Pyk2 is up-regulated in bladder cancer tissues.

<p>(A) Pyk2 expression on a bladder cancer tissue microarray was determined by immunofluorescence and AQUA analysis using the AQUA PM-2000 system (HistoRx, Inc). Automated quantification and statistics on the different types of bladder cancer tumor tissues (B) and in the cytoplasmic and nuclear fractions of urothelial carcinoma cells (C) was calculated by AQUA Software. (B) *<i>P</i><0.05. **<i>P</i><0.01 compared to normal tissue controls. (C) *<i>P</i> = 0.012 compared to non-neoplastic nuclear fraction.</p

Hypoxia influence the effect of metformin on Ewing sarcoma.

<p><b>A</b>) <i>In vivo</i> treatment with metformin in association or not with vincristine (VCR) against TC-71 xenografts in athymic nude mice. Treatments began when tumors started being measurable at day +7 after subcutaneous (s.c.) cell injection. <i>Left</i> graph, <i>in vivo</i> growth curves of TC-71 tumor volume (cm3) after s.c. injection of 5×10⁶ TC-71 cells in groups of 5 mice (treated groups) or 8 mice (Vehicle group). Vehicle, mice treated with PBS intratumor or water alone; metformin in drinking water, mice receiving metformin 200 mg/Kg in drinking water; metformin intratumor, mice receiving intratumor injection of metformin 200 mg/Kg daily, 5 days weekly. <i>Right</i> graph, <i>in vivo</i> growth curves of TC-71 tumor volume after s.c. injection of 7.5×10⁶ TC-71 cells in groups of 5 mice. Vehicle, mice treated with PBS; Metformin, mice treated daily, 5 days weekly with 500 mg/kg Metformin p.o. (gavage); VCR, mice treated with 1 mg/kg/d i.p. for 2 consecutive days; Metformin+VCR, combined therapy with Metformin 500 mg/Kg p.o. and VCR 1 mg/Kg/d i.p. <b>B</b>) Decrease responsiveness to metformin in hypoxia respect to normoxia environment. In hypoxia experiments, TC-71 and ASP-14 Ewing sarcoma cells were pre-treated for 18h with hypoxia mimetic agent CoCl2 (200 uM) than exposed for 48h to different concentration of metformin (10–30 mM) or left untreated. In combination experiments, TC-71 Ewing sarcoma cells were pre-treated for 18h with CoCl2 (200 uM) than exposed for 48h to metformin (10 mM) alone or in combination with different concentrations of vincristine (0.3–10 ng/ml). Effects of metformin on cell growth were assessed by trypan bleu assay and shown as percentage of survival over untreated control. *P < 0.05, ** P<0.01 statistically significant differences by Student’s t test. <b>C</b>) Western blotting analysis of AMPKα phosphorylation levels under hypoxia and normoxia conditions after metformin (10–30 mM) treatment. TC-71 and ASP-14 EWS cells were pre-treated for 18h with CoCl2 (200 uM) than exposed for 4h to different concentration of metformin (10–30 mM) or left untreated. Accumulation of HIF-1α protein confirmed the induction of hypoxia; β-actin was used as loading control. <b>D</b>) Immunohistochemical evaluation of HIF-1α and phospho-AMPKα in hypoxic (left panel) and normoxic areas (righ panel) in xenografts. Representative figures are shown (magnification X100).</p

IGF-I-activated Pyk2 is critical for IGF-IR-dependent motility of invasive urothelial cancer cells.

<p>(<i>A</i>) Serum-starved 5637 cells were stimulated with 50 ng/ml of IGF-I for the indicated time points. Pyk2 phosphorylation was detected by immunoblot using anti-phospho-Pyk2 (Tyr402) antibodies, while total Pyk2 protein level was assessed using anti-Pyk2 polyclonal antibodies. Blot is representative of two independent experiments. (<i>B</i>) Migration of 5637 cells transiently transfected with either Flag-tagged wild type (PYK2 WT) or a dominant negative (KD PYK2) Pyk2 proteins was assessed after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. ** <i>P</i><0.01. (<i>C</i>) Expression levels of transiently transfected Pyk2 proteins were assessed by immunoblot with anti-flag M2 antibodies. Blot is representative of two independent experiments.</p

Pyk2 is critical for IGF-IR-induced motility, invasion and signaling of invasive urothelial cancer cells.

<p>(A) 5637 cells were transfected with the Pyk2 siGenome pool or control. After 72 hours Pyk2 and FAK expression was detected by immunoblot with specific antibodies. Blot is representative of three independent experiments with an average Pyk2 depletion level of 92.6±3 (arbitrary units) as assessed by densitometric analysis (B and C) Migration and invasion of 5637 cells were assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040148#s4" target="_blank">Materials and Methods</a> after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. *<i>P</i><0.05; **<i>P</i><0.01. (<i>D</i>) Pyk2-depleted 5637 cells were tested for the activation of the Akt and MAPK pathways after 10 min of IGF-I stimulation using a mix of phospho-specific antibodies (PathScan Cocktail I). eIF4E monitors protein loads. Blot is representative of three independent experiments.</p

<i>In vitro</i> combination study of metformin with conventional and targeted drugs in sarcoma cells.

<p><i>In vitro</i> combination study of metformin with conventional and targeted drugs in sarcoma cells.</p

FAK is not important for IGF-I-mediated motility and signaling of invasive urothelial cancer cells.

<p>(A) 5637 cells were transfected with the FAK siGenome pool or control oligos. After 72 hours FAK and Pyk2 expression was detected by immunoblot with specific antibodies. Blot is representative of three independent experiments with an average FAK depletion level of 93.3±3.5 (arbitrary units) as assessed by densitometric analysis (B and C) Migration and invasion assays of 5637 cells were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040148#s4" target="_blank">Materials and Methods</a> and assessed after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. *<i>P</i> = 0.046. (<i>D</i>) FAK-depleted 5637 cells were tested for Akt and MAPK activation after 10 minutes of IGF-I stimulation using a mix of phospho-specific antibodies (PathScan Cocktail I). eIF4E monitors protein loads. Blot is representative of three independent experiments.</p

Analysis of metformin targets in Ewing sarcoma cell lines.

<p><b>A</b>) protein expression levels of LKB1 in a panel of Ewing and osteosarcoma cell lines. β-Actin was used as loading control. <b>B</b>) <i>Left</i> Graph showing cellular ATP levels (RLUs) as a percentage of control in control and metformin treated ES cells. <i>Right</i> ES cells were treated as described in material and methods, and cellular lactate levels were measured using a colorimetric assay kit (Abcam). Graph shows L-lactate concentration. Values are expressed as mean ± SEM and significance (*) p<0.05; (**) p<0.01 as indicated. <b>C</b>) Metformin activity on AMPK and mTOR signaling on Ewing sarcoma cell lines. Western Blotting analysis of metformin-treated cells TC-71, SK-N-MC and 6647. Cells were treated with metformin (10 mM) for the indicated time points (30min-24h). Immunoblot analysis was carried out using antibodies against phosphorylated AMPKα (Thr¹⁷²), AMPKα, phosphorylated pS6K (Ser^240/244), pS6, and β-actin as normalization. <b>D</b>) Analysis of Metformin (10 mM) effects on cell cycle over 48h treatment in three Ewing sarcoma cell lines (TC-71, SK-N-MC, 6647), together with two anti-IGF1R agents resistant cells (TC/AVE, TC/AEW). <i>Left panel</i>, mean percentage of cells in different cell cycle phases as determined by flow cytometry analysis; <i>Righ panel</i>, Ki-67 positive cells (Ki-67 labeling index). Data are shown as percentages of mean of three independent experiments and significance (*) p<0.05 as indicated.</p

Metformin effects are independent to IGF system activation.

<p><b>A</b>) Cell growth was assessed by MTT assay after 72h exposure to Metformin (mM) in cells resistant to anti-IGF-1R agents (TC/AVE, TC/AEW, TC/CP) where recently we demonstrated an increased expression of IR-A with a concomitant downregulation of IGF-1R and displayed as a percentage of survival over controls. <i>Points</i>, mean of two independent experiments; <i>bars</i>, SE; <b>B</b>) After overnight starvation, TC-71 and 6647 EWS cells were exposed to indicate concentration of metformin and/or IGF-1 and/or Insulin in IMDM containing 1% FBS for 48h. Cell growth was assessed by trypan blue assay and shown as percentage of growth over untreated controls. <b>C</b>) Starved cells were exposed to metformin (10 mM) for 4h in IMDM plus 1% FBS than stimulated with IGF-1 (50 ng/ml) or Insulin (10 nM) for 15min. After harvesting, cells were lysed and prepared for immunoblot analyses for p-ERK and total ERK. β-Actin was used as loading control.</p

Lack of correlation between IGF-1R and IR expression and sensitivity to metformin in pediatric sarcomas.

<p>^a Spearman’s test r =  0.448, <i>P</i>>0.050.</p><p>^b Spearman’s test r =  –0,402, <i>P</i>>0.050.</p><p>^c Spearman’s test r =  0.457, <i>P</i>>0.050.</p

<i>In vitro</i> sensitivity of Metformin in sarcomas.

<p><b>A</b>) Cell growth was assessed by MTT assay after 72h exposure to Metformin (mM) in seven Ewing sarcoma, four Osteosarcoma, and three Rhabdomyosarcoma cell lines and displayed as a percentage of survival over controls. <i>Points</i>, mean of two independent experiments; <i>bars</i>, SE. <b>B</b>) Effects of metformin on sarcoma cells in anchorage independent conditions. Number of colonies was determined after 7–10 days of growth in IMDM 10% FBS. <i>Columns</i>, mean of three independent experiments; <i>bars</i>, SE. Bars: mean of three experiments ± SE. *P < 0.05, ** P<0.01, ***P < 0.005 statistically significant differences by Student’s t test. Representative pictures of the effects of the inhibitor on colonies formation in soft agar. Magnification x40; <b>C)</b> IC50 values of metformin in Ewing sarcoma (TC/Doxo8) and osteosarcoma (U-2OS/Doxo35) chemoresistant cell lines.</p