels of DDX3 protein in a panel of immortalized mammary epithelial cells and breast cancer cell lines. (c) Relative DDX3 mRNA expression level quantified by real-time PCR in an identical panel of cell lines as in (b) right. BPDE, benzo[a]pyrene diol epoxide; con, control cells; THF, tetrahydrofuran. Figure 2: Phenotypic characterization of MCF 10A-DDX3 cells. (a) Photomicrographs of MCF 10A-DDX3 cells (spindle-shaped) as compared to the parental MCF 10A cells (cuboidal). (b) Immunocytochemistry using anti-DDX3 antibody on MCF 10A, MCF 10A-DDX3 and MDA-MB-231 (positive control) cells. Secondary antibody against anti-DDX3 was labeled with AlexaFluor 488 (green fluorescence). Nuclei were stained with 4,6-diamidino-2-phenylindole (blue fluorescence). (c) Growth characteristics of MCF 10A, MCF 10A-DDX3 and MDA-MB-231 on Matrigel. Arrows indicate the morphology of MCF 10A-DDX3 after a 4-week growth period. Calcein AM staining (green) was performed to identify live cells. (d) Colony-forming ability of MCF 10A-DDX3 cells on soft agar relative to the parental cell line. Arrows indicate growing colonies. Figure 3: Stable expression of DDX3 in MCF 10A cells increased motility and invasion of MCF 10A cells. (a) Bar graph represents motility and invasion of MCF 10A-DDX3 as compared to the parental MCF 10A cells. Results shown are the means (s.d.) of three independent experiments. (b) In vitro wound-healing/scratch assay. A clearing, as demarcated by the horizontal black lines, through confluent monolayers of MCF 10A and MCF 10A-DDX3 cells was generated with a sterile pipet tip. Photomicrographs were obtained at the indicated time points with the indicated magnifications. The strikingly different phenotypes of MCF 10A and MCF 10A-DDX3 cells are readily seen at the edge of the 'wound' with 40 magnification (bottom images). Figure 4: Overexpression of DDX3 represses E-cadherin expression in breast cells. (a) Left: immunoblot analyses for E-cadherin and DDX3 levels in MCF 10A and MCF 10A-DDX3 cells. Actin was scored for the loading control. Right: immunocytochemistry for E-cadherin (red) and -catenin (green) in MCF 10A and MCF 10-DDX3 cells. Colocalization of the two signals is shown as yellow-green in the merged image. (b) Luciferase (Luc)-based reporter assay. At the left are schematic representations of the E-cadherin promoter constructs (E1–E6), which ranged from -995 to -195 bp upstream of transcription start site to 135 bases downstream of the transcription start site. The bar graph to the right indicates the fold repression of Luc activity in the presence of DDX3. All experiments were done in replicates and repeated at least three times. (c) In vivo binding of DDX3 to the E-cadherin in MCF 10A-DDX3 cells was analysed using E-cadherin promoter-specific primers by PCR. Top panel: schematic representation of the E-cadherin promoter. Arrows represent the region amplified by PCR for the chromatin immunoprecipitation experiment. Bottom panel: lanes 1 and 7—molecular weight (MW) marker; lane 2—total input chromatin; lane 3—acetyl histone H3 precipitation; lane 4—DDX3 antibody; lane 5—nonspecific antibody; and lane 6—no antibody precipitation. Identical volumes from the final precipitate were used for PCR (except for the input chromatin, which was diluted 100 ). PCR was performed with primers (5'-CAACATGGTGAAACCCCGTCTG-3', and antisense, 5'-GTGAGCCATGAGCCACTGAGCT-3') spanning the -893 to -641 region of the E-cadherin promoter. Figure 5: Knockdown of DDX3 by shRNA promotes E-cadherin expression. Stable shRNA clones against DDX3 were generated in MCF 10A-DDX3 cells. (a) Morphology of the shRNA clone as compared to the parental MCF 10A-DDX3 cells. (b) Immunoblot showing E-cadherin and DDX3 protein levels in the shRNA clone. (c) Morphology of MCF-7 cells overexpressing DDX3. (d) Protein levels of E-cadherin and DDX3 in the transgenic MCF-7-DDX3 clone. shRNA, short hairpin RNA. Figure 6: Lack of correlation between DDX3 and p21 levels in breast cells. (a) Histogram showing relative fold expression (2-(Ct)) values of p21 mRNA in MCF 10A and MCF 10A-DDX3 cell lines. The p21 expression levels were downregulated in MCF 10A-DDX3 relative to MCF 10A cell line. (b) Quantitative real-time PCR analyses of DDX3 and p21 mRNA levels (2-(Ct)) in a panel of immortalized mammary epithelial cells and breast cancer cell lines. (c) p21 promoter-reporter assays with DDX3 as the effector molecule. The luciferase activities were analysed from the cell lysates transfected with DDX3 and the p21 promoter-reporter constructs. Reporter activity was measured as relative light units. White columns represent 2.3 Kb and black columns represent 0.16 Kb of the p21 promoter region, respectively.
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