Pharmacological blockade of LPA₁<i>in vivo</i> inhibits HB-EGF secretion by human PC3 xenographs.

<p>PC3 tumor cells were injected subcutaneously in the right flank of male BALB/C nude mice. At day 35, post-tumor cell injection animals were randomized into two groups and treated with Ki16425 (25 mg/kg) or the vehicle for 5 d. (A) Representative photographs of primary tumors at day 40 (upper panels). Box plot represent tumor volumes (in mm³) (lower panels). Bar represents 10 mm. (B) <i>LPAR1</i>, <i>LPAR2</i> and <i>LPAR3</i> expressions were measured by real-time quantitative PCR and normalized to housekeeping <i>L32</i> gene values (Veh: Vehicle; Ki: Ki16425) (C) Box plot represents expression of HB-EGF mRNA expression detected by real-time quantitative PCR from total RNAs isolated from tumors of animals treated with vehicle or Ki16425. Values were normalized to housekeeping L32 gene. ¶: <i>p</i><0.05, using unpaired Student t-Test. (D) Box plot represents HB-EGF concentration detected by ELISA in the serum of animals treated with Ki16425 or vehicle. ¶: <i>p</i><0.05, using unpaired Student t-Test.</p

Pharmacological blockade of LPA₁<i>in vivo</i> inhibits HB-EGF secretion by human PC3 xenographs.

<p>PC3 tumor cells were injected subcutaneously in the right flank of male BALB/C nude mice. At day 35, post-tumor cell injection animals were randomized into two groups and treated with Ki16425 (25 mg/kg) or the vehicle for 5 d. (A) Representative photographs of primary tumors at day 40 (upper panels). Box plot represent tumor volumes (in mm³) (lower panels). Bar represents 10 mm. (B) <i>LPAR1</i>, <i>LPAR2</i> and <i>LPAR3</i> expressions were measured by real-time quantitative PCR and normalized to housekeeping <i>L32</i> gene values (Veh: Vehicle; Ki: Ki16425) (C) Box plot represents expression of HB-EGF mRNA expression detected by real-time quantitative PCR from total RNAs isolated from tumors of animals treated with vehicle or Ki16425. Values were normalized to housekeeping L32 gene. ¶: <i>p</i><0.05, using unpaired Student t-Test. (D) Box plot represents HB-EGF concentration detected by ELISA in the serum of animals treated with Ki16425 or vehicle. ¶: <i>p</i><0.05, using unpaired Student t-Test.</p

Determination of LPA₁-specific early genes upregulated by LPA.

<p>(A) Fluorescent values (Y-axis) of Affimetrix probe sets corresponding to each LPA receptor (X-axis) generated using total RNAs isolated from PC3, MDA-MB-231 and MCF-7 cells. (B) Relative expression levels of LPA receptors in PC3, MDA-MB-231 and MCF-7 cells extrapolated from Affimetrix fluorescent values presented in A). (C) Heat map of genes significantly upregulated in both MDA-MB-231 (MDA-231) and PC3 cells and not in MCF-7 cells stimulated by LPA (1 µM) for 45 min. Color scale corresponds to fold increase.</p

Expression of HB-EGF is mediated through functional LPA₁<i>in vitro</i>.

<p>(A,B,D,E) Expression of HB-EGF mRNA was measured by real-time quantitative PCR and normalized to housekeeping L32 gene in (A) PC3 cells treated for 45 min with LPA (1 µM) or fetal bovine serum (FBS, 10% w/v) in absence or presence of Debio0719 (Debio), (B) PC3 and MDA-MB-231 cells treated for 45 min with FBS (10% w/v) in absence or presence of Ki16425 (10 µM), (D) MDA-B02/GFP-βGal, MDA-B02/shLPA1 and MDA-B02/LPA1 cells culture in presence of FBS 10%, and (E) PC3 cells treated for 24 h with LPA (1 µM) in absence or presence of Ki16425 (10 µM). (C) Expression of LPA₁ mRNA was measured by real-time quantitative PCR and normalized to housekeeping L32 gene in MDA-B02/GFP-βGal, MDA-B02/shLPA1 and MDA-B02/LPA1 cells cultured in the presence of 10% FBS. (F) Quantification of HB-EGF concentration in the conditioned culture media of PC3 cells treated for 24 h with LPA (1 µM) in absence or presence of Ki16425 (10 µM). All values were the mean±SD of at least three experiments. *p<0.05; **p<0.01; ***p<0.001 using one-way ANOVA with a Bonferroni post-test.</p

Increased expression of HB-EGF linked to high expression of LPA₁ in human primary tumors of breast, prostate, lung and colon.

<p>(A) Total RNAs were extracted from 234 human primary breast tumor biopsies. Expression of LPA₁ mRNA was measured by real-time quantitative PCR and normalized to housekeeping L32 gene values. LPA₁ relative expression values were distributed into quartiles (Q) dividing the 234 primary tumors into four equal groups with equal frequencies. HB-EGF relative expression values in each LPA₁ subgroups were represented in box plot. All values are the mean±SD of each quartile. *<i>p</i><0.05; ***<i>p</i><0.001 vs. Q4 using Kruskal-Wallis with Dunn's post-test. (B) Scatter plot was constructed showing the correlation between LPA₁ and HB-EGF (R Spearman = 0.25; <i>p</i><0.0001) in the same RT-QPCR data. Scatter plots of LPA₁ and HB-EGF expression were constructed with the Log2 tranformed values extracted from publically available databases using R2 genomics analysis and visualization platform for (C) Prostate Tumor (GSE2109; n = 72; R Spearman = 0.45; <i>p</i><0.0001); (D) Lung Tumor (GSE43580; n = 150; R Spearman = 0.29; <i>p</i><0.0001) and (E) Colon Tumor (GSE21510; n = 148; R Spearman = 0.27; <i>p</i><0.0001).</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

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

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