Pim1 kinase is upregulated in glioblastoma multiforme and mediates tumor cell survival

Abstract

Background: The current therapy for glioblastoma multiforme (GBM), the most aggressive and common primary brain tumor of adults, involves surgery and a combined radiochemotherapy that controls tumor progression only for a limited time window. Therefore, the identification of new molecular targets is highly necessary. Inhibition of kinases has become a standard of clinical oncology, and thus the oncogenic kinase Pim1 might represent a promising target for improvement of GBM therapy.

Methods: Expression of Pim1 and associated signaling molecules was analyzed in human GBM samples, and the potential role of this kinase in patients' prognosis was evaluated. Furthermore, we analyzed the in vivo role of Pim1 in GBM cell growth in an orthotopic mouse model and examined the consequences of Pim1 inhibition in vitro to clarify underlying pathways.

Results: In comparison with normal brain, a strong upregulation of Pim1 was demonstrated in human GBM samples. Notably, patients with short overall survival showed a significantly higher Pim1 expression compared with GBM patients who lived longer than the median. In vitro experiments with GBM cells and analysis of patients' GBM samples suggest that Pim1 regulation is dependent on epidermal growth factor receptor. Furthermore, inhibition of Pim1 resulted in reduced cell viability accompanied by decreased cell numbers and increased apoptotic cells, as seen by elevated subG1 cell contents and caspase-3 and -9 activation, as well as modulation of several cell cycle or apoptosis regulatory proteins.

Conclusions: Altogether, Pim1 could be a novel therapeutic target, which should be further analyzed to improve the outcome of patients with aggressive GBM.

Keywords: epidermal growth factor receptor; glioblastoma multiforme; pim1 kinase.

Fig. 1.

Expression of Pim1, Akt1, EGFR, Bad, and HSP90 in GBM tissue and GBM cell lines. (A) Protein expression of Pim1S, Pim1L, HSP90, Akt, phosphorylated Bad (pBADSer112), and phosphorylated EGFR (pEGFRTyr1173) in various human and murine GBM cell lines (upper panel), patient-derived glioma lines, and xenografts of a rat (lower panel) analyzed by immunoblotting. GAPDH was used as loading control. (B) Relative mRNA expression levels of Pim1, Akt1, EGFR, and c-myc in frontal/temporal lobes of nonneoplastic brain (control) and glioblastoma patients' samples (GBM: primary tumor vs first and second relapse) analyzed by quantitative real-time PCR with normalization to 18S rRNA and shown as box plots representing the median as horizontal bars as well as the 5th and 95th percentiles. (C) Cryosections of nonneoplastic brain and glioblastoma tissue were immunostained with antibodies against the respective proteins. Nuclei are counterstained by the use of DAPI. Note that Pim1S, Pim1L, and pEGFR are strongly stained in glioblastoma, whereas no or little staining is observed in nonneoplastic brain tissue (frontal/temporal lobes). Scale bar represents 20 µm. (D, left) Illustration of a significant correlation between Pim1 and c-myc gene expression determined by Spearman's nonparametric correlation in 72 GBM samples (r = 0.63, P < .0001). (D, right) Illustration of a significant correlation between Pim1 and EGFR gene expression determined by Spearman's nonparametric correlation in 72 GBM samples (r = 0.28, P = .0195). (E) Expression of c-myc and EGFR in GBM dependent on the Pim1 mRNA level (Pim1 mRNA ≤median vs >median expression) analyzed by quantitative real-time PCR with normalization to 18S rRNA and shown as box plots representing the median as horizontal bars as well as the 5th and 95th percentiles. *P < .05, **P < .005, ***P < .001. (F) Representative immunoblots of total protein lysates from nonneoplastic frontal/temporal lobes (NB) and glioblastoma patients' samples (GB) analyzed for protein expression of Pim1S, Pim1L, phosphorylated Akt1 (pAkt1Ser473), pEGFRTyr1173, and pBadSer112. The membranes were stripped and reprobed with anti–β-actin antibody as loading control (not shown because the figures represent several different immunoblots).

Fig. 2.

Expression of Pim1, Akt1, EGFR, and c-myc in glioma tissue and GBM cell lines. (A) Cryosections were immunostained with antibodies against Pim1 and GFAP ((left panel) or platelet endothelial cell adhesion molecule (PECAM)/CD31 (right panel). Nuclei are counterstained by the use of DAPI. Note that Pim1 and GFAP are strongly colocalized in glioblastoma cells, whereas no staining of Pim1 was observed in PECAM-positive endothelial cells. (B) Immunofluorescence staining of Pim1S, Pim1L, phosphorylated EGFR (pEGFRTyr1173), and Akt1 (pAkt1Ser473) in glioblastoma cells. Paraformaldehyde fixed LN18 glioblastoma cells stained with antibodies against Pim1S and Pim1L, as well as pAkt1 and pEGFR. Nuclei are counterstained by the use of DAPI. Scale bar represents 20 µm in (A) and (B). (C) Comparison of Pim1, Akt1, EGFR, and c-myc mRNA expression in glioblastoma and low-grade astrocytoma (grades I–III) analyzed by quantitative real-time (RT-)PCR with normalization to 18S rRNA and shown as box plots representing the median as horizontal bars as well as the 5th and 95th percentiles. (D) Regulation of Pim1 mRNA by EGF (1 and 10 ng/mL, 48 h) in LN18 cells was determined by quantitative RT-PCR, mean ± SD of 3 independent experiments. Pim1 mRNA level was normalized to 18S rRNA and is expressed relative to the PBS treated control, which was given a value of 1. (E) Total cell lysates of nontreated and EGF-treated (1 and 10 ng/mL, 48 h) LN18 cells were immunoblotted and stained with antibodies against Pim1S, Pim1L, and β-actin (upper panel) followed by densitometric analysis of signals with normalization of Pim1 protein expression to β-actin (lower panel, Pim1: β-actin ratio). Pim1 protein level is expressed as a proportion of the PBS-treated control cells, which was given a value of 1 (n = 3), mean ± SD. (F) Influence of AG1478 (100 nM), LY294002 (25 µM), and PD98059 (10 µM) on Pim1 expression determined by quantitative RT-PCR. Pim1 mRNA level was normalized to 18S rRNA and is expressed relative to the PBS-treated control, which was given a value of 1 (n = 3), mean ± SD. (G) Representative samples of the analysis of EGFRwt or deletion variant EGFRvIII expression in GBM tissue determined by conventional PCR. (H) Association analysis of Pim1 mRNA level with the expression of EGFRwt or EGFRvIII in glioblastoma shown as box plots representing the median as horizontal bars as well as the 5th and 95th percentiles. *P < .05, **P < .005, ***P < .001.

Fig. 3.

Growth analysis of murine and human GBM cell lines after application of different inhibitors. (A and B) Time-dependent effects of PI3K/Pim1 inhibitor LY294002 (LY, 5 and 50 µM), Pim inhibitor QT (5 and 50 µM), selective Pim1 inhibitor TCS Pim 1 (5 and 50 µM), and HSP90 inhibitor AGA (0.5 and 5 µM) on cell viability of human U87MG (A) and LN18 (B). Cell viability was determined 48, 72, or 96 h after application of inhibitors using the resazurin assay (n = 3), mean ± SD. (C) Comparison of cell viability curves of serum starved and control LN18 cells treated with DMSO (as solvent), LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM). Cell viability was determined using the resazurin assay. Half-maximal inhibitory concentration values were determined by nonlinear regression analysis and compared with Wilcoxon signed-rank test (n = 3), mean ± SD. (D) Crystal violet staining of LN18 and GL261 cells 72 h after application of DMSO (as solvent), LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), AGA (0.5 and 5 µM), and 100 µM temozolomide (n = 3), mean ± SD. (E) Determination of cell viability using the resazurin assay after treatment of HepG2 and Caco2 cells with DMSO (as solvent), LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM) for 72 h (n = 3), mean ± SD. (F) Silencing of Pim1 and EGFR by gene-specific analysis. LN18 cells were transfected with either control-, Pim1-, or EGFR-siRNA using Lipofectamine 2000, and 48 h afterward the Pim1 mRNA expression was determined by quantitative real-time PCR with normalization to 18S rRNA. Mean + SD of 4 independent experiments. (G) Cell viability of LN18 cells 72 h after transfection with either control-, Pim1-, or EGFR-siRNA. Cell viability was determined using the resazurin assay (n = 4), mean ± SD. *P < .05, **P < .005, ***P < .001 vs control.

Fig. 4.

Analysis of the cytotoxic potential of HSP90 and Pim1 inhibitors. (A and B) Fluorescence activated cell sorting–based cell cycle analysis using ethanol fixed and propidium stained LN18 and GL261 cells 48 h after application of DMSO (as solvent), LY294002 (LY, 5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM). (A) Distribution of cells in the subG1 and (B) in the G1 cluster. Mean + SD, n = 4. (C) Determination of caspase-3 activity using a commercially available assay in LN18 and GL261 cells after treatment with DMSO (as solvent), LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM) for 48 h. Mean + SD, n = 4. (D) Determination of caspase-9 activity using a commercially available assay in LN18 and GL261 cells after treatment with DMSO (as solvent), LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM) for 48 h. Mean + SD, n = 4. (E and F) Protein extracts of LN18 cells treated with different inhibitors were analyzed by immunoblotting. Forty-eight hours after application of LY (5 and 50 µM), QT (5 and 50 µM), TCS (5 and 50 µM), and AGA (0.5 and 5 µM) LN18 cells were harvested, and (E) protein extracts were immunoblotted with antibodies against pBad (pBADSer112), phosphorylated Myt1 (pMyt1Ser83), phosphorylated Wee1 (pWee1Ser53), phosphorylated CDC2 (pCDC2Thr14/Tyr15), p21, p27, PCNA, XIAP, Bcl-xL, Bak, and GAPDH as loading control. Representative blots of 4 independent experiments. (F) Densitometric analysis of 4 independent experiments analyzed for the respective proteins by immunoblotting. Each target protein level was normalized to GAPDH (target protein:GAPDH ratio), mean + SD. *P < .05, **P < .005, ***P < .001 vs control.

Fig. 5.

Association between Pim1 mRNA or protein expression and survival time of patients with GBM. (A) mRNA expression of Pim1, c-myc, EGFR, and Akt1 was determined with quantitative real-time (RT-)PCR. Patients wer divided into 2 subgroups according to their OS time: patients with survival equal to or below the median time (≤ median OS) vs patients with survival above the median time (> median OS). Box plots represent the median mRNA expression as horizontal bars as well as the 5th and 95th percentiles, Mann–Whitney U test. (B) Screening of TCGA (caINTEGRATOR2_V1_4_RC2) for an association between Pim1 gene expression and survival of patients with GBM. Kaplan–Meier survival curves of AgilentG4502A_07_single ADF data (at the top) and of REMBRANDT data (at the bottom), both based on microarray analyses. Green lines: Pim1 overexpressing patients; blue lines: Pim1 underexpressing patients; red lines: Pim1 intermediate expressing patients. (C–E) Kaplan–Meier survival curves for patients with GBM based on their Pim1 mRNA expression (C), Pim1 and c-myc mRNA expression (D), and Pim1 and EGFR expression (E). Patients were divided into 2 subgroups depending on gene expression as determined by quantitative RT-PCR. Left panels represent the complete patient cohort, right panels demonstrate survival data for a 365-day observation time span. The broken vertical lines denote the current survival times (12 and 15 mo) of patients with GBM treated with standard therapy (surgical removal, combined radio- and chemotherapy). *P < .05, **P < .005.

Fig. 6.

Analysis of Pim1 as a therapeutic target in an orthotopic in vivo mouse model. Female wild-type C57BL/6 mice were immobilized in a stereotactic head holder in the flatskull position; 1 μL (2.5 × 10⁴ cells/μL) of GL261 cells in PBS was inserted 1 mm anterior and 2 mm lateral to bregma to a depth of 3 mm from the dural surface. Twelve days postinjection tumor development was assessed in isoflurane-anesthetized mice by MRT to start the treatment at a tumor volume >1.5 mm³. Tumor volume was calculated with OsiriX software in coronar and axial gadolinium-enhanced T1-sections. Afterward, mice were treated with either 5% dextrose (control animals, n = 5) or 75 mg/kg TCS (n = 5) as Pim1 inhibitor every second day by oral gavage until the end of the study (12 d treatment). Tumor size was measured using MRT 12 days after starting the treatment. (A) Mean value of tumor size of control (5% dextrose) and TCS-treated animals at the day of beginning the pharmacological intervention (day 0) and at day 12. (B) Tumor sizes of each individual animal from the control and TCS-treated group at the day of beginning pharmacological intervention (day 0) and at day 12. (C) Representative gadobutrol-enhanced T1-weighted images (coronar) for 2 animals of the control (5% dextrose) and the TCS-treated group at the day of beginning pharmacological intervention (day 0) and at day 12. **P < .01 vs TCS treated animals (day 12) and ^§P < .05 vs the respective day 0 group.