Targeted inhibition of ubiquitin signaling reverses metabolic reprogramming and suppresses glioblastoma growth

Abstract

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.

Fig. 1. Expression analysis of praja2 in glioma tissues.

a Tissue sections from human astrocytoma grade II and GBM lesions were immunostained with anti-praja2 antibody and analyzed by stereological microscope. The images are representative of praja2 staining in low-grade (astrocytoma II) and high-grade (GBM) gliomas. IDH1 gene mutation status in tumor lesions is indicated. Images were acquired at 100X magnifications. Scale bar = 50 μm. b Graph represents the levels of praja2 in different histological types of glioma and are expressed as the density of positive cells for microscope fields. P value: ** = 0.002; ** = 0.007; *** = 0.00015. The IHC analysis was carried out on a total of 20 gliomas (ten with wild-type IDH1 and ten gliomas with IDH1-R132 mutation).

Fig. 2. Assigning interaction of praja2 with other proteins possibly related to GBM.

a Praja2 protein–protein interaction (PPI) network. Praja2 complexes were purified from cell lysates of HEK293 cells transiently transfected with Flag-praja2rm vector or flag vector (control), which were independently subjected to an enrichment step with an anti-Flag derivatized resin, whose eluates were finally subjected to proteomic analysis. Identified praja2-binding partners involved in metabolic pathways and those reported in available databases were used to generate a PPI network. Connections are colored based on the interaction source: interactors identified in this study (green edges), CPTAC-GBM interactors (red edges), Intact interactors (blue edges), and STRING interactors (gray edges). The complete list of protein interactors is reported in Supplementary Data 1. b Schematic representation of praja2 constructs (lower panel) used in co-immunoprecipitation assays (upper panel). Lysates from HEK293 cells transiently transfected with Myc-tagged KSR2 and Flag-praja2 vectors were subjected to immunoprecipitation with an anti-Myc antibody. Precipitates and an aliquot of lysates were immunoblotted with anti-Flag and anti-Myc antibodies. c In vitro translated, [³⁵S] labeled KSR2 was subjected to pull-down assay with GST and GST-praja2 polypeptides. d U87MG cells were immunostained with anti-Myc and anti-praja2 antibodies and further analyzed by confocal microscopy. Scale bar = 5 μm. The Pearson’s coefficient value of KSR2 and praja2 signals is shown (lower, right panel). e A trimeric complex composed of praja2, KSR2, and AMPKα1 was isolated from lysates of HEK293 cells transiently expressing Flag-praja2 and KSR2-Myc and subjected to immunoprecipitation with anti-Flag. f HEK293 cells transfected with HA–ubiquitin and Flag-praja2 or Flag-praja2rm and KSR2-Myc were treated for 6 h with 10 μM MG132. Lysates were immunoprecipitated with an anti-KSR2 antibody. Lysates and precipitates were immunoblotted with anti-HA, anti-Flag, and anti-KSR2 antibodies. g Lysates from growing HEK293 cells transiently transfected with HA–ubiquitin, KSR2-Myc, and control siRNA (siRNAc) or siRNAs targeting praja2 (siPraja2) were immunoprecipitated with anti-KSR2 antibody. Lysates and precipitates were immunoblotted with anti-HA, anti-praja2, and anti-KSR2 antibodies. h Cells were transiently transfected with flag-praja2 or flag-praja2rm for 24 h. Lysates were immunoblotted with the indicated antibodies. i Quantitative analysis of data shown in panel h. A mean value ± SEM of three independent experiments is reported. P value: * = 0.037; ** = 0.0026. j U87MG cells were transfected with a control vector or with vectors encoding for flag-praja2 or flag-praja2rm. Where indicated, cells were pretreated with MG132 for 6 h before harvesting. Lysates were subjected to immunoblot analysis with the indicated antibodies. k Quantitative analysis of data shown in panel j. A mean value ± SEM of four independent experiments is reported. P value: * = 0.011; ** = 0.0020.

Fig. 3. Praja2 restrains AMPK signaling and supports the glycolytic pathway.

a U87MG cells were transiently transfected with siRNAs (siRNAc or siPraja2). Twenty-four hours later, cells were left untreated (time point 0) or deprived of glucose for the indicated times. Lysates were immunoblotted with anti-pThr172-AMPKα, anti-AMPKα1, and anti-praja2 antibodies. b Quantitative analysis of the experiments shown in panel a. A mean value ± SEM of three independent experiments is reported. P value: *** = 0.00070; * = 0.028. c Same as in a, with the exception that glucose-supplemented cells were treated with 1 mM AICAR for the indicated times. d Quantitative analysis of the experiments shown in panel c. A mean value ± SEM of three independent experiments is reported. P value: ** = 0.0042;* = 0.014. e U87MG cells were transiently transfected with siRNAs (siRNAc, siPraja2 or siPraja2, and siKSR2). Twenty-four hours later, cells were left untreated (time point 0) or cells were treated with 1 mM AICAR for the indicated times. Lysates were immunoblotted with indicated antibodies. f Quantitative analysis of the experiments shown in panel e. A mean value ± SEM of three independent experiments is reported. P value: * = 0.014; * = 0.034. g Oxygen consumption rate (OCR) in U87MG cells transiently transfected with the indicated siRNAs. Reported data are the mean values ± SEM of four independent experiments. OCR was measured in real-time, under basal conditions, or in the presence of the indicated mitochondrial inhibitors: oligomycin, FCCP, antimycin A plus rotenone. h Indices of mitochondrial respiratory function, as calculated from the OCR profile of U87MG cells transiently transfected with the indicated siRNAs: basal OCR, maximal respiration, spare respiratory capacity, ATP production. Reported data were the mean values ± SEM of four measurements deriving from four independent experiments. P value: * = 0.011; * = 0.025; ** = 0.0069; ** = 0.0093; ** = 0.0061; ** = 0.0.0058 ; ** = 0.0026. i Oxygen consumption rate (OCR) in U87MG cells transiently transfected with reported siRNAs. When indicated, cells were pretreated with the AMPK inhibitor SBI-0206965 (5 μM for 4 h). Reported data were the mean values ± SEM of four independent experiments. j Indices of mitochondrial respiratory function, as calculated from the OCR profile of praja2-silenced and control U87MG cells: basal OCR, maximal respiration, spare respiratory capacity, ATP production. When indicated, cells were pretreated with the AMPK inhibitor SBI-0206965 (5 μM for 4 h). Reported data were the mean values ± SEM of four independent experiments. P value *** = 0.00014; * = 0.039; *** = 0.0.00013; ** = 0.0035; *** = 0.000090; * = 0.012.

Fig. 4. Transcriptional reprogramming of praja2-silenced GBM cells.

a MA-plot from RNA-Seq data analysis showing the transcriptome differences after praja2 silencing in U87 cells, compared to control. Red dots represent upregulated transcripts with fold-change ≥1.5 and adjusted p value ≤0.10, while blue dots represent downregulated transcripts with fold-change ≤−1.5 and adjusted p value ≤0.10. Gray dots represent those transcripts with −1.5< fold-change <1.5 and/or with an adjusted p value >0.10. The x-axis represents the log2 of mean expression, while the y-axis represents the log2 fold-change, as computed by DESeq2. Dashed lines highlight the fold-change cutoff of ≥1.5 or ≤−1.5. b Histogram showing the distribution of adjusted p values for the 725 differentially expressed genes. More than 81.5% of differentially expressed genes are associated with an adjusted p value ≤ 0.05. c Histogram showing NES (normalized enrichment score) values of the molecular signatures statistically significant (FDR ≤0.25) involving the differentially expressed transcripts, as computed by the GSEA tool. d Heatmap summarizing expression data for the differentially expressed transcripts involved in the molecular signature of indicated pathways, as computed with GSEA, in siPraja2 versus siRNAc conditions. Normalized expression values in log2 scale and centered on the median value. Immunoblot analysis of praja2 in siRNA-transfected U87MG cells before RNA preparation is also shown. e IPA upstream regulator analysis of master upstream regulatory factors (PPARGC1A, PPARGC1B, and NRF1) on their target genes. Data were expressed as the ratio of the normalized read counts in siPraja2 vs siRNAc samples.

Fig. 5. Generation of SANPs-siRNA targeting praja2.

a Schematic representation of self-assembling nanoparticles (SANPs) conjugated with transferrin and encapsulating control siRNA or siRNA targeting praja2. b Cells were perfused for 72 h with different preparations of SANPs-siRNAs targeting praja2 (F1: SANP1-siPraja2; F2: SANP2-siPraja2; F3: SANP3-siPraja2; F4: SANP4-siPraja2; F5: SANP5-siPraja2; F6: SANP6-siPraja2) or control SANPs (SANPs-siRNAc). Lysates were immunoblotted with anti-praja2 and anti-HSP90 antibodies. c Quantitative analysis of the experiments shown in panel b. Reported data were the mean values ± SEM of three independent experiments. P value: * = 0.0167; * = 0.0169. d Growth curves of U87MG cells treated with vehicle, SANPs-siRNAc, SANPs-siPraja2. At indicated time points, cells were harvested and counted. Three independent experiments were performed and the corresponding mean values ± SEM are shown. P value: ** = 0.0054. e, f FACS analysis of U87MG cells treated with siRNAc or siPraja2 (e), and SANPs-siRNAc or SANPs-siPraja2 (f) for 72 h. Cell cycle distribution (G0/G1, S, and G2/M) of treated cells is indicated as a percentage of total cells scored.

Fig. 6. Systemic delivery of SANPs-siPraja2 inhibits GBM growth.

a Schematic view of the experimental procedures. U87MG cells were stereotaxically implanted into the brain of nude mice (time 0). One week post-implantation, SANPs-siRNAs were i.v. injected into the caudal vein every 48 h, for a total of 14 days of treatment. At 3 weeks post-implantation, mice were sacrificed and tumor lesions isolated and further characterized. b Brain distribution of rhodamine-labeled SANPs-siPraja2 by fluorescence analysis at 9 h after i.v. injection. GBM lesions were identified by immunostaining the same brain sections with an anti-human vimentin antibody. Nuclei were stained with DAPI. Representative images are shown. Scale bar, 50 μm. c Tissue sections from tumor lesions were stained with hematoxylin/eosin. d Quantitative analysis of the tumor volume is expressed as a mean value ± SEM. Three independent experiments were performed. P value: ** = 0.0014. e Tumor sections were stained with hematoxylin/eosin or immunostained for Ki-67. Scale bar, 50 μm. f Quantitative analysis of Ki-67-positive cells in tumor lesions from control and SANPs-siPraja2 treated mice. The data were expressed as a mean value ± SEM from three independent experiments. P value: ** = 0.0055. g U87MG-Luc cells were injected into the brain of 6 weeks old CD1 mice. Three hours following implantation, bioluminescent intensity (BLI) was measured by intraperitoneal injection of 150 mg/kg d-Luciferin potassium salt. At 1-week post-injection, based on BLI measurement, mice were randomized into two experimental groups of 12 animals, and each group was treated by tail vein injection with SANPs-siPraja2 (GP1) and SANPs-siRNAc (GP2), respectively. Treatments were repeated twice a week for 4 weeks, then four mice for each group were sacrificed and organs collected. BLI analysis was performed every week and quantitative data were collected. A representative set of animals for each experimental group is shown. h Quantitative and cumulative analysis of BLI scores. *** <0.001. i Kaplan–Meier curve of treated animals. At 52 days from U87MG implantation, all the animals from SANPs-siRNAc group died. In contrast, about 40% of SANPs-siPraja2 mice were still healthy, but the experiment was terminated in accordance with Authorities guidelines. j Immunostaining analysis for praja2, KSR2, pThr172-AMPKα, and AMPKα1 in tumor sections from control and SANPs-siPraja2 treated mice. Scale bar, 50 μm.