Despite abundant evidence implicating receptor tyrosine kinases (RTK), including the platelet-derived growth factor receptor (PDGFR), in the pathogenesis of glioblastoma (GBM), the clinical use of RTK inhibitors in this disease has been greatly compromised by the rapid emergence of therapeutic resistance. To study the resistance of proneural gliomas that are driven by a PDGFR-regulated pathway to targeted tyrosine kinase inhibitors, we utilized a mouse model of proneural glioma in which mice develop tumors that become resistant to PDGFR inhibition. We found that tumors resistant to PDGFR inhibition required the expression and activation of the insulin receptor (IR)/insulin growth-like factor receptor (IGF1R) for tumor cell proliferation and survival. Cotargeting IR/IGF1R and PDGFR decreased the emergence of resistant clones in vitro. Our findings characterize a novel model of glioma recurrence that implicates the IR/IGF1R signaling axis in mediating the development of resistance to PDGFR inhibition and provide evidence that IR/IGF1R signaling is important in the recurrence of the proneural subtype of glioma in which PDGF/PDGFR is most commonly expressed at a high level. Mol Cancer Ther; 16(4); 705–16. ©2017 AACR.

Receptor tyrosine kinases (RTK) recognize extracellular signals and activate intracellular adaptor proteins, which in turn mediate downstream effectors such as phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) or mitogen-activated protein kinases (MAPK)/extracellular signal–regulated kinases (ERK1/2) to modulate important pathologic properties of tumors including proliferation, resistance to apoptosis, and cell motility (1). Activation of RTKs has been linked to the initiation, maintenance, and progression of many different tumor types including glioblastoma (GBM; refs. 2, 3). GBM is one of the most common malignant intracranial tumors in adults (4). Despite optimal medical management, the median survival of patients with GBM is only 12 to 15 months (4). Surgery, chemotherapy, and radiation can contribute to an initial response, but most GBMs progress despite therapy or become resistant to therapy and recur. Therefore, significant efforts are currently under way to find more effective approaches to treat GBM including the use of tyrosine kinase inhibitors (TKI).

TKIs have been used in the clinic to treat tumors in which there was evidence for the pathologic activation of the RTK that they target, and in some cases this approach has dramatically improved patient survival (1, 2). Examples of this strategy currently being used for treatment include epidermal growth factor receptor (EGFR) inhibition for lung cancer, human epidermal growth factor 2 (HER2) inhibition for breast cancer, and breakpoint cluster region (BCR)-Abelson (ABL) inhibition for chronic myeloid leukemia (CML; ref. 5). Recent transcriptomic analysis of high-grade gliomas resulted in the characterization of four clinically relevant subtypes: proneural, neural, classical, and mesenchymal glioblastoma (6). Each of these subtypes is defined by a specific molecular signature (6). Genetic alterations resulting in increased platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) signaling are a characteristic feature of the proneural subgroup of gliomas (6–8). The importance of PDGF signaling in the proneural subgroup of high-grade gliomas, which is the least responsive to therapy, provided a strong rationale for clinical trials to evaluate PDGFR inhibitors in the treatment of this type of glioma (9). However, the clinical efficacy of RTK inhibitors has been shown to be compromised by the rapid emergence of resistance in both glioma (4, 10) and other tumor types (11).

Multiple mechanisms of resistance to RTK-targeted therapy, including mutation of the active site, amplification of the targeted receptor, and activation of alternative RTKs, have been identified in a variety of tumors (12). Given this diversity of mechanisms known to contribute to the development of RTK resistance, we sought to characterize therapeutic resistance to PDGF/PDGFR inhibition so that novel clinical strategies to inhibit these key drivers of tumor progression could be developed. This is especially important in the context of proneural gliomas where the current standard of therapy has shown the least benefit relative to the other subtypes of glioma (6).

Cell cultures

Primary mouse tumor sphere cells (TSCs) from CNS glioma or from flank allografts were isolated as previously described (13). The first two passages of TSCs were maintained in EGF-containing (20 ng/mL) DMEM/F12 with 1X B27 supplement with insulin. TSCs were then propagated in DMEM/F12 with 1X B27 supplement with or without insulin, in a humidified 5% CO2 atmosphere at 37°C.

Mouse allograft

To establish doxycycline-resistant allografts from doxycycline-sensitive TSCs, we injected 100,000 doxycycline-sensitive TSCs into Nu/Nu or C57BL/6J syngeneic mice. Following formation of a palpable tumor, animals were treated with oral doxycycline and were observed for regression in allograft size. Oral doxycycline was always administered at 1 g/L in the animal's drinking water. All tumors initially regressed. After three months of treatment, recurrent flank allografts were excised and cultured. All animal care was in accordance with approved IACUC-approved protocols.

Basso mouse scale assessment

To assess neurological deficits in tumor-bearing mice, functional hind limb strength is evaluated using the Basso mouse scale as previously described (14).

Drugs

AG1295 (Sigma-Aldrich; ref. 15,), OSI-906 (Selleckchem; ref. 16), Imatinib (Sigma-Aldrich), LY294002 (Sigma-Aldrich; ref. 17) and UO126 (Qiagen; ref. 18) stock solutions were prepared in DMSO and stored in −20°C. Doxycycline (Santa Cruz Biotechnology) is diluted in PBS as a stock and stored in −20°C. All drugs were diluted into fresh media immediately before experimentation.

SiRNA transfections

TSCs were seeded at 50,000 cells/mL and transfected with Neofx (Ambion) per the manufacturer's manual. Pooled siRNA targeting IR (Thermo Scientific), IGF1R (Thermo Scientific), and the control siRNA siGlo (Thermo Scientific) were transfected at 25 nmol/L final concentration.

Cell growth and cell death

TSCs were seeded into 6 or 96 well plates at a concentration of 50,000 cells/mL and grown for 7 days. As a surrogate measurement of cell number, total ATP was examined in lysed cells based on the luminescence produced by the reaction of ATP with luciferase and D-luciferin (ATPlite, PerkinElmer). For some experiments, cell counts were determined on a Nexolom cell counter. Cell viability was monitored by fluorescence-activated cell-sorting (FACS) analysis of propidium iodide (PI) exclusion assay (19).

Western blotting and RTK antibody array

Cells lysed in RIPA buffer were size fractionated in 8% SDS-PAGE gels and subsequently transferred to PVDF (Millipore). Antibodies detecting cleaved and uncleaved PARP, total PDGFRα/β, phospho-PDGFRα/β, total AKT, phospho-AKT, total ERK1/2, phospho-ERK1/2, phospho-ErbB2, total ErbB2, phospho-IGF1R/IR, total IR, and total IGF1R were purchased from Cell Signaling Technology. Each antibody was diluted at 1:1,000 in TBST containing 5% BSA for overnight immunoblotting. An HRP-conjugated actin antibody (Sigma) was used at 1:50,000 to detect β-actin as a loading control. The RTK Antibody Array was purchased from R&D Systems, Inc. Following treatment of TSC-S1 and TSC-R1 with doxycycline (1 μg/mL) for 48 hours, whole-cell lysates were used to probe RTK antibody arrays, upon which a series of antibodies to RTKs were spotted in duplicate.

Immunohistochemistry

Tumor tissues were perfusion fixed by immersion in 4% PFA overnight, and then embedded in paraffin. Immunohistochemical analysis was performed by the Norris Cotton Cancer Center Research Pathology Shared Resource (Lebanon, NH) using standard techniques. Antibodies detecting PDGFRα/β and IR/IGF1R were purchased from Cell Signaling Technology. Each was diluted at 1:500.

Real-time PCR

RNA was isolated using the RNeasy Kit (Qiagen) and reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). To examine mRNA levels, we utilized the iQ SYBR Green Supermix (Bio-Rad) and MyIQ Real-Time PCR system (Bio-Rad). Expression data were analyzed relative to untreated or DMSO control and normalized to 18S Ct values. Primer sequences are provided in Supplementary Table S2.

Generation and application of RTK signature

The RTK signature was derived from previously published data from our lab (20). The microarray profile of untreated GFAP/tTA:TRE/hPDGFB cells was compared with doxycycline-treated cells by taking the log2-fold change of all genes in the array. A P value was computed for each gene and –log10 transformed. These values were used to generate an upregulated and downregulated profile; in the upregulated profile, all downregulated genes were assigned a zero value and vice versa for the downregulated profile. The BASE algorithm was used to calculate an RTK score for each patient by taking the tumor gene expression profile and the upregulated and downregulated signatures as input (21).

Data queries and statistical analysis

All data are representative of three or more independent experiments, presented as the mean ± SEM. We used the two-tailed student t test to evaluate differences between two groups, and one-way ANOVA to evaluate differences in multiple groups, followed by the Tukey correction. Dose–response curves were compared using two-way ANOVA, and each time point was compared with the one-way ANOVA and corrected using the Tukey correction. The frequency of recurrence in doxycycline and OSI-906 treated TSCs was analyzed using a contingency table. Gene expression data of human GBM or human proneural GBM were obtained through The Cancer Genome Atlas (TCGA). Heat map and hierarchical clustering were performed by GENE-E software. All statistical analyses were performed by Graphpad Prism 5 software.

Seeking an in vivo model to study the mechanism of resistance to RTK inhibition in high-grade gliomas, we took advantage of the PDGF/PDGFR-driven mouse model of proneural glioma developed previously in our laboratory (22). This model uses the glial fibrillary acidic protein (GFAP) promoter to drive the expression of the tetracycline transactivator (tTA) which regulates expression of a transgene encoding human platelet-derived growth factor B by binding to a tetracycline responsive element that is located just upstream of the transgene (TRE; ref. 11). This GFAP/tTA:TRE/hPDGFB mouse model produces tumors that were characterized in our laboratory to faithfully model the proneural subtype of glioma (20) as defined by Verhak and colleagues (6). Expression of the hPDGFB ligand in the GFAP/tTA:TRE/hPDGFB mouse drives tumorigenesis. PDGFB is recognized by both PDGFRA and PDGFRB homodimers or heterodimers (23). Exposure to either tetracycline or doxycycline inhibits transcription of the hPDGFB transgene to decrease PDGFR signaling, thereby mimicking important aspects of the therapeutic activity of RTK inhibitors (19, 20, 22).

To study the effectiveness of targeted RTK inhibition on established PDGF/PDGFR-driven high-grade proneural gliomas, we treated GFAP/tTA:TRE/hPDGFB mice showing evidence of glioma. We withdrew oral doxycycline administration from adult GFAP/tTA:TRE/hPDGFB mice to allow hPDGFB production. Then, when these mice exhibited symptoms of a CNS tumor, we reexposed them to oral doxycycline. In this mouse model, progressive paralysis is an effective surrogate for tumor development (22), and it can be measured using the Basso Mouse Scale for Locomotion (14, 22, 24). We evaluated mice during the course of oral doxycycline administration and found that even though all mice (n = 30) treated with doxycycline exhibited evidence of tumor regression, some animals relapsed after a period of remission (n = 8) developing disabilities in locomotion we could recognize as evidence of tumor progression (Supplementary Fig. S1A and S1B). Tumors arising in these animals that became resistant to treatment were used to prepare TSCs, designated TSC-R1, TSC-R2, and TSC-R3 (Supplementary Table S1). These cultures were evaluated in subsequent experiments.

We also developed an allograft model to study resistance to inhibition of PDGFR in glioma driven to grow by PDGF/PDGFR. We prepared TSCs from individual tumors that arose in GFAP/tTA:TRE/hPDGFB animals that had not been exposed to doxycycline. The growth of these TSC cultures was sensitive to doxycycline, and they were designated TSC-S1 and TSC-S2 (Fig. 1A; Supplementary Fig. S1A and Supplementary Table S1). Subsequently, we injected these TSCs into the flank of syngeneic animals. Once flank tumors were approximately 300 mm3, animals bearing these tumor allografts were treated with oral doxycycline (Supplementary Fig. S1C and S1D). As we observed in the spontaneous model described above, we found that all mice treated with doxycycline exhibited tumor regression (n = 17), but some animals (n = 13) relapsed after a period of remission of 1 to 3 months (Supplementary Fig. S1D). These recurrent tumors grew despite continued oral doxycycline treatment. TSCs from these relapsed tumors provided a second set of cellular reagents from resistant tumors, designated TSC-R4, TSC-R5, and TSC-R6 (Supplementary Table S1) with which to pursue these studies of therapeutic resistance.

Figure 1.

TSCs derived from recurrent GFAP/tTA:TRE/hPDGFB tumors are resistant to hPDGFB suppression. A, Relative cell growth measured by ATP content of TSC-Ss and TSC-Rs cultures treated with doxycycline for 7 days. Data points represent five independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-Ss lines to all TSC-Rs lines, P < 0.0001. B, Relative cell growth measured by ATP content of TSC-Ss and TSC-Rs cultures treated with imatinib for 7 days. Data points represent five independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-Ss lines to all TSC-Rs lines, P < 0.0001. C, Cell death measured by FACS analysis of PI positivity in TSCs grown in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 72 hours. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM; **, P < 0.01; ***, P < 0.001. D, Cell death measured by FACS analysis of PI positivity in TSCs grown in the presence (+) or absence (−) of AG1295 (10 μmol/L) for 72 hours. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM; ***, P < 0.001. E, Western blot analysis of PARP cleavage in representative sensitive and resistant TSCs in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 48 hours.

Figure 1.

TSCs derived from recurrent GFAP/tTA:TRE/hPDGFB tumors are resistant to hPDGFB suppression. A, Relative cell growth measured by ATP content of TSC-Ss and TSC-Rs cultures treated with doxycycline for 7 days. Data points represent five independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-Ss lines to all TSC-Rs lines, P < 0.0001. B, Relative cell growth measured by ATP content of TSC-Ss and TSC-Rs cultures treated with imatinib for 7 days. Data points represent five independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-Ss lines to all TSC-Rs lines, P < 0.0001. C, Cell death measured by FACS analysis of PI positivity in TSCs grown in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 72 hours. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM; **, P < 0.01; ***, P < 0.001. D, Cell death measured by FACS analysis of PI positivity in TSCs grown in the presence (+) or absence (−) of AG1295 (10 μmol/L) for 72 hours. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM; ***, P < 0.001. E, Western blot analysis of PARP cleavage in representative sensitive and resistant TSCs in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 48 hours.

Close modal

TSCs derived from recurrent proneural hPDGFB-driven tumors are resistant to PDGF/PDGFR inhibition

While hPDGFB suppression mediated by doxycycline exposure (Supplementary Fig. S2A) dramatically inhibited the growth of TSC-S lines, it had no significant effect on the growth of TSC-R cultures, regardless of whether they were derived from de novo hPDGFB-driven CNS glioma or hPDGFB-driven flank allografts (Fig. 1A and Supplementary S2A). Further validating the resistance of TSC-Rs to PDGF/PDGFR signaling inhibition, TSC-Rs exhibited a significantly decreased sensitivity to treatment with Imatinib, a pharmacologic inhibitor of PDGFR that readily suppressed the growth of TSC-Ss (Fig. 1B; ref. 25). To examine the effect of PDGF/PDGFR signaling on the viability of these TSCs, we used a dye-exclusion assay. After exposing TSCs to doxycycline or AG1295, a chemical inhibitor of PDGFR, we incubated these cultures with propidium-iodide (PI) and evaluated them. Inhibition of PDGFR using either doxycycline or AG1295 induced cell death in TSC-S cultures but not in TSC-R cultures (Fig. 1C and 1D). To confirm the induction of apoptotic cell death, we examined PARP cleavage following inhibition of PDGF/PDGFR in the doxycycline-sensitive and resistant TSCs (Fig. 1E). Cleaved PARP, which provides evidence of apoptosis, was detected only in TSC-Ss and not in TSC-Rs following the inhibition of PDGF/PDGFR signaling. This provides additional evidence that cell survival of TSC-Rs was not affected by inhibition of PDGF/PDGFR signaling (Fig. 1C–E). These in vitro studies indicate that TSCs derived from our in vivo models retain the PDGF sensitivity of the tumors from which they were derived.

Proneural TSCs resistant to PDGF/PDGFR inhibition require PI3K/AKT and MAPK/ERK signaling for cellular proliferation

Because the function of the PI3K/AKT and MAPK/ERK1/2 signaling pathways in driving cellular proliferation is well characterized (26, 27), we examined whether PDGFR activation regulated these second messengers in TSCs. As expected, treatment of all TSC lines with doxycycline reduced hPDGFB mRNA to undetectable levels (Supplementary Fig. S2A) and consequently decreased the levels of PDGFR phosphorylation as shown in several representative cultures (Fig. 2A). Also, treatment with AG1295, a PDGFR inhibitor, resulted in a marked reduction of PDGFR phosphorylation in all treated cell cultures as shown in several representative cultures (Fig. 2B; ref. 15). In sensitive lines, doxycycline, as well as pharmacologic inhibition of PDGFR with AG1295 or imatinib, suppressed AKT and ERK1/2 phosphorylation as shown for several representative cultures (Fig. 2A and B), reduced cellular growth, and increased cell death (Fig. 1A–D). Consistent with these data, expression of a constitutively activated AKT (myristoylated-AKT) rescued a representative sensitive TSC line, TSC-S2, from doxycycline-mediated growth suppression (Supplementary Fig. S3A) illustrating the importance of AKT activation in PDGF/PDGFR-mediated growth of TSC-Ss. In TSC-R cultures, however, PDGFR inhibition had less of an effect on the canonical PDGFR downstream targets, AKT and ERK1/2 (Fig. 2A and B). Whether the PDGFR inhibition was mediated by doxycycline treatment or pharmacological inhibition, it had less or no detectable effect on the phosphorylation of AKT and ERK1/2 as well as no significant effect on cellular growth in TSC-Rs (Figs. 1A and 2A). These findings indicate that TSC-Rs are able to maintain AKT and ERK1/2 activation in the absence of PDGFR signaling. This loss of PI3K/AKT and MAPK/ERK regulation by PDGF/PDGFR provides evidence that PI3K/AKT and MAPK/ERK may still be key mediators of proliferation and survival in TSC-Rs. Moreover, it suggests TSC-Rs have developed an alternative mechanism for maintaining the activation of these pathways, thereby conferring resistance to PDGFR inhibition.

Figure 2.

The proliferation of TSCs resistant to hPDGFB suppression is repressed by inhibition of the PI3K or MAPK pathway. A, Western blot analysis of phosphorylated PDGFR and phosphorylated and total AKT and ERK1/2 in TSCs grown in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 48 hours. B, Western blot analysis of phosphorylated PDGFR and phosphorylated and total AKT and ERK1/2 in TSCs grown in the presence (+) or absence (−) of AG1295 (10 μmol/L) for 48 hours. C, Relative cell growth measured by ATP content of TSCs treated with DMSO, AG1295, LY294002, or UO126 at 10 μmol/L for 120 hours. DMSO was used as a control. Data represent three or more independent experiments conducted in triplicate presented as the mean +/− SEM. *, P < 0.05; **, P < 0.01. D, Western blot analysis of phosphorylated and total AKT and ERK1/2 in TSC-R4 grown in the presence (+) or absence (−) of LY294002 (10 μmol/L) or UO126 (10 μmol/L) for 48 hours. E, RTK Antibody Array (R&D Systems, Inc.). Following treatment of TSC-S1 and TSC-R1 with doxycycline (1 μg/mL) for 48 hours, whole-cell lysates were used to probe RTK antibody arrays, upon which a series of antibodies to RTKs were spotted in duplicate. Expressed RTKs are framed and numbered. The corresponding RTK is listed on the right.

Figure 2.

The proliferation of TSCs resistant to hPDGFB suppression is repressed by inhibition of the PI3K or MAPK pathway. A, Western blot analysis of phosphorylated PDGFR and phosphorylated and total AKT and ERK1/2 in TSCs grown in the presence (+) or absence (−) of doxycycline (1 μg/mL) for 48 hours. B, Western blot analysis of phosphorylated PDGFR and phosphorylated and total AKT and ERK1/2 in TSCs grown in the presence (+) or absence (−) of AG1295 (10 μmol/L) for 48 hours. C, Relative cell growth measured by ATP content of TSCs treated with DMSO, AG1295, LY294002, or UO126 at 10 μmol/L for 120 hours. DMSO was used as a control. Data represent three or more independent experiments conducted in triplicate presented as the mean +/− SEM. *, P < 0.05; **, P < 0.01. D, Western blot analysis of phosphorylated and total AKT and ERK1/2 in TSC-R4 grown in the presence (+) or absence (−) of LY294002 (10 μmol/L) or UO126 (10 μmol/L) for 48 hours. E, RTK Antibody Array (R&D Systems, Inc.). Following treatment of TSC-S1 and TSC-R1 with doxycycline (1 μg/mL) for 48 hours, whole-cell lysates were used to probe RTK antibody arrays, upon which a series of antibodies to RTKs were spotted in duplicate. Expressed RTKs are framed and numbered. The corresponding RTK is listed on the right.

Close modal

To evaluate whether the persistent activation of PI3K/AKT and MAPK/ERK1/2 mediates the resistance of these glioma-derived TSCs to PDGFR inhibition, we assessed the growth of these cells after treatment with AG1295, LY294002, or UO126, pharmacologic agents that specifically target PDGFR, PI3K, or ERK1/2, respectively (Fig. 2C; refs. 1, 26, 27). In TSC-S cultures, we observed growth inhibitory effects with PDGFR, PI3K, or ERK1/2 inhibitors, as expected. In TSC-S cultures, the most significant effect of these various inhibitors on growth was observed following AG1295 inhibition of PDGFR (Fig. 2C), most likely because it leads to the inhibition of both the PI3K/AKT and MAPK/ERK1/2 axis (Fig. 2B). In TSC-R cultures, however, we observed a different growth response following such drug treatments (Fig. 2C). None of the resistant lines were sensitive to PDGFR inhibition, though all but one of them (TSC-R3) were sensitive to the growth inhibitory effect of the PI3K inhibitor, LY294002, and the ERK1/2 inhibitor, UO126 (Fig. 2C). The activity of these drugs in a representative TSC-R cell line is demonstrated in Figure 2D. The growth inhibitory effect of LY294002 and UO126 was statistically significant in all TSC-R lines except for TSC-R3 (Fig. 2C). These findings support a model of resistance to RTK inhibition in which the molecular alterations maintaining proliferation are upstream of the PI3K/AKT and MAPK/ERK1/2 axis.

IR/IGF1R is required for proliferation of proneural hPDGFB-driven TSCs after they become resistant to PDGF/PDGFR inhibition

To determine if an alternative RTK upstream of AKT and ERK1/2 may be responsible for the maintenance of AKT and ERK1/2 activation and the proliferation of TSC-R cultures, we examined the phosphorylation of a panel of RTKs in representative TSC cultures (Fig. 2E). We found increased phosphorylation of three RTKs in the resistant line: proto-oncogene Neu (ErBB2), muscle specific kinase (MuSK), and insulin receptor (IR)/insulin growth-like factor receptor (IGF1R; Fig. 2E). However, the increase in MuSK and ErBB2 could not be validated by Western blot in these cell lines or others.

Based on these data and previously reported findings supporting a role for IR/IGF1R in maintaining the activation of AKT and ERK in GBM (3), we conducted a histologic analysis to characterize the expression of IR/IGF1R throughout the process of remission and recurrence in response to doxycycline-mediated hPDGFB suppression in our allograft model. Recurrent tumors that grew despite hPDGFB suppression invariably expressed IR/IGF1R uniformly throughout the tissue analyzed and at a generally higher level than seen in tissue from primary tumors (Supplementary Fig. S4A–S4C). Consistent with this observation, the expression of IR and IGF1R was elevated in TSC-R cultures compared with the levels in the parental TSC-S cultures from which they were derived, as demonstrated in pairs of such cell lines when examined for the steady-state level of encoding mRNA (Supplementary Fig. S5A and S5B) or in a representative pair of such TSCs when examined for expression of pro-IR, total IR, or IGF1R protein (Fig. 3A). We also observed that total protein levels of IR and IGF1R decreased when insulin was present (Fig. 3A), consistent with the increased protein turnover of RTKs following ligand binding (1). These data illustrate that IR/IGF1R is expressed in some cells within sensitive tumors and in TSC-Ss, but it becomes much more abundant in resistant tumors and TSC-Rs. This enhanced expression of IR/IGF1R in vitro and in vivo suggests that activation of a preexisting IGF1R pathway might contribute to the development of resistance to PDGFR inhibition in these proneural glioma cells.

Figure 3.

Sensitivity to IGF1R/IR inhibition of TSCs derived from recurrent tumors. A, Western blot analysis of pro-IR and phosphorylated and total IGF1R/IR, AKT, and ERK1/2 in TSC-S2 and TSC-R6 in the presence (+) or absence (−) of Insulin (200 ng/mL) for 48 hours in serum-free medium. B, Relative cell growth measured by ATP content of TSCs cultured in the presence (+) or absence (−) of insulin (200 ng/mL) for 96 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; ***, P < 0.001. C, Cell death measured by FACS analysis of propidium iodide (PI) positivity of TSCs in the presence (+) or absence (−) of insulin (200 ng/mL) for 72 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. **, P < 0.01; ***, P < 0.001. D, Western blot analysis of phosphorylated and total IR/IGF1R, AKT, and ERK1/2 in TSC-R1 and TSC-S2 treated with DMSO or OSI-906 (5 μmol/L) for 48 hrs in serum-free medium. E, Relative cell growth measured by ATP content of TSCs treated with an IR/IGF1R inhibitor, OSI-906, at the indicated concentrations for 96 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-S lines to all TSC-R lines, P < 0.0001. F, Cell death measured by FACS analysis of PI positivity of TSC-S2 and TSC-R6 treated with DMSO or OSI-906 (5 μmol/L) for 72 hrs in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 3.

Sensitivity to IGF1R/IR inhibition of TSCs derived from recurrent tumors. A, Western blot analysis of pro-IR and phosphorylated and total IGF1R/IR, AKT, and ERK1/2 in TSC-S2 and TSC-R6 in the presence (+) or absence (−) of Insulin (200 ng/mL) for 48 hours in serum-free medium. B, Relative cell growth measured by ATP content of TSCs cultured in the presence (+) or absence (−) of insulin (200 ng/mL) for 96 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; ***, P < 0.001. C, Cell death measured by FACS analysis of propidium iodide (PI) positivity of TSCs in the presence (+) or absence (−) of insulin (200 ng/mL) for 72 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. **, P < 0.01; ***, P < 0.001. D, Western blot analysis of phosphorylated and total IR/IGF1R, AKT, and ERK1/2 in TSC-R1 and TSC-S2 treated with DMSO or OSI-906 (5 μmol/L) for 48 hrs in serum-free medium. E, Relative cell growth measured by ATP content of TSCs treated with an IR/IGF1R inhibitor, OSI-906, at the indicated concentrations for 96 hours in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. When comparing all TSC-S lines to all TSC-R lines, P < 0.0001. F, Cell death measured by FACS analysis of PI positivity of TSC-S2 and TSC-R6 treated with DMSO or OSI-906 (5 μmol/L) for 72 hrs in serum-free medium. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; **, P < 0.01.

Close modal

We used insulin as a tool to determine if there was a functional role in TSC-Rs for the increased expression of IR and IGF1R, which respond to both insulin and IGF (28–32). We found that in the absence of insulin, cell death increased and relative cell growth decreased in average by more than 50% in TSC-Rs (Fig. 3B and 3C). Consistent with these data, insulin withdrawal resulted in the inhibition of the canonical RTK downstream target, AKT, in TSC-Rs, and a representative culture demonstrates this in Figure 3A. The proliferation of TSC-R3, which did not require the activation of PI3K or MAPK (Fig. 2C), was unaffected by insulin treatment (Fig. 3B). Importantly, doxycycline-sensitive TSC-Ss, which were isolated and cultured in the same manner as TSC-Rs, were insensitive to insulin withdrawal (Fig. 3B and 3C). We observed no altered cellular growth or changes in cell death following insulin withdrawal from the TSC-S cultures (Fig. 3B and 3C), and in the absence of insulin, the downstream growth regulatory mediators, AKT and ERK1/2, were not activated in TSC-Ss, as demonstrated in a representative TSC-S culture in Figure 3A. These findings suggest a functional role for IR and IGF1R in the proliferation and survival of TSC-Rs.

To examine further the effect of IR/IGF1R inhibition on TSC-Rs, we evaluated the response of TSCs to OSI-906, which inhibits IR and IGF1R activation (ref. 16; Fig. 3D). Comparing the relative cell growth and cell death of TSC-Rs and TSC-Ss in the presence of OSI-906, we found that TSC-Rs are much more sensitive to OSI-906 than the TSC-Ss from which they were derived (Fig. 3E and F, and Supplementary Table S1). We confirmed this finding utilizing pooled siRNAs to modulate IR expression. We found that inhibition of IR expression led to a dramatic growth inhibitory effect in TSC-Rs when compared with the effect seen in a representative TSC-S culture (Supplementary Figs. S5C and S5D). The same siRNAs were used in Supplemental Figs. 5C and 5D. Moreover, inhibition of phosphorylation of IR/IGF1R using OSI-906 also resulted in inhibition of AKT and ERK1/2 activation in TSC-R1 but not in TSC-S2 (Fig. 3D). These findings are consistent with the observation that upon PDGF/PDGFR inhibition sensitive TSCs stop growing and die, while the resistant TSCs do not (Fig. 1A–E). Resistant cells are clearly no longer dependent on PDGF/PDGFR signaling, because they have acquired an alternative RTK, IR/IGF1R, to drive proliferation and survival through AKT and ERK1/2 activation.

Proliferation and survival modulation by IR/IGF1R activation is not evident in the doxycycline-sensitive cultures, TSC-Ss (Fig. 3B and 3C), despite the clear presence of IR/IGF1R in these sensitive cells (Fig. 3A; Supplementary Figs. S5C and S5D). Therefore, we hypothesized that the functional potential of IR/IGF1R signaling may be masked during robust PDGF/PDGFR growth stimulatory pathway signaling in the sensitive TSCs. To test this hypothesis, we incubated sensitive cells, TSC-Ss, in a dose range (25–100 pg/mL) of doxycycline that we determined in preliminary experiments was adequate to inhibit growth but did not cause cell death. We then evaluated the effect of insulin on the proliferation of these cells. Although IR/IGF1R signaling did not enhance the proliferation of sensitive TSC when the PDGF/PDGFR pathway was active (Fig. 3B), when PDGF/PDGFR activation was diminished the effect of insulin on proliferation of TSC-S became obvious (Fig. 4A and 4B). Consistent with these observations, we found that when hPDGFB expression was suppressed in the sensitive TSCs, insulin contributed to maintaining the phosphorylation levels of downstream mediators shared by the PDGFR and IR/IGF1R pathways, including SHP2, AKT, and ERK1/2 (Fig. 4C). We observed that AKT was particularly sensitive to IR/IGF1R inhibition during doxycycline-mediated hPDGFB suppression, as indicated by the sharp decrease in its phosphorylation seen following insulin-withdrawal from TSC-Ss in the presence of doxycycline (Fig. 4C). Unlike our observation that insulin partially rescues the inhibition of cell growth mediated by doxycycline (Fig. 4B), the inhibition of cell growth mediated by the PI3K/AKT inhibitor LY294002 was unaffected by insulin in TSC-Ss and is demonstrated here in a representative line in Figure 4D. These findings suggest that the IR/IGF1R pathway is already expressed and is either active or capable of being activated in TSCs from sensitive tumors. During suppression of PDGFR, the driver RTK, IR/IGF1R is able to mediate proliferation of these sensitive cells through the activation of downstream mediators shared with PDGFR.

Figure 4.

The emergence of TSCs resistant to hPDGFB suppression is decreased by IGF1R/IR inhibition during in vitro drug treatment. A, Phase-contrast microscopy (40×) of TSC-S2 cultured at the indicated concentrations of doxycycline in the presence (+) or absence (−) of insulin (200 ng/mL) for 48 hours. B, Relative cell growth measured by counting TSC-S2 cells cultured in the presence (+) or absence (−) of doxycycline (100 pg/mL) and insulin (200 ng/mL) for 7 days. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; **, P < 0.01. C, Western blot analysis of phosphorylated SHP2 as well as total and phosphorylated AKT and ERK1/2 in TSC-S2 grown in the presence (+) or absence (−) of insulin (200 ng/mL) at the indicated concentrations of doxycycline for 48 hours. D, Relative cell growth measured by counting TSC-S2 cells cultured in the presence (+) or absence (−) of LY294002 (10 μmol/L) and insulin (200 ng/mL) for 7 days. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. ***, P < 0.001. E, TSC-S2 cells were plated into 24-well plates (5,000 cells per well) and maintained in the presence (+) or absence (−) of doxycycline (1 μg/mL) and OSI-906 at the indicated concentrations. The presence of spheres was examined at 6 weeks. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. **, P < 0.01.

Figure 4.

The emergence of TSCs resistant to hPDGFB suppression is decreased by IGF1R/IR inhibition during in vitro drug treatment. A, Phase-contrast microscopy (40×) of TSC-S2 cultured at the indicated concentrations of doxycycline in the presence (+) or absence (−) of insulin (200 ng/mL) for 48 hours. B, Relative cell growth measured by counting TSC-S2 cells cultured in the presence (+) or absence (−) of doxycycline (100 pg/mL) and insulin (200 ng/mL) for 7 days. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. *, P < 0.05; **, P < 0.01. C, Western blot analysis of phosphorylated SHP2 as well as total and phosphorylated AKT and ERK1/2 in TSC-S2 grown in the presence (+) or absence (−) of insulin (200 ng/mL) at the indicated concentrations of doxycycline for 48 hours. D, Relative cell growth measured by counting TSC-S2 cells cultured in the presence (+) or absence (−) of LY294002 (10 μmol/L) and insulin (200 ng/mL) for 7 days. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. ***, P < 0.001. E, TSC-S2 cells were plated into 24-well plates (5,000 cells per well) and maintained in the presence (+) or absence (−) of doxycycline (1 μg/mL) and OSI-906 at the indicated concentrations. The presence of spheres was examined at 6 weeks. Data represent three independent experiments conducted in triplicate presented as the mean ± SEM. **, P < 0.01.

Close modal

To further characterize the role of IR/IGF1R in the development of resistance to PDGFR inhibition in hPDGFB-driven proneural TSCs, we examined the effect of IR/IGF1R inhibition on the emergence of TSC populations resistant to PDGFR inhibition (Fig. 4E). We plated 5,000 freshly dispersed hPDGFB-driven proneural cells in 24-well plates and incubated them in the presence of insulin for 6 weeks in increasing doses of OSI-906 alone or in combination with doxycycline using DMSO as the vehicle. The concentrations of doxycycline and OSI-906 used were previously shown to significantly inhibit the proliferation of TSC-Ss and TSC-Rs, respectively (Fig. 1A and 3D). After this prolonged incubation, we observed the emergence of tumor spheres in most of the cultures exposed to doxycycline alone. The addition of OSI-906 to doxycycline, however, resulted in a very significant reduction in the frequency with which tumorspheres appeared (Fig. 4E). The effect of adding OSI-906 to the doxycycline-mediated hPDGFB suppression was dose dependent and this addition of OSI-906 achieved approximately a 90% reduction of tumorsphere formation at a dose of 0.5 μmol/L when compared with the DMSO control. OSI-906 alone, however, had no effect on tumorsphere formation. We interpreted these findings to indicate that OSI-906–mediated IR/IGF1R inhibition suppressed the expansion of the subset of cells in the TSC-S cultures that were able to develop resistance to doxycycline.

IGF1R is associated with survival and RTK activity in a subgroup of proneural GBMs with lower PDGFRA and PDGFRB levels

In an initial effort to explore the clinical relevance of our current findings, we queried TCGA database seeking to identify evidence within the proneural subgroup of human GBM, suggesting a pathologic role for IGF1R. Utilizing Verhak's gene expression-based molecular classification of GBM, we identified 95 proneural GBMs with complete mRNA data in TCGA provisional dataset (6). Euclidian hierarchical clustering, based on the expression of the 58 known human RTKs, identified two distinct patient groups: cluster “A” and cluster “B” (Fig. 5A). Interestingly, cluster “B” has a 2.4-fold lower average expression of PDGFRA (P < 0.0001) and a 1.5-fold lower average expression of PDGFRB (P < 0.0001) than was observed in cluster “A.”

Figure 5.

IGF1R is associated with survival and RTK activity in a subgroup of proneural GBMs with lower PDGFRA levels. A, Heat map and dendogram show hierarchical clustering of 95 proneural samples from the TCGA database based on RTK expression. B, Kaplan–Meier survival curves comparing IGF1R high (z > 0.5) and IGF1R low (z ← 0.5) proneural GBM patients within cluster B shown in A. Log-rank test comparing the survival curves has a P value of 0.0408.

Figure 5.

IGF1R is associated with survival and RTK activity in a subgroup of proneural GBMs with lower PDGFRA levels. A, Heat map and dendogram show hierarchical clustering of 95 proneural samples from the TCGA database based on RTK expression. B, Kaplan–Meier survival curves comparing IGF1R high (z > 0.5) and IGF1R low (z ← 0.5) proneural GBM patients within cluster B shown in A. Log-rank test comparing the survival curves has a P value of 0.0408.

Close modal

To develop a measure of RTK activity for each GBM sample, we derived a “RTK signature” from the expression profile of TSC-Ss treated with and without doxycycline. We assumed that RTK drivers share common downstream pathways, and used this signature to derive a RTK activity score for each patient. This activity score was positively correlated with the expression of PDGFRA (P < 0.0001) and PDGFRB (P < 0.0001) in the proneural GBMs analyzed, which asserts its validity as a measure of RTK activity.

To test RTKs that could potentially be driving tumor progression in these clusters, we correlated the mRNA expression level of each RTK with RTK activity and survival. While many RTKs are differentially expressed between clusters “A” and “B”, we found that in cluster “B,” which has lower PDGFRA and PDGFRB levels than were observed in cluster “A,” IGF1R was the only RTK amongst all 58 known human RTKs examined, whose expression was positively correlated with both RTK activity (Spearman r = 0.2305, P = 0.0384) and decreased survival (Spearman r = −0.3326, P = 0.0068). Furthermore, in this cluster, IGF1R expression was associated with shorter patient survival (Fig. 5B). We interpret these data as being consistent with the possibility that IGF1R-mediated RTK signaling might serve as an alternative pathway driving proneural GBM progression in the absence of high PDGF/PDGFR signaling.

IR and IGF1R are homologous proteins (3, 30, 32, 33) which share common ligands and common downstream targets (28–32). These RTKs, which are widely coexpressed, are known to heterodimerize activating various downstream targets important for growth and survival (28, 34–38). Previous reports on IR/IGF1R in the context of therapeutic resistance to targeted RTK therapy have recognized the sensitization of various tumors to RTK therapy that results when an inhibitor of IR/IGF1R is used in combination with a second TKI, including PDGFR inhibitors (39–48). For example, preclinical studies have shown that dual targeting of IGF1R and PDGFRα/β act synergistically to inhibit the growth of high-grade gliomas (39–41). Similarly, it has been reported that that coinhibition of IR/IGF1R and EGFR improves the treatment of subcutaneous GBM xenografts in a synergistic fashion (46). Consistent with these reports, insulin and IGF1 have been shown to antagonize EGFR inhibition in EGFR-dependent GBM patient-derived mouse xenografts (46). In the clinic, coinhibition of IR/IGF1R and EGFR has been suggested to improve the response of lung, breast, and head and neck cancers to RTK therapy in the clinic (41–48). However, these recent reports suggesting a role for IR/IGF1R in the sensitization of tumors to various TKIs including PDGFR (39–48) have not addressed the role of IR/IGF1R in acquired resistance and recurrence.

Our studies demonstrate that activation of IR/IGF1R is a novel bypass RTK associated with the emergence of resistance to PDGFR inhibition in PDGFR-driven proneural GBMs. These studies provide a strong rationale for deciphering the mechanism underlying IR/IGF1R activation in this setting. Unlike the mechanism proposed by Akhavan, in which transcription of the bypass RTK is suppressed by activation of the driver RTK in EGFRvIII mutant GBMs (49), we found that IR/IGF1R was expressed regardless of PDGFR activity in our model of proneural glioma (Supplementary Figs. S4 and S6). IR/IGF1R was expressed and was either active or capable of becoming activated in TSCs from primary, drug sensitive tumors (Fig. 4). As resistance to PDGF/PDGFR inhibition emerges and IR/IGF1R becomes the new driver of proliferation, tumor cells which can respond to insulin become a dominant feature of the recurrent tumor (Fig. 3; Supplementary Fig. S4). In resistant tumor cells, the original driver RTK, PDGFR, continues to be expressed and is phosphorylated in the presence of PDGF (Fig. 2; Supplementary Fig. S6A and S6B), but it no longer mediates viability and proliferation through AKT and ERK1/2 activation (Figs. 1 and 2). Rather, there is an apparent loss of dependence of AKT and ERK1/2 activation from PDGFR signaling as a new association between IR/IG1R and AKT and ERK1/2 becomes more prominent.

These observations might be explained by the selection of a subpopulation of cells, from within the primary sensitive PDGFR-driven tumors that has acquired a growth dependence on IR/IGF1R and thereby mediates resistance to PDGF/PDGFR inhibition upon treatment. Going forward, the development of TKI combination therapies, targeting not only the driver RTKs, but also the bypass RTKs, may significantly enhance treatment outcomes in the proneural subgroup of GBM.

No potential conflicts of interest were disclosed.

Conception and design: D.A. Almiron Bonnin, C. Ran, M.C. Havrda, H. Liu, M.A. Israel

Development of methodology: D.A. Almiron Bonnin, C. Ran, M.C. Havrda, H. Liu, M.A. Israel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.A. Almiron Bonnin, C. Ran, Z. Zhang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.A. Almiron Bonnin, C. Ran, M.C. Havrda, C. Cheng, M. Ung, M.A. Israel

Writing, review, and/or revision of the manuscript: D.A. Almiron Bonnin, C. Ran, M.C. Havrda, M.A. Israel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.A. Almiron Bonnin, C. Ran, Y. Hitoshi, M.A. Israel

Study supervision: D.A. Almiron Bonnin, C. Ran, M.C. Havrda, Z. Zhang, M.A. Israel

The authors wish to thank Drs. D. Compton, L. Witters, and A. Eastman for suggestions and assistance and Tabatha Richardson for her administrative support. The authors also wish to thank Dr. T. Tosteson for his consultation on statistical analysis.

Support was generously provided by the Theodora B. Betz Foundation (M.A. Israel), the Jordan and Kyra Memorial Foundation (M.A. Israel), and the Andrea Clark Nelson Medical Research Endowment (M.A. Israel).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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