PI3K-p110α dependent regulation of LIFRα expression.

<p>Expression of LIFRα was analyzed in DAOY cells transfected with siRNA against <i>PIK3CA or PIK3CD</i> (a), at protein level by Western blot and (b) at mRNA level by RT-PCR after 48h transfection. Protein and mRNA expression of <i>LIFR</i>α was also measured upon 24h treatment with YM024 (5 μM). The expression of PI3K-p110α, total and phospho-S6 (Ser235-236) are also shown (c). pcDNA3 vector containing active PI3K-p110α (PIK3CA CAAX) or not were transfected into DAOY cells. 48h post transfection samples were analyzed for p110α and LIFRα expression by qPCR (d-e) or Western blot (f). The expression levels of total and phospho-S6 (Ser240/244) were also measured (c-f). (a, c, f) were quantified by ImageJ analysing software.</p

Expression of LIFRα and LIF in medulloblastoma cell lines and primary brain tumors.

<p>(a) LIFRα protein levels in medulloblastoma cell lines and normal brain (NB) tissue were evaluated by Western blot and compared with β-actin as the loading control. (b) <i>LIF</i> and <i>LIFR</i> expression in medulloblastoma cell lines was analyzed by semi quantitative RT-PCR. (c) Expression of <i>PIK3CA</i> and <i>LIFR</i> derived from the transcriptomic analysis of primary medulloblastoma, grouped according to molecular disease variants. Data from Northcott et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123958#pone.0123958.ref032" target="_blank">32</a>] are shown. Gene expression levels were compared using the Kruskal-Wallis test and the p-value is shown at the bottom right of the illustration. As this global test was significant, Dunn's post hoc tests were used to compare the groups. P-values < 0,05, as determined by Dunn's post hoc tests, are indicated in order to illustrate the differences. (d) Analysis of cDNA microarray data performed on <i>PTCH1</i>^<i>+/-</i>; <i>TP53</i>^<i>+/-</i> or <i>PTCH1</i>^<i>+/-</i><i>; TP53</i>^<i>+/+</i> mice. The table represents a summary of fold change (logFC), average expression values (AveExpr), p values obtained by comparison of the two murine strains from Data set GEO accession number GSE37316). (e) Cell lysates of DAOY cells stimulated with or not with 5, 10, 25, 50 and 100 ng/ml of LIF for 10 min were analyzed for expression and phosphorylation of AKT and ERK pathway downstream targets. Treatment of 10 ng/ml of OSM for 10 min was used as a positive control.</p

LIFRα inhibition induces reduction in Medulloblastoma tumor volume.

<p>(a) Tumors formed on (CAM) were treated with 20μg/ml of rh-LIFα recombinant or phosphate-buffered saline for 4 consecutive days. Quantification of tumor volume changes before and after treatment is shown. Lines indicate the mean of each group, *P<0.05 compared with control treatment. (b) The expression of LIFRα downstream targets was analyzed upon treatment for 10, 30, 240 min with 20 μg/ml of rh-LIFRα. β-actin was used as a loading control.</p

Effects of LIFRα down-regulation on medulloblastoma cell proliferation and survival.

<p>(a) Protein expression level of LIFRα in DAOY cells transfected with siRNA against <i>LIFR</i> compared to the control siRNA transfected cells. (b) The effects on cell viability of the siRNA-mediated target down-regulation of LIFRα (white bars) compared to control siRNA (black bars) in DAOY and UW228 cells was assessed for the indicated time points and expressed as percentages. (c) The protein expression of LIFRα downstream targets was analyzed upon treatment for 10 or 30 min with 20μg/ml of anti-LIFRα neutralizing antibody. β-actin was used as a loading control. (d) Cell viability was evaluated in DAOY, UW228, PNET5 and PFSK cells treated for 48h with an anti-LIFRα neutralizing antibody at the indicated concentrations.</p

Gene expression analysis.

<p>(a) DAOY cells were transfected with siRNA against <i>PIK3CA</i> or <i>PIK3CD</i> and analyzed 48h post transfection for gene down-regulation at mRNA level by real-time PCR and at protein level by Western blot. (b) Heat map representing the expression of <i>PIK3CA</i> and <i>PIK3CD</i> regulated genes. (c) GeneGo Metacore analysis of transcriptional networks. The set of genes deregulated by <i>PIK3CA</i> silencing in DAOY cells were analyzed via GeneGo Metacore software. The most affected network comprising the oncogene c-Myc and some its downstream targets is shown.</p

p110α regulates LIFR expression through c-Myc.

<p>The protein expression levels of c-Myc and LIFRα were evaluated after 12h, 24h and 30h of 100nM PIK75 exposure by Western blot (a) in DAOY-derived cell clones overexpressing c-Myc (DAOY M2.1) and (b) in UW228-Myc-ER cells induced for c-Myc expression by tamoxifen for 24 h, 48 h and 72 h. (c) LIFRα mRNA levels were analyzed by qPCR and protein levels were analyzed by Western blot in the inducible UW228-Myc-ER cells treated independently, simultaneously or in alternate manner with the p110α inhibitor PIK75 (100 nM) and/or with tamoxifen (500 nM) for 24 h or 48 h. UW228-Myc-ER were simultaneously exposed to both drugs for 24h (bar 4/lane 4), alternately for 48 h, bar 4/lane 4 24 h tamoxifen (500 nM) exposure followed by 24 h PIK75 (100 nM) and bar 5/lane5 24 h PIK75 (100 nM) treatment followed by 24h tamoxifen (500 nM) induction.</p

c-Myc regulates LIFR expression via miR-125b.

<p>Expression of c-Myc (a) and LIFRα (b) were analyzed by qPCR and Western Blot in DAOY M2.1 cells, overexpressing c-Myc, transfected with siRNA against c-Myc. (c) miR-125b expression levels were measured by qPCR after 24h and 48h of 4-OHT treatment in UW228-Myc-ER cells. MiR-125b fold change was calculated via normalization to U6 snRNA expression levels in the corresponding condition (d) miR-125b expression was analyzed by qPCR after transfection with mimic or antagomir for miR-125b in DAOY cells. MiR-125b fold change was calculated as in (c) (e) LIFRα expression level was analyzed by qPCR and Western blot after transfection with mimic or antagomir for miR-125b. (b) and (e) were quantified by ImageJ analysis software.</p

PI3K p110α-specific inhibition of T98G tumors formed on the chick chorioallantoic membrane impairs tumor growth.

<p>(A) Representative pictures of T98G tumors formed on the CAM three days post cell application (10 × magnification). Tumors were treated with PIK75 as indicated for four consecutive days. (B) Quantification of changes in tumor volume before and after PIK75 or control treatment. Lines indicate the mean of each group. *: p = 0.02 compared to control treatment as determined by two-sided, two-sample Student’s <i>t</i>-test.</p

Inhibition of the PI3K isoform p110α with isoform-specific inhibitor PIK75 induces apoptosis.

<p>(A) Western blot analysis of apoptosis markers of GBM cells following treatment with increasing concentrations of PI3K p110α-specific inhibitors YM024 or PIK75 (6 h). (B) Flow cytometry analysis of T98G cells after treatment with PI3K p110α-specific inhibitor PIK75 (8 h) stained with Annexin-V and PI. (C) Quantification of Annexin-V-positive/PI-negative T98G cells after treatment with PI3K p110α-specific inhibitors YM024, A66, PIK75, or PI3K p110β-specific inhibitor TGX221 (8 h). Bars represent the means of three or two individual experiments ± standard deviation for PI3K p110α- or PI3K p110β-specific inhibitors, respectively; ***: p≤0.001 compared to 0.0 μM inhibitor as determined by two-sided, one-sample Student’s <i>t</i>-tests.</p

Class I_A PI3K isoforms p110α and p110β play a role in GBM cell migration.

<p>(A) Analysis of the migratory potential of GBM cells by means of wound healing assays in the presence of PI3K p110α or PI3K p110β-specific inhibitors (18 h). Bars represent the means of three individual experiments ± standard deviation; *: p≤0.05, **: p≤0.01, ***: p≤0.001 compared to 0.0 μM inhibitor as determined by two-sided, one-sample Student’s <i>t</i>-tests. (B) Migration speed of T98G cells in a random migration experiment following downregulation of class I_A PI3K isoforms by siRNA. *: p≤0.05 compared to SCR non-targeting siRNA as determined by two-sided, one-sample Student’s <i>t</i>-test. (C) Distance of migration in a random migration experiment of T98G cells transfected with non-targeting control (SCR) or PI3K p110β-targeting siRNA (PIK3CB). (D) Migration speed of T98G cells in a random migration experiment following downregulation of PI3K/Akt signaling pathway molecules by siRNA. *: p≤0.05, **: p≤0.01 compared to SCR non-targeting siRNA as determined by two-sided, two-sample Student’s <i>t</i>-tests.</p