Fig 2: PI3K/AKT/mTOR pathway-specific differentially regulated gene expression in the Ptch1+/-/Tis21KO MB mouse model. As described in our previously published data, we performed a functional genomic analysis by microarray of GCPs isolated from the EGL at P7 of Ptch1+/+/Tis21WT, Ptch1+/+/Tis21KO, Ptch1+/-/Tis21WT and Ptch1+/-/Tis21KO mice (27). Through further analysis of these data, we now identify 13 genes, shown here, encoding for proteins involved in the PI3K/AKT/mTOR pathway (see Figure 1), whose expression significantly differs in the pairwise comparison Ptch1+/-/Tis21KO versus Ptch1+/-/Tis21WT (column D). The deregulation of these genes could explain, at least in part, the large increase in MB frequency observed in Ptch1+/-/Tis21KO relative to Ptch1+/-/Tis21WT mice. Of note, among the 13 PI3K/AKT/mTOR pathway-specific genes, the majority of them showed concordant expression changes between the pairwise comparison Ptch1+/+/Tis21KO vs. Ptch1+/+/Tis21WT (column B) and the pairwise comparison Ptch1+/-/Tis21WT vs. Ptch1+/+/Tis21WT (column A), and opposite expression changes with respect to the pairwise comparison Ptch1+/-/Tis21KO versus Ptch1+/-/Tis21WT (column C). This suggests that the activation of the PI3K/AKT/mTOR signaling is the result of a synergy between the Ptch1+/- and Tis21KO genotypes. The data in columns (A–C) are expressed in terms of log2 fold-changes of gene expression; the colors indicate overexpression (pink) or down-expression (light blue) of genes, relative to the median value. The p values of differentially expressed genes in the pairwise comparison Ptch1+/-/Tis21KO versus Ptch1+/-/Tis21WT, reported in column (D), are calculated as described in the section Microarray Analysis.

Fig 3: In Ptch1+/-/Tis21KO MBs, PI3K/AKT/mTOR activation is associated to increased proliferation and survival of tumor cells. (A) Representative confocal images of proliferating MB cells (labeled with the antibody against Ki67, in red) in tumors from Ptch1+/-/Tis21WT and Ptch1+/-/Tis21KO mice. Sections are counterstained with Hoechst 33258 to visualize the nuclei. Scale bar, 50 µm. The white box areas are shown with 2× digital magnification. (B) Quantification of mean ± SEM percentage ratio of cells positive for Ki67 to the total number of cells (Hoechst-positive; proliferation index) in MBs from Ptch1+/-/Tis21WT and Ptch1+/-/Tis21KO mice. ****p < 0.0001, Mann-Whitney U-test. MBs analyzed: n = 4 for each genotype; total fields analyzed: n = 320. (C) Representative images by confocal microscopy showing the apoptotic MB cells, identified as activated Caspase-3+ cells (in red), in Ptch1+/-/Tis21WT and Ptch1+/-/Tis21KO tumors. Nuclei were stained with Hoechst 33258. Scale bars: 50 µm. Cells in boxed areas are shown at higher digital magnification (2×). (D) Analysis in Ptch1+/-/Tis21WT and Ptch1+/-/Tis21KO MBs of the number of apoptotic cells, measured as mean ± SEM percentage ratio between number of activated Caspase-3+ cells and total number of cells (visualized by Hoechst 33258; apoptotic index). ****p < 0.0001, Mann-Whitney U-test. MBs analyzed: n = 4 for each genotype; total fields analyzed: n = 327.

Fig 4: OSC inhibition blocks in vitro and in vivo PI3K/Akt pathway activation in endothelial and cancer cells.(A, B) Akt, ERK and P70S6K protein phosphorylation levels were evaluated by Western blot analysis of lysates from ECs upon treatment with Ro 48-8071 (1 µM) for 9, 18 and 24 h. (A) OSC inhibition was effective in decreasing Akt and P70S6K phosphorylation starting from 18 hours after incubation with Ro 48-8071, while p-ERK was not affected. (B) Relative protein levels of p-Akt and p-ERK were compared to total Akt and ERK, respectively, and normalized to ß-tubulin. Values are mean ±SD (*p < 0.05 for Akt, for #p < 0.05 pP70S6 kinase; ANOVA test). Western blot analysis of ß-tubulin was used as loading control. The images shown are representative of 5 independent experiments. (C,E) Tumors from RIP-Tag2 (C) and HCT116 (D) and HPAF-II (E) xenografts treated with Ro 48-8071 and vehicle were subjected to confocal analysis, and Akt activation and localisation were assessed by co-staining of anti p-Akt with Meca32. Activated Akt was highly expressed in both vessels (arrows) and tumor cells in baseline conditions, and it was strongly inhibited after Ro 48-8071 treatment. The image presents confocal microscopy of 5 fields per mouse. (F) Akt activation level was quantified by fluorescence intensity mean (MFI) (**P < 0.01 with the Student T-test). (G, H) Western blot analysis on HCT116 and HPAF-II cancer cells showed an inhibition of Akt/P70S6K pathway and of ERK phosphorylation after 9 hours of incubation with Ro 48-8071(1 µM). Relative protein levels were normalised to ß-tubulin. Values are mean ±SD, (*p < 0.05 for Akt, #p < 0.05 for pP70S6 kinase, §p < 0.05 for ERK; ANOVA test).

Fig 5: PKC, Akt/GSK3ß, and N-methyl-D-aspartate receptor (NMDAR)/ calmodulin-dependent kinase II (CaMKII) signaling pathways in Black Swiss (BS) mice. Total levels and phosphorylated forms of the proteins were determined by SDS-PAGE/western-blot analysis in frontal cortical tissue obtained from BS Taconic Farms (BStac) and BS Charles River (BScr) mice. The immunosignal corresponding to the BScr control was assigned an arbitrary value of 1.The assay was repeated thrice on samples from different mice that were not previously subjected to behavioral testing. *Significantly different from the BScr control group with ANOVA, Holm-Sidak multiple comparisons, P<.05. Representative blots are shown.