Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo

Abstract

Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.

Figure 1

TMZ treatment and RPPM analysis of GSCs. (a) A collection of GSCs and two commercially available GBM cell lines have been treated with increasing doses of TMZ and viability was measured at 72 h. Data are represented as mean±S.E.M. from at least three independent experiments. (b) Hierarchical clustering of RPPM data (mean of three technical replicates) obtained by assaying a panel of antibodies mainly directed against components of the EGF-R and PI3-K/AKT/mTOR pathways on protein lysates of selected GSC lines treated either with vehicle (DMSO) or with 50 μM TMZ for either 12 or 72 h. Colored clusters were selected by cutting the dendrogram at the height indicated in the upper-right hierarchy plot. A summary of the phosphoproteins present in each cluster is available as Supplementary Table S2

Figure 2

Kinase inhibitor library screening and titration assays of positive hits and analogs in GSCs. (a) Point chart of the viability of two GSCs and two commercial GBM cell lines treated with the kinase inhibitor library each compound at 5 μM for 72 h. Normalized viability of each cell line is plotted as mean±S.D. from three independent experiments. Horizontal dashed lines represent reference lines for 100% (dark green) and 50% (red) viability. (b) Parallel plots of fitted 4PL curves and correspondent hierarchical clustering maps of pEC₅₀ values (see Materials and Methods section for details) derived from titration curves of GSCs treated for 72 h with positive hits from the library screening and (c and d) analog compounds. Fitting was performed on individual dose-response curves obtained from three independent experiments

Figure 3

Immunoblot analysis of UCN-01 targets. (a) GSC lines selected from each sensitivity group were exposed to increasing doses of UCN-01 and the phosphorylation levels of PKC, PDK1, CDC25C, and CDK1 (CDC2) were evaluated at 72 h. Representative blots are shown for three GSC lines (Supplementary Figure S4 displays the blots performed on the other tested GSC lines in each sensitivity group). (b) Normalized intensities of the immunoblot bands from each antibody tested are plotted for all three GSC lines in each sensitivity group as percentage over the untreated control. No statistically significant difference between sensitivity groups at various UCN-01 concentrations was found after two-way ANOVA and Bonferroni post test

Figure 4

RPPM analysis on GSCs treated with UCN-01. (a) Annotated hierarchical clustering heatmaps of RPPM data obtained on nine GSCs treated with DMSO (untreated or 0) or increasing concentrations of UCN-01. Most analytes detected are part of the DNA damage response, cell cycle, and PI3K/AKT/mTOR pathways. Data for each GSCs represent the mean of three technical replicates. (b) Normalized intensity plots of end points extracted from the RPPM data and involved in the PI3K/AKT/mTOR, DNA-damage response, and cell cycle pathways. Mean and SD (n=3) for each sensitivity group are connected by solid, colored lines at increasing concentrations of UCN-01. The name of the analyte and the time point are reported on top of every single plot

Figure 5

Effects of UCN-01 on the growth of intracerebral GFP-expressing GSC xenografts. (a) By 8 weeks after grafting, control mice showed tumor cells in the homolateral striatum, piriform cortex, corpus callosum, anterior commissure, internal capsule, optic tract, septal nuclei, and fimbria-hippocampus. (Left panel) Photomontage of two adjacent coronal brain sections 240 μm apart (scale bars, 1100 μm); (middle panel) schematic drawings of three adjacent sections 120 μm apart showing area demarcation for calculating tumor volume; (right panel) density of tumor cells in the grafted striatum (upper picture) and anterior commissure (lower picture) (scale bars, 140 μm). (b) Brain specimen of UCN-01-treated mouse showing inhibited growth and infiltrative potential by GSCs (left panel, scale bars, 1100 μm; right panel, scale bars, 140 μm). The brain area injected with UCN-01 showed autofluorescent cell debris without remarkable changes of the brain parenchyma (upper right panel). (c and d) Diagrams showing that both the volume of the brain region invaded by the GFP-expressing GSCs and the density of these cells in the grafted striatum are significantly smaller in UCN-01-treated mice as compared with controls (**P<0.0001)

Figure 6

Effects of UCN-01 treatment on established subcutaneous xenografts. (a) Subcutaneous nodules in a control and UCN-01-treated mouse by 3 weeks after the end of treatment. (b) Growth curves of tumor xenografts in control and UCN-01-treated mice. The tumor size reached 1 week before starting UCN-01 administration (arrows) is indicated at the time point −1. The time point 0 corresponds to the beginning of treatment. Values are expressed as means±S.E.M. *P<0.01. (c) Histological pattern of control and UCN-01-treated tumors. Control tumors showed the typical GBM pattern with foci of necrosis, perinecrotic pseudopalisades, angiogenic phenomena, and frequent mitotic figures (arrows). In UCN-01-treated tumors, clusters of tumor cells with elongated morphology were separated by septa of fibrous tissue. The density of tumor cells and mitosis was much lower than in control tumors. Mitotic index was significantly lower in UCN-01-treated tumors than in controls (1.47±0.17 versus 3.09±0.37, mean ±S.E.M., P<0.002). Scale bars, 140 μm (upper panels), 50 μm lower panels)