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

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

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

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

Stimulation of osteoclasts and osteoblasts by PC3c cells <i>in vitro</i>.

<p>(<b>A</b>) Primary mouse bone marrow cells were cultured in the presence of RANKL and M-CSF and treated or not (Ct) with conditioned medium obtained from PC3 and PC3c cells. More OCs (white arrow) were formed in cultures treated with PC3c conditioned medium compared to cultures treated with PC3 conditioned medium and Ct (ANOVA, p<0.0001). (<b>B</b>) Primary mouse calvaria cell cultures were treated from day 1-21 with conditioned medium obtained from PC3 and PC3c cell. Mineralized bone nodules were present and visualized by von Kossa staining at day 21 (see mineral in black, white arrows). Mineralized bone nodule formation was decreased when primary cells were treated with conditioned medium from any of the PC3/PC3c cells (compared with non-treated (Ct) cells); the decrease was less when PC3c cell conditioned medium was used (compared with PC3) (ANOVA, p<0.001 versus Ct and versus PC3). (<b>C</b>) PC3 conditioned media stimulated the expression of OPG and RANKL in primary OBs compared with non-treated (Ct) while PC3c conditioned media only inhibits the expression of OPG compared with Ct leading to an higher RANKL/OPG ratio in PC3c conditions. (<b>D</b>) Detection by real-time PCR of SOST, DMP1, OPG and RANKL mRNA expression in MLO-Y4 cells treated with PC3 and PC3c conditioned medium. Results are plotted as the mean number of OC ± SD and OB nodules ± SD of three wells for controls and each condition and are representative of two independent experiments. Genes expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0.05; **p<0,001, ***p<0,0001.</p

Expression of pro-osteoblastic factors by PC3c cells.

<p>Detection by real-time PCR of AR mRNA expression in PC3, PC3c and VCAP cancer cells lines (A), AMACR, PAP (B) and DKK1, ET-1, FGF9, Noggin, OPN, OPG, Runx2 and TGFβ mRNA expression (C and D) in PC3 and PC3c cancer cells lines. Genes expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0.05; **p<0,001, ***p<0,0001.</p

Induction of lytic and mixed bone lesions by PC3 and PC3c cells respectively after intratibial injection.

<p>(<b>A</b>) PC3 and PC3c cells were inoculated into male SCID mice; 10 weeks post inoculation, radiography revealed pure osteolytic lesions in mice injected with PC3 cells (n=6) (<b>E</b>) and mixed lesions in mice injected with PC3c cells (n=8) (<b>I</b>) compared to mice injected with PBS (n=10) (<b>A</b>) (see *(lysis) and white arrows (formation)). (<b>B,C- </b>F,G-J,K) Three-dimensional micro-CT reconstructions of tibiae and (<b>D</b>, <b>H</b>, <b>L</b>) histology after Goldner’s Trichrome staining confirmed the radiography results. T: Tumor; NB: New Bone.</p

PC3c cells highly expressed type I collagen.

<p>(<b>A</b>) Detection by real-time PCR of type I collagen mRNA expression in PC3c and PC3 cells cultured in normal conditions. Gene expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 *p<0,05. (<b>B</b>) Visualization of type I collagen in PC3c cells cultured on glass coverslip by electron microscopy (see black arrows).</p

PC3c cells osteomimicry properties.

<p>(<b>A</b>) Immunodetection of OPN in OB in bone metastases induced by PC3c cells (see OBs <b>a</b> and <b>b</b> magnifications of <b>a</b>; see black arrows) (<b>A</b>) and in tumors cells (B a). Similarly to OPN, BSP expression is detected in tumors cells <i>in </i><i>vivo</i> (<b>B</b>, <b>b</b>). (<b>C</b>) Similarly to primary mouse calvaria cells, PC3 and PC3c were cultured in osteogenic conditions for 21 days. ALP (a, c) and von Kossa staining (b, d) show high expression of ALP (c) and mineralization (white arrow) (d) in PC3c cells while no ALP expression (a) and mineralization were detected in PC3 cells (b). (<b>D</b>) Detection by real-time PCR of OPN, ALP and OCN mRNA expression in PC3c and PC3 cells cultured in osteogenic conditions for 21 days. Gene expression was assessed by real-time PCR on triplicate samples and normalized against that of the ribosomal protein gene L32 **p<0,001. Bar=200µm T: Tumor; OB: osteoblasts; NB: New Bone.</p