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First published online 21 September 2005
doi: 10.1242/jcs.02574

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Netrin-1 regulates invasion and migration of mouse mammary epithelial cells overexpressing Cripto-1 in vitro and in vivo

¹ Mammary Biology and Tumorigenesis Laboratory, NCI/CCR, 37 Convent Drive, Building 37, Bethesda, MD 20892, USA
² Cell Biology and Preclinical Models Unit, INT-Fondazione Pascale, 80131 Naples, Italy
³ Department of Biology, University of California at Santa Cruz, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA

View larger version (83K): [in a new window]   Fig. 1. Expression of Netrin-1 and its receptors, Neogenin and UNC5H1 in mouse mammary epithelial cells overexpressing Cripto-1. (A) Western blot analysis of expression of Netrin-1, Neogenin and UNC5H1 in the wild type (WT) and Cripto-1 (Cr-1)-overexpressing EpH4 and HC-11 cells. Detection of actin in bottom panel acts as a loading control. (B) Photomicrographs (40x magnification) of immunofluorescence show expression of Netrin-1 and Neogenin in HC-11 cells. Nuclei are stained blue by DAPI. (C) Representative photomicrographs (20x magnification) of histological sections from MMTV-CR-1 transgenic mice mammary tumors containing anaplastic mesenchyme-like tumor cells and stained by immunohistochemistry show low expression of Netrin-1 relative to Neogenin. Sections were counterstained with hematoxylin.

View larger version (48K): [in a new window]   Fig. 2. Reduction of Cripto-1 expression or inhibition of Cripto-1 signaling rescues Netrin-1 expression in mammary epithelial cells overexpressing Cripto-1. (A) Results from reverse transcriptase-PCR and Western blot experiments show reduction of Cr-1 expression in HC-11/Cr-1 cells transfected with a specific anti-Cr-1 siRNA. (B) Western blot analysis of lysates from Cripto-1 (Cr-1)-overexpressing HC-11 cells treated with anti-Cr-1 siRNA (Cr-1+siRNA) shows increase in expression of Netrin-1 and decrease in expression of Neogenin to levels comparable to those detected in wild-type (WT) HC-11 cells. Cr-1/cntrl, control HC-11/Cr-1 cells not treated with anti-Cr-1 siRNA. (C) Representative western blot analysis shows an increase in expression of Netrin-1 and a decrease in expression in Neogenin in HC-11/Cr-1 cells treated with the c-Src inhibitor (PP2) or with the PI-3K/Akt inhibitor (LY) compared to non-treated control HC-11/Cr-1 cells. (D) Histograms summarize results from densitometric analyses of western blots performed in the experiments described in C. Error bars indicate s.d. Treatment of HC-11/Cr-1 cells with a MAPK inhibitor (PD) had no significant effect on expression of Netrin-1 or Neogenin. (*P<0.05 compared to levels in the control group).

View larger version (54K): [in a new window]   Fig. 3. Exogenous Netrin-1 reverses Cripto-1-dependent epithelial-to-mesenchymal transition. (A) Western blot analysis of lysates from EpH4/Cr-1 cells cultured in growth medium containing 50 ng/ml of exogenous rmNetrin-1 (+NTN) showing increased expression of E-cadherin (E-cad), reduced expression of vimentin (Vim) and reduced expression of phosphorylated-Akt (P-Akt) compared to EpH4/Cr-1 cells cultured in growth medium alone (control). (B) Histogram summarizes results from invasion assays showing increased invasion of EpH4/Cr-1 and HC-11/Cr-1 cells as compared to wild-type cells. Addition of rmNetrin-1 (25 ng/ml and 50 ng/ml) significantly reduced invasion of both Cr-1 overexpressing cell lines. Values are the mean±s.d. of three separate experiments (*P<0.05 compared to levels in the relevant control group). (C) Western blot analysis of lysates from Cr-1 overexpressing cells shows that Neogenin expression decreases and UNC5H1 expression increases when these cells are treated with exogenous soluble rmNetrin-1 (+Netrin-1) as compared to untreated control cells. (D) Histogram summarizes results from invasion assays showing that the significant inhibition of invasion of EpH4/Cr-1 cells in the presence of 25 ng/ml of exogenous rmNetrin-1 is restored to control level when EpH4/Cr-1 cells are pretreated with neutralizing antibody against human UNC5C (-UNC5) but not when pretreated with neutralizing antibody against Neogenin (-NEO). Values are the mean±s.d. of three separate experiments (*P<0.05 compared to levels in the control group).

View larger version (47K): [in a new window]   Fig. 4. Formation of colonies in 3D extracellular matrix by mouse mammary epithelial cells overexpressing Cripto-1 and ductal elongation in mammary glands from transgenic mice overexpressing Cripto-1 are both reduced in proximity to a Netrin-1 source. (A) Colony formation of EpH4/Cr-1 cells was assessed in Matrigel containing disks that were preabsorbed with PBS or rmNetrin-1 (200 ng/ml). Colonies were quantified in areas situated proximal (P), medial (M) or distal (D) from the sources as illustrated on the microphotographs (5x magnification). (B) Detail shows the area of growth inhibition (arrows) proximal to the disk preabsorbed with rmNetrin-1 (100 ng/ml) surrounded by a ring of EpH4/Cr-1 cells. (C) Summary of the results obtained from quantification of the colonies formed by EpH4/Cr-1. The mean number of colonies of at least 500 µm in diameter (=500 µm) was significantly reduced only in the areas in proximity to the rmNetrin-1 source (*P<0.05 compared to levels in the control group). White bars represent the number of EpH4/Cr-1 colonies in Matrigel containing PBS source and black bars, the number of colonies formed in Matrigel containing rmNetrin-1. Values are the mean ± s.d. of four separate experiments. (D) Whole mount morphology (10x magnification) of cholesterol pellets (*) releasing 25 ng/day of rmNetrin-1 shows a significantly reduced ductal elongation in the mammary gland of MMTV-CR-1 transgenic mice compared to cholesterol-only control pellets. The Netrin-1-releasing pellets did not affect ductal elongation in mammary glands of FVB/N mice. L, lymph node (E) Histogram summarizing the results of ductal elongation in mammary glands containing Netrin-1-releasing pellets. Distance was measured from the center of the mammary gland lymph node to the tip of the farthest growing duct and values are the mean with error bars indicating the s.d. from experiments in FVB/N (n=4) and MMTV-CR-1 (n=5) mice. A significant difference in elongation distance was observed between the two groups (P=0.008).

View larger version (123K): [in a new window]   Fig. 5. Immunostaining for human Cripto-1 in mammary glands of MMTV/CR-1 transgenic mice. Increased expression of E-cadherin and UNC5H1 is observed in MMTV/CR-1 mammary glands containing Netrin-1-releasing pellets relative to expression in the control. Peripheral staining for UNC5H1 in the terminal end buds of MMTV/CR-1 control mammary gland was detected compared to staining of the entire terminal end bud in MMTV/CR-1 mammary glands containing the Netrin-1 pellets. Reduced intensity of immunostaining of P-Akt in the epithelial structures, and almost no staining of the stromal component was detected in MMTV/CR-1 mammary glands containing Netrin-1 pellets compared to the strong staining for P-Akt detected in both epithelial and stromal components of MMTV/CR-1 mammary glands implanted with control pellets. Magnification, 40x.