Regulation of E-cadherin and migration.

<p>A. Real time RT-PCR for E-cadherin. levels. 94-10-FR1 cells were cultured in the presence of heparin or heparin and FGF2 with or without U0126, BAPTA-AM or BIRB for 72 h. mRNA was harvested, cDNA made and used for real-time PCR. Levels were normalised to SDHA and standardised to heparin control. B. The effect of the inhibitors on migration was measured by transwell assay. Cells were seeded in the upper chamber, pretreated with inhibitors for 1 hr and fixed and stained after 24 h. Values represent percentage intensity of DAPI stained migrated cells compared to cells cultured with FGF2. C. Western blot showing activation of pathways in 94-10-FR1 and 94-10-Y766F expressing cells. Cells were cultured with either heparin or heparin and FGF2 for 10 min. D. Transwell assay for 94-10 vector control, 94-10-FR1 and 94-10-Y766F cells cultured with heparin and FGF2. Values represent percentage intensity of DAPI stain in the lower chamber compared to 94-10-FR1 cells. E. 94-10-FR1 and 94-10-Y766F cells were cultured with heparin or heparin and FGF2 for 2 h. Cells were stained with DAPI and Phalloidin to examine changes in FGF2-induced actin cytoskeleton (bars = 30 µm). F. FGFR1 and Y766F cells were grown on matrigel in a transwell for 96 h before imaging. FGF2 was used as a chemoattractant in the lower chamber. Arrows indicate regions of invasion (bars = 0.5 mm).</p

FGFR1 activation promotes EMT in 94-10 cells.

<p>A. 94-10-FR1 cells were cultured with heparin or heparin and FGF2 for 72 h. Images were taken at 72 h (bars = 100 µm). B. At 72 h 94-10-FR1 cells cultured with heparin or heparin and FGF2 were fixed and stained with DAPI and Phalloidin-Alexa 488 (bars = 30 µm). Images represent a merged picture of DAPI and Phalloidin-Alexa 488 staining. C. Transwell assays were used to assess changes in migration in 94-10-FR1 and control cells. Cells which had migrated (cells on the lower part of the transwell) were stained at 36 hr with haematoxylin. D. Invasion assays were performed on 94-10-FR1 cells seeded onto a layer of 50% matrigel containing heparin or heparin and FGF2. At 96 h they were fixed, stained with Phalloidin and imaged using confocal microscopy (bars = 0.5 mm). E. Western blots for E-cadherin and tubulin (loading control) on 94-10-FR1 and 94-10 vector control cells cultured with heparin or heparin and FGF2 for 72 h. F. Real time RT-PCR for E-cadherin on 94-10-FR1 cells cultured with heparin or heparin and FGF2 for 72h. SDHA was used an internal control.</p

FGFR1 induces expression of COX-2.

<p>A. Real time RT-PCR for COX-2 on cell lines expressing ectopic FGFR1 cultured with heparin and FGF2 for 24 h. Values were standardised to cells cultured with heparin alone and represent fold increase compared to controls. B. Real-time RT-PCR for COX-2 in 94-10-FR1 cells cultured with heparin and FGF2 for the indicated time points. Results are represented as fold change compared to untreated cells. C. Transwell assays comparing the effects of COX-2 and EP4 inhibitors on FGFR1-induced migration. Values represent relative percentage of migrated cells (as measured by DAPI stain) compared to 94-10-FR1 cells cultured with heparin and FGF2. D. Real time RT-PCR for COX-2 in 94-10-FR1 cells cultured with heparin or heparin and FGF2 for 1 h. Cells were pre-treated for 1h with and without U0126 and results are represented as a fold-change compared to heparin alone. E. Real-time RT-PCR for COX-2 expression in 94-10-Y766F cells cultured with heparin and FGF2 for 1 h. All real time RT-PCR experiments were normalised to SDHA. F. PGE2 levels were measured by an enzyme immunoassay in lysates (black bars) and media (gray bars) from cells cultured with heparin; heparin and FGF2; heparin, FGF and NS-398; or arichidonic acid (AA) for 4 h.</p

FGFR1 activation induces EMT in multiple UC cell lines.

<p>A. J82, 96-1, and LUCC3 expressing ectopic FGFR1 were cultured with heparin or heparin and FGF2. Images were taken at 72 h (Bars = 100 µm). B. Real time RT-PCR for E-cadherin on cell lines cultured with heparin or heparin and FGF2 for 72 h. Levels were normalised to SDHA and represented relative to untreated controls. C. Transwell assays were used to assess changes in migration. Migrated cells were stained with DAPI, imaged and total staining quantified using Volocity software. Values represent relative intensity of DAPI stain compared to the heparin only treated cells.</p

Signaling downstream of FGFR1 in 94-10 cells.

<p>A. The Human Phospho-Kinase Array was performed to examine FGFR1 signalling. Boxes 1, 2 and 3 represent activated proteins from the MAPK, STAT and downstream effectors of MAPK induced signaling cascades, respectively. B. Confirmation by Western blotting of the phosphorylated proteins identified in the Human Phospho-Kinase Array. Vector controls and FGFR1-expressing cells were cultured with heparin and FGF2 for indicated time periods (minutes). Lysates were harvested and used for Western blotting. C. Western blots showing inhibition of specific pathways using small molecule inhibitors. 94-10-FR1 cells were cultured with BAPTA-AM, U0126 or BIRB for 1 hr prior to addition of FGF2 for 10 min.</p

Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.

<p>Expression of ERβ1 and miR–92 are not inversely correlated in patients with primary breast cancer. Staining patterns of ERβ1 showed nuclear expression in the normal breast (A) epithelium (examples shown by red arrows) with no significant difference in nuclear staining during cancer progression (B). A shift in the localisation of ERβ1 staining was observed with a significant increase in cytoplasmic staining (green arrows) during progression to DCIS (C, E) and invasive breast cancer (D, E). Blue arrows show ERβ1-negative nuclei. Images of breast tissues show the staining patterns for patient 4 as a representative example. Horizontal lines represent the mean. Range ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01.</p

Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.

<p>Down-regulation of miR92 expression in normal fibroblasts (NFs), but not cancer associated fibroblasts (CAFs), significantly enhances the invasive capacity of breast cancer epithelial cells. Matched NFs and CAFs were reverse transfected with either an inhibitor of miR92 or negative control (A) and a Matrigel™ invasion assay was used to assess effects on the behaviour of breast cancer epithelial cells 48 hours post-transfection. Down-regulation of miR92 significantly increased the invasion of MCF7 (B) and MDA-MB–231 (C) cells. Error bars are ± S.D. *denotes significance of p<0.05; **denotes significance of p<0.01; ***denotes significance of p<0.001.</p

<i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.

<p><i>In silico</i> analyses using the Oncomine platform showed that expression of the ESR2 gene in the breast tumour microenvironment does not correlate with patient outcome (p = 0.9337) (A). Expression of TGFBR2 (B) and BMPR2 (C) showed a trend towards significance (p = 0.1046 and p = 0.2029) suggesting that high expression levels of these genes are associated with lower rates of patient survival.</p

<i>In silico</i> analysis using BreastMark [26] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).

<p><i>In silico</i> analysis using BreastMark [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139698#pone.0139698.ref026" target="_blank">26</a>] shows luminal A breast cancer patients (n = 154) who express miR–92 have improved disease free survival (DFS) rate compared to patients with low expression levels with a hazard ratio of 0.49 (95% confidence intervals 0.28–0.84; p = 0.008).</p

Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.

<p>Laser micro-dissection (LMD) was used to isolate areas of epithelium in normal (A), DCIS (B) and invasive (C) breast tissue from the same tissue section. Breast tissue images show examples of the areas captured for patient 4 as a representative example. The expression of miR–92 decreased during breast cancer progression with highest levels observed in normal breast epithelium, decreasing in DCIS and being lowest in invasive breast tissue (D). *denotes significance of p<0.01.</p