CREB signaling activity correlates with differentiation and survival in medulloblastoma

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteom

Growth-Factor-Driven Rescue to Receptor Tyrosine Kinase (RTK) Inhibitors through Akt and Erk Phosphorylation in Pediatric Low Grade Astrocytoma and Ependymoma

Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present study will extend our previous observations that vascular endothelial growth factor receptor (VEGFR)-2, platelet derived growth factor receptor (PDGFR) beta, Src, the epidermal growth factor receptor (ErbB) family, and hepatocyte growth factor receptor (HGFR/cMet) are potentially drugable targets in pediatric low grade astrocytoma and ependymoma with investigations concerning growth-factor-driven rescue. This was investigated in pediatric low grade astrocytoma and ependymoma cell lines treated with receptor tyrosine kinase (RTK) inhibitors e.g. sorafenib, dasatinib, canertinib and crizotinib. Flow cytometry analyses showed high percentage of cells expressing VEGFR-1, fibroblast growth factor receptor (FGFR)-1, ErbB1/EGFR, HGFR and recepteur d'origine nantais (RON) (respectively 52-77%, 34-51%, 63-90%, 83-98%, 65-95%). Their respective inhibitors induced decrease of cell viability, measured with WST-1 cell viability assays. At least this was partially due to increased apoptotic levels measured by Annexin V/Propidium Iodide apoptosis assays. EGF, HGF and FGF, which are normally expressed in brain (tumor) tissue, showed to be effective rescue inducing growth factors resulting in increased cell survival especially during treatment with dasatinib (complete rescue) or sorafenib (partial rescue). Growth-factor-driven rescue was less prominent when canertinib or crizotinib were used. Rescue was underscored by significantly activating downstream Akt and/or Erk phosphorylation and increased tumor cell migration. Combination treatment showed to be able to overcome the growth-factor-driven rescue. In conclusion, our study highlights the extensive importance of environmentally present growth factors in developing tumor escape towards RTK inhibitors in pediatric low grade astrocytoma and ependymoma. It is of great interest to anticipate upon these results for the design of new therapeutic trials with RTK inhibitors in these pediatric brain tumors

Crizotinib and canertinib does not affect RTK and growth factor expression levels in medulloblastoma cell lines.

<p><b>A-B</b> Barplots, showing the cell surface expression levels of various RTK’s in MFI using flow cytometry analyses in <b>A</b> RES256 and <b>B</b> UW473 cells treated with crizotinib, canertinib or control. Viable cells were stained with fluorescently labeled RTK-specific antibodies or isotype control antibody. Log values represent the MFI of RTK-expressing cells, subtracted by the MFI of their isotype controls. <b>C-D</b> Barplots, showing the protein expression levels of VEGF-A, PDGF-AB, HGF, FGF-2 basic and EGF in <b>C</b> RES256 and <b>D</b> UW473 cell treated with crizotinib, canertinib or control. Mean signal intensities of individual growth factors were normalized to control. <b>E-F</b> Cell proliferation assays, showing the effect of MET knockdown on cell count in <b>E</b> RES256 and <b>F</b> UW473 cells using crystal violet staining. Cells were seeded at day 4 after transduction for 7 days at a density of 2 x 10³ cells/well in DMEM/F12 containing 5% FCS. Tumor cell count was calculated using an internal calibrator.</p

Effects of the addition of growth factors on tumor cell count in medulloblastoma cell lines.

<p><b>A-E</b> Barplots, showing the effects of <b>A</b> VEGF-A, <b>B</b> PDGF-AB, <b>C</b> HGF, <b>D</b> FGF-2 and <b>E</b> EGF on cell count after 48h using crystal violet staining. Tumor cell count was calculated using an internal calibrator. Experiments were performed in sextuple. <b>F</b> Barplot, showing the protein expression levels of VEGF-A, PDGF-AB, HGF, FGF-2 basic and EGF in 5 medulloblastoma cell lines. Mean signal intensities of individual growth factors were normalized to a medium control. *p<0.05, **p<0.01</p

HGF significantly increases tumor cell viability in canertinib-treated medulloblastoma cell lines.

<p>Cell viability assays, showing the effects of different concentrations of <b>A</b> crizotinib and <b>B</b> canertinib on tumor cell viability in 5 medulloblastoma cell lines after 48h. <b>C</b> Cell viability assay, showing the effect of exogenous HGF in addition to canertinib on tumor cell viability in cell line UW473. The difference in area under the curve (AUC) is depicted by the grey area. <b>D-M</b> Cell viability assays showing the effects of exogenous VEGF-A, PDGF-AB, HGF, FGF-2 and EGF in addition to crizotinib or canertinib in medulloblastoma cell lines. Cells were stimulated with growth factor after 1h of RTK inhibition for 48 hours. Lines depict the relative AUC in percentages between inhibitor and growth factor-treated cells and inhibitor-treated cells only (100%). Asterisks indicate enhanced tumor cell viability and was defined by an increase in the relative AUC after subtraction of the standard deviations of all data points of both curves. A nonparametric wilcoxon matched-pairs signed rank test indicated significantly enhanced cell viability (*p<0,05) between HGF-stimulated cell lines compared to non-HGF stimulated medulloblastoma cell lines during canertinib treatment.</p

Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines

<div><p>Recent clinical trials investigating receptor tyrosine kinase (RTK) inhibitors showed a limited clinical response in medulloblastoma. The present study investigated the role of micro-environmental growth factors expressed in the brain, such as HGF and EGF, in relation to the effects of hepatocyte growth factor receptor (MET) and epidermal growth factor receptor family (ErbB1-4) inhibition in medulloblastoma cell lines. Medulloblastoma cell lines were treated with tyrosine kinase inhibitors crizotinib or canertinib, targeting MET and ErbB1-4, respectively. Upon treatment, cells were stimulated with VEGF-A, PDGF-AB, HGF, FGF-2 or EGF. Subsequently, we measured cell viability and expression levels of growth factors and downstream signaling proteins. Addition of HGF or EGF phosphorylated MET or EGFR, respectively, and demonstrated phosphorylation of Akt and ERK1/2 as well as increased tumor cell viability. Crizotinib and canertinib both inhibited cell viability and phosphorylation of Akt and ERK1/2. Specifically targeting MET using shRNA’s resulted in decreased cell viability. Interestingly, addition of HGF to canertinib significantly enhanced cell viability as well as phosphorylation of Akt and ERK1/2. The HGF-induced bypass of canertinib was reversed by addition of crizotinib. HGF protein was hardly released by medulloblastoma cells itself. Addition of canertinib did not affect RTK cell surface or growth factor expression levels. This manuscript points to the bypassing capacity of exogenous HGF in medulloblastoma cell lines. It might be of great interest to anticipate on these results in developing novel clinical trials with a combination of MET and EGFR inhibitors in medulloblastoma.</p></div

HGF and EGF phosphorylate MET and EGFR, respectively.

<p><b>A-B</b> Immunoblots, showing the effects of <b>A</b> HGF stimulation (0-100ng/ml) in combination with crizotinib on MET (MET: 145kDa, pro-MET: 170kDa) phosphorylation and <b>B</b> EGF stimulation (0-100ng/ml) in combination with canertinib on EGFR (175kDa) phosphorylation in cell lines RES256 and UW473. Bands of MET represent MET (145kDa) and pro-MET (170kDa). Total-MET and total-EGFR were used as loading controls. <b>C-D</b> Quantification analysis of immunoblots showing the <b>C</b> normalized phospho-MET expression and <b>D</b> normalized phospho-EGFR expression. Quantitations are representative of 2 to 3 independent experiments. Student’s t-test: *p<0.05. <b>E-F</b> Quantitative analysis of the effects of <b>E</b> HGF and <b>F</b> EGF using human phospho-kinase arrays in cell lines RES256 and UW473. The plot displays the phosphorylation sites of the proteins present on the proteome array that are categorized upon different signal transduction pathways. Mean pixel intensities were calculated and subtracted by the mean background intensity of the array. Bars depict the difference between growth factor-stimulated cells and control cells. Positive values indicate growth factor-induced phosphorylation of the kinase.</p

Combined RTK inhibition blocks HGF-enhanced downstream signaling and tumor cell viability.

<p><b>A-B</b> Cell viability assays showing the effects of combined treatment with crizotinib and/or canertinib in medulloblastoma cell lines relative to single treatment in addition to <b>A</b> EGF or <b>B</b> HGF after 48 hours. Lines depict the percentage AUC of growth factor in addition combined RTK inhibition relative to growth factor in addition to single RTK inhibition (100%). A nonparametric wilcoxon matched-pairs signed rank test indicated significantly enhanced tumor cell viability (*p<0,05) between HGF-stimulated cell lines compared to non-HGF stimulated medulloblastoma cell lines in addition to canertinib. <b>C-D</b> Immunoblots showing the effects of <b>C</b> EGF and <b>D</b> HGF during single and combined treatment with crizotinib and/or canertinib on downstream signaling effectors Akt (S473) (60kDa) and ERK1/2 (44 kDa,42 kDa). β-actin was used as a loading control. Growth factors were added after 1 hour of inhibitor treatment. Lysates were made after 2 hours of inhibitor treatment.</p