Figure 1.
Determination of LPA1-specific early genes upregulated by LPA.
(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.
Figure 2.
Expression of HB-EGF is mediated through functional LPA1 in vitro.
(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.
Figure 3.
Increased expression of HB-EGF linked to high expression of LPA1 in human primary tumors of breast, prostate, lung and colon.
(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. *p<0.05; ***p<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; p<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; p<0.0001); (D) Lung Tumor (GSE43580; n = 150; R Spearman = 0.29; p<0.0001) and (E) Colon Tumor (GSE21510; n = 148; R Spearman = 0.27; p<0.0001).
Figure 4.
Pharmacological blockade of LPA1 in vivo inhibits HB-EGF secretion by human PC3 xenographs.
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) LPAR1, LPAR2 and LPAR3 expressions were measured by real-time quantitative PCR and normalized to housekeeping L32 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. ¶: p<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. ¶: p<0.05, using unpaired Student t-Test.