Characterization of silencing autotaxin expression in mouse breast carcinoma 4T1 cells.

<p>(A) RT-PCR amplification products for LPA receptors, LPA₁ (1), LPA₂ (2), LPA₃ (3), LPA₄ (4), LPA₅ (5), GPR87, P2Y5, and autotaxin (ATX) from 4T1 cells total RNAs were analyzed on a 2% agarose gel. MW, molecular weight marker. (B) Cell invasion was stimulated with increased LPA concentrations used as chemoattractant. Results are the mean ± SD of cells of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². (C) Autotaxin expression in 3 clones of 4T1 cells transfected with a pStrike vector coding for either irrelevant small hairpin RNAi (sbATX, clones no. 14, no. 16, no. 20) or specific small hairpin RNAi (siATX, clones no. 1, no. 17, no. 52). (Upper panel) Immunoblotting using anti-ATX polyclonal antibody or anti-?tubulin as loading control. (Lower panel) lysoPLD activity (pmol LPA/ml) measured in cell culture conditioned media. (D) Cell proliferation assessed by BrdU incorporation of 4T1 cells and a pool of three 4T1-sbATX clones (no. 14, no. 16, no. 20) or three 4T1-siATX clones (no. 1, no. 17, no. 52), in response to increased concentrations of LPC. Results are expressed in mean ± SD of 6 replicates and are representative of 3 separates experiments. (E) Invasion assay. Cells were placed in presence or absence of LPA (0.1–1 µM) in the upper chamber and FBS, used as chemoattractant, was placed in the lower chamber. Results are the mean ± SD of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². *, <i>P</i><0.05.</p

Distribution of ATX mRNA in different subsets of cases defined by classical prognostic parameters in primary tumors of metastastic patients with bone metastases and of non metastatic patients without metastasis recurrence.

<p>Data are expressed as the median of ATX/L32 mRNA ratio.</p>*<p><i>p</i> values were obtained using the non parametric Mann & Whitney test.</p>**<p>in ductal carcinomas only and <i>p</i> values were obtained using the non parametric Kruskall-Wallis test.</p

Characterization of forced expression of autotaxin in human breast cancer MDA-B02 cells.

<p>(A) Cells transfected with bidirectional expression vectors pBiL-ATX or pBil-NPP1 were plated with (+) or without (-) doxycycline (Dox). Proteins from conditioned media (CM) or lysates of tumor cells (CL) of two stable clones (no. 30 and no. 38 to ATX, no. 10.5 and no. 42 to NPP1) were electrophorezed then immunoblotted with an anti-ATX antibody (Left panel) or anti-Myc antibody (Right panel). (B) Quantifications of luciferase activity (Left panel), lysoPLD activity (Middle panel) and PDE activity (Right panel) in each clone and parental MDA-B02 cells. (C) Cell proliferation was stimulated with LPC (10 µM) in absence or presence Ki16425 (10 µM). Results are expressed as the % of BrdU incorporation compared to unstimulated MDA-B02 parental cells. Data correspond to the mean ± SD of 6 replicates and are representative of at least 3 independent experiments. (D) Cell invasion was stimulated with 10% FBS used as chemoattractant. Results are the mean ± SD of cells of 3 replicates and are representative of at least 3 independent experiments. Data are expressed as the number of cells/mm². *, <i>P</i><0.05. **, <i>P</i><0.01</p

Effect of forced expression of autotaxin <i>in vivo</i> on MDA-B02 cells increased the formation osteoclasts at the bone metastatic site.

<p>(Upper left panels) Representative immunohistological examination of proximal tibia sections from metastatic animals 29 days after tumor cell inoculation, using the anti-ATX antibody 4F1. T indicates tumor cells. (Lower left panels) Representative histological examination of TRAP-stained proximal tibia sections from metastatic animals. T indicates tumor cells. Bone is stained in dark blue and osteoclats are stained in red (arrows). (Right panel) Quantification of active-osteoclast resorption surface per trabecular bone surface (Oc.S/BS). Results are the mean ± SE of 8–9 animals per group. *: <i>P</i><0.05. Scale bars: 200 µm.</p

Effect of autotaxin expression in orthotopic primary tumor growth and spontaneously metastasis dissemination of mouse 4T1 cells.

<p>4T1 parental cells, 4T1-sbATX clones and 4T1-siATX clones were injected in the mammary gland of normal syngenic female BALB/C mice. At day 14, primary tumors were resected, and weighed. (A) Box plots represent tumor weight (in mg). (B) Primary tumors were embedded in paraffin. Tumor tissue sections were analysed by mmunohistochemistry using a specific antibody directed against the nuclear ki-67 antigen. The mitotic index (numbers in each panel) was calculated as the percentage of nuclei positive for ki-67 (results are the mean ± SD, scale bar: 50 µm). (C) Animals were sacrificed 35 days after tumor cell injection and lungs were collected to quantify spontaneously metastasis formation of 4T1 cells. (Upper panels) representative photographs of lung tissue sections stained with eosin. (Lower panel) Quantification of lung metastasis foci. The number of metastatic foci was enumerated under microscope. P<0,05. T indicates metastatic foci. Scale bar: 200 µm.</p

Effect of forced expression of autotaxin on osteolytic bone metastasis formation of MDA-B02 cells.

<p>(A) (Left panels) Representative radiographs of hind limbs from metastatic mice bearing MDA-B02 cells or MDA-B02-ATX clone no. 30 or MDA-B02-NPP1 clone no. 42, 29 days after tumor cell inoculation. Osteolytic lesions are indicated by arrows (scale bar: 0.5 cm). (Right panel) Quantification of osteolytic lesion areas on radiographs in metastatic animals. Data correspond to the mean ± SE of two independent experiments of 7 to 10 animals per group. (B) (Left panels) Representative bone histology of Goldner's trichrome-stained tibial metaphysis from metastatic animals. Bone is stained in blue; bone marrow and tumor cells are stained in red. (scale bar: 1 mm). (Right panel) Histomorphometric analysis of metastatic hindlimbs using the bone volume/tissue volume ration (BV/TV, black bars and left axis) and the tumor volume/tissue volume ratio (TumV/TV, open bars and right axis) as referents. Values are the mean ± SE of 7–10 animals per group representative of two independent experiments. *, <i>P</i><0.05.</p

Schematic representation of LPA/autotaxin effects on the progression of osteolytic bone metastases.

<p>Bone-residing breast cancer cells (1) induce platelet (2) aggregation and the release of LPA and LPA precursors (LPC) from activated platelets. Platelet-derived LPA and LPA-derived from autotaxin (ATX) lysoPLD activity secreted by cancer cells can act on tumor cells to stimulate both tumor growth and the production of IL-6 and IL-8, which in turn induce the expression of RANK-L by osteoblasts (3) that stimulate osteoclast precursors (4) differentiation and osteoclast-mediated bone resorption. Platelet-derived LPA and LPA derived from ATX activity can act directly on osteoblasts to stimulate migration and proliferation, and on osteoclast precursors to stimulate osteoclastogenesis. Doted arrows indicate unknown origin.</p

Effect of lysoPLD activity of autotaxin on osteoclastogenesis.

<p>(A) Bone marrow cells were cultured in presence of FBS (10%), mouse M-CSF, RANK-L and MDA-B02 cell or MDA-B02-ATX clone #30 or MDA-B02-NPP1 clone #10.5 conditioned media. (B) Bone marrow cells were cultured in presence of FBS (10%), mouse M-CSF, RANK-L and recombinant ATX, in absence (-) or presence of increasing concentrations of ATX inhibitor, vpc8a202. (C) Bone marrow cells were cultured in presence of mouse M-CSF, RANK-L and FBS (Control) or lipid-depleted FBS (Dep. FBS) in absence or presence of 1-Oleoyl-LPA (1 µM). (Left panels) Representative images of multinucleated cells stained for the TRAP activity. (Right panels) Quantification of osteoclast number was based on the multinucleation of TRAP-positive cells. Results are the mean ± SD of 3 separate experiments. *: <i>P</i><0.05. Scale bars: 200 µm.</p

Expression of autotaxin mRNA in primary tumors of breast cancer patients.

<p>Total RNA were extracted from primary breast tumor biopsies of patients without or with metastases at the time of diagnosis. Soft and Bone represent subsets of metastatic patients with soft tissue only and bone metastases, respectively. W/O Rec represent a subset of non metastatic patients with no recurrence of metastasis during a five year period. Soft Rec and Bone Rec represent subsets of patients with recurrence of metastases to soft tissue only and to bone over a five year period, respectively. n indicates the numbers of patients in each group. Expression of Enpp2/ATX mRNA was measured by real-time quantitative by real time PCR. Quantifications were normalized to corresponding L32 RNA values. Data are given as box plots with the median. The box encompasses the 25^th to 75^th percentiles. The 5^th percentiles are displayed as error bars.</p