Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases

Overexpression of CD9 in human breast cancer cells promotes the development of bone metastases.

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental MDA-MB-231 breast cancer cell line. Here, we investigated the putative relationship between CD9 expression and the osteotropic phenotype. MATERIALS AND METHODS: Overexpression of CD9 was analyzed by immunoblotting in different cell lines. Immunohistochemistry was used to assess CD9 expression in primary tumors and metastatic lesions. In vivo experiments were conducted in mice using a monoclonal antibody against CD9. RESULTS: CD9 overexpression was confirmed in osteotropic cells. CD9 was significantly overexpressed in bone metastases versus primary tumors and visceral metastatic lesions. Finally, in vivo experiments showed that an antibody against CD9 delays homing of B02 cells in bone marrow, slowing down bone destruction. CONCLUSION: Our study reveals a potential implication of CD9 in the formation of bony metastases from breast cancer cells

Automated liver volumetry in orthotopic liver transplantation using multiphase acquisitions on MDCT.

International audienceOBJECTIVE: The aim of this study was to test a new automated hepatic volumetry technique by comparing the accuracies and postprocessing times of manual and automated liver volume segmentation methods in a patient population undergoing orthotopic liver transplantation so that liver volume could be determined on pathology as the standard of reference. CONCLUSION: Both manual and automated multiphase MDCT-based volume measurements were strongly correlated to liver volume (Pearson correlation coefficient, r = 0.87 [p < 0.0001] and 0.90 [p < 0.0001], respectively). Automated multiphase segmentation was significantly more rapid than manual segmentation (mean time, 16 ± 5 [SD] and 86 ± 3 seconds, respectively; p = 0.01). Overall, automated liver volumetry based on multiphase CT acquisitions is feasible and more rapid than manual segmentation

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

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

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

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