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Proc Natl Acad Sci U S A. Jul 10, 2007; 104(28): 11736–11741.
Published online Jul 2, 2007. doi:  10.1073/pnas.0703145104
PMCID: PMC1913900
Medical Sciences

Insulin-like growth factor binding protein 2 promotes glioma development and progression

Abstract

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression in many types of human cancer. In this study we used a glial-specific transgenic mouse model to examine the active role of IGFBP2 in tumorigenesis and progression. Our studies show that IGFBP2 coexpression results in progression to a higher-grade glioma in platelet-derived growth factor β (PDGFB)-driven tumors. These anaplastic oligodendrogliomas are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. Combined expression of IGFBP2 or Akt with K-Ras was required to form astrocytomas, indicating that activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells coexpressing IGFBP2 versus PDGFB expression alone. Thus, this study provides definitive evidence that IGFBP2 plays a key role in activation of the Akt pathway and collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma.

Keywords: glial-specific transgenic mouse model, oligodendroglioma

Cancer genomic profiling studies have recently identified many genes as candidate cancer markers. One such gene, IGFBP2, was found to be overexpressed in high-grade gliomas, glioblastoma multiforme (GBM) (1, 2), and anaplastic oligodendroglioma (3). Insulin-like growth factor binding protein 2 (IGFBP2) is also up-regulated in other high-grade tumors such as prostate (4), ovarian (5), adrenocortical (6), breast (7), and colorectal carcinomas (8) and leukemia (9), as well as in drug-resistant tumors (10). However, gene–phenotype association studies do not provide evidence of a causal relationship between candidate genes and cancer development. In this study we sought to determine the role of IGFBP2 in cancer development and progression using glioma as a model tumor.

Human diffuse gliomas comprise a family of primary brain tumors that result in 13,000 deaths annually in the United States alone. Patients with the most advanced type of glioma, GBM (WHO grade IV), have a meager median survival of less than a year, which has not improved significantly over the last four decades (11, 12). There are two major subtypes of diffuse glioma, oligodendroglioma and astrocytoma, which may arise from transformed oligodendrocytic or astrocytic precursor cells, respectively, or from neural stem cells (11). A number of genetic and molecular alterations have been identified in gliomas, such as loss of p16INK4a (13), loss of PTEN (14), overexpression of EGFR (EGF receptor) (15), and overexpression of PDGFR (platelet-derived growth factor receptor) (16). The role of some of these genes in gliomagenesis has been recapitulated in animal models (17, 18). Among the models currently available, somatic cell gene transfer with RCAS (replication-competent avian leukemia virus splice acceptor) vectors into transgenic mice has proven to be particularly useful in parsing the key events that lead normal neural progenitor cells into the cancerous state (19). In the RCAS/N-tva system, avian virus receptor (tva) is exclusively expressed in glial cells via the glial-specific nestin promoter. Genes of interest are cloned into an avian RCAS vector, and viral particles are expanded in DF-1 avian fibroblasts. DF-1 cells are subsequently injected into the transgenic mice, which results in chronic signaling of the genes of interest (19). A major advantage of this model is its ability to determine whether a gene identified through clinical marker correlation studies plays an active tumorigenesis role. Previous studies have shown that chronic platelet-derived growth factor β (PDGFB) signaling leads to development of oligodendroglioma (20), and the combined delivery of Akt and activated K-Ras leads to astrocytoma development (21). Another advantage of the RCAS/N-tva system is the ability to breed otherwise wild-type N-tva mice with mice of various genetic backgrounds, e.g., INK4a-ARF null, to determine the effect of oncogene activation in the context of tumor suppressor loss. In INK4a-ARF null/N-tva mice, progression of PDGFB-induced oligodendroglioma (22) and Akt/K-Ras-induced astrocytoma to GBM was observed (23). PTEN is mutated or deleted in a large variety of cancers, including GBM, endometrial cancer, and prostate cancer, and has been well characterized as a tumor suppressor (2427). In the RCAS/N-tva system, PTEN loss is associated with Akt up-regulation and progression to GBM (28). Thus, the RCAS/N-tva system has proven to be valuable for elucidating specific components of signaling pathways that are essential for glioma development and progression and can give insight as to how particular pathways synergize in gliomagenesis.

IGFBP2 was initially discovered through genomic studies to be a gene that is overexpressed in high-grade gliomas, and its overexpression is associated with poor patient survival (13). In vitro studies showed that IGFBP2 promotes glioma cell migration and invasion by forming a complex with integrin α5 protein and activating expression of the matrix metalloproteinase 2 gene (29, 30). However, it has not been determined whether IGFBP2 plays a causal role in glioma development and progression in vivo. If IGFBP2 does play a key driving role in the formation and progression of tumors, we would expect to see tumor initiation and/or progression from low-grade tumor to high-grade tumor resulting from IGFBP2 alone or from IGFBP2 in combination with a minimal number of other genes in an otherwise wild-type background.

Results

We cloned IGFBP2 cDNA into the RCAS vector and confirmed its expression first in avian fibroblasts after transfection and subsequently in mouse fetal glial cells after infection. We then tested the role of IGFBP2 in glioma development and progression using an RCAS/N-tva somatic cell gene transfer model, with examination of the key signaling pathways. Mice were killed at the first sign of hydrocephalus, distress, or other ill effect of the tumor, beginning at 3 weeks after injection. Mice that remained healthy in appearance with or without the development of tumor were killed at 13 weeks, which served as an endpoint for the experiment. GFP was delivered alone by the nontransforming RCAS vector, which served as a negative control. Inflammation induced by the injection was a rare event, occurring in <10% of injected mice, and no tumors arose in negative control mice, as was expected and shown in previous reports [supporting information (SI) Fig. 4] (19, 21, 31). RCAS-GFP was also coinjected in all experiments to serve as an injection marker (SI Fig. 5 E and F).

Combination of IGFBP2 and K-Ras Leads to Development of Astrocytomas.

Similar to the previous observation that neither activated K-Ras nor Akt alone is sufficient to induce glioma formation in the RCAS model (20, 21), delivery of IGFBP2 alone also failed to lead to glioma development. We then evaluated the effect of two-gene combinations. IGFBP2 delivered in combination with activated K-Ras led to the formation of astrocytomas that were histologically similar to the astrocytomas that resulted from the combined delivery of Akt and K-Ras (elongated, fibrillary cytoplasm and oval, elongated nuclei) (Fig. 1 A–E). Overall, we saw astrocytoma formation in 17.4% of K-Ras-IGFBP2-injected mice and in 18% of K-Ras-Akt-injected mice (Table 1). No tumor formed when Akt and IGFBP2 were delivered simultaneously, suggesting that IGFBP2 and Akt likely lie in the same pathway or in converging pathways that are important for gliomagenesis.

Fig. 1.
IGFBP2 up-regulation leads to astrocytoma initiation and oligodendroglioma progression. H&E staining of several representative tumors from N-tva mice shows RCAS-GFP vector control-injected gray matter (A), astrocytoma resulting from the combined ...
Table 1.
IGFBP2 coinjections result in astrocytoma initiation and in progression of oligodendroglioma

IGFBP2 Promotes Oligodendroglioma Progression.

Previous studies showed that PDGFB overexpression is a driving event for the development of oligodendrogliomas in the RCAS/N-tva model (20). Previous data have also shown that IGFBP2 overexpression is frequently found in primary anaplastic oligodendrogliomas (3). Consistently, our results confirm that PDGFB alone leads exclusively to the formation of low-grade oligodendrogliomas with very high penetrance (Fig. 1 F–J); overall, oligodendrogliomas developed in 90.1% of injected mice (Table 1). These tumors are characterized by a uniform population of cells with regular, round nuclei surrounded by perinuclear halos, diffusely infiltrating as well as focal nodular growth patterns, and secondary structures of Scherer (the same features as are observed in human oligodendroglioma). Strikingly, anaplastic oligodendrogliomas formed in 37.9% of animals injected with PDGFB plus IGFBP2 (Fig. 1 K–O) (Table 1). Compared with low-grade oligodendroglioma, these higher-grade tumors are characterized by the presence of increased cellular density, increased mitotic activity, vascular proliferation, and foci of necrosis. The mouse tumors are morphologically indistinguishable from human anaplastic oligodendrogliomas. Reactive astrocytes are detected on H&E-stained tissue sections and visualized in detail by GFAP immunostaining (SI Fig. 5 A, B, G, and H), recapitulating another feature seen in human anaplastic oligodendrogliomas. Cellular proliferation of both injection sets was analyzed and quantified by phosphohistone H3 immunostaining for mitotic figures. Three representative tumors were selected from each injection group for quantification. Increased mitotic activity was seen in the tumors resulting from IGFBP2–PDGFB coinjection (Fig. 2). Immunostaining for RCAS-PDGFB-HA and RCAS-GFP injection markers is shown in SI Fig. 5 C–F.

Fig. 2.
IGFBP2 up-regulation increases tumor cell proliferation. PDGFB- and PDGFB–IGFBP2-driven tumors were stained with phosphohistone H3 antibody, which recognizes mitotic figures. Three representative tumors from each injection set were photographed, ...

IGFBP2–PDGFB Codelivery Leads to AKT Pathway Activation.

PDGFB has been previously shown to initiate oligodendroglioma formation; however, these tumors did not arise through activation of the Akt pathway (20, 32). We observed that both IGFBP2 and Akt could collaborate with activated K-Ras to form astrocytomas, whereas the IGFBP2–Akt combination did not (Table 1). This observation led us to hypothesize that IGFBP2 at least partially activates the Akt pathway to promote progression when delivered in combination with PDGFB. To test this, we immunohistochemically examined PDGFB-induced oligodendrogliomas and PDGFB–IGFBP2-induced anaplastic oligodendrogliomas for Akt, phosphorylated Akt (pAkt), and the active form of the Akt downstream protein S6K (pS6K). Phosphorylation of Thr308 and Ser473 of Akt by its upstream kinase(s) or by autophosphorylation is critical for optimal activation of its kinase activity; therefore, we stained with antibodies specific for both phosphorylation sites. The protein kinase S6 kinase (S6K) is a key node of the signaling pathways from Akt to the ribosome. When phosphorylated and activated in an Akt-dependent manner, pS6K in turn phosphorylates and thereby activates the S6 subunit of ribosomes, which increases protein translation (33). Up-regulation and activation of the Akt pathway are important for cellular growth and survival (34). The results illustrated in Fig. 3 A–J show that, indeed, higher levels of Akt, pAkt(Thr308), pAkt(Ser473), and pS6K are present in PDGFB–IGFBP2-induced anaplastic oligodendrogliomas but not in PDGFB-induced oligodendrogliomas.

Fig. 3.
IGFBP2 delivery results in up-regulation and activation of the Akt pathway, the inhibition of which results in reduced viability of IGFBP2 coinfected cultures. (A–J) Immunostaining of PDGFB-driven tumors (A–E) and PDGFB–IGFBP2-driven ...

We further tested this signaling pathway ex vivo. We first collected and cultured the primary glial progenitor cells from newborn N-tva mice. Sterilely filtered RCAS viral particles encoding PDGFB were added to these cells alone or in combination with RCAS viral particles encoding IGFBP2. RCAS viral particles encoding GFP were transferred to all primary cultures as an imaging control for transfer efficiency. Total proteins were isolated 3 weeks after infection and analyzed by Western blotting for signaling molecules in the Akt pathway. PDGFB–IGFBP2 cultures showed an increase in total Akt, pAkt(Ser473), and pAkt(Thr308) (SI Fig. 6). We also detected up-regulation of the Akt pathway downstream molecules S6K and pS6K. These results reconfirmed Akt pathway activation results shown by immunohistochemistry in mouse tumor tissue.

IGFBP2 Expression Leads to Increased Akt Pathway Signaling and Dependence on Akt Signaling for Cell Survival.

The above ex vivo experiments showed that the Akt pathway is activated in IGFBP2–PDGFB-infected cells but not in cells infected with PDGFB alone. This led to our prediction that the IGFBP2–PDGFB-infected cells should be selectively inhibited by Akt inhibitors. Infected primary cultures were treated with an Akt inhibitor (Akt Inhibitor IX; Calbiochem, San Diego, CA) in increasing concentrations (24 and 48 μM), and cell viability was analyzed by Trypan blue assay at 2, 4, and 6 days. A statistically significant increase in cell death was seen in PDGFB–IGFBP2 coinfections versus PDGFB and control cells by the sixth day of drug treatment (Fig. 3 K and L).

Discussion

In the last decade, cancer genomic and proteomic studies have led to the identification of a large number of candidate genes that are associated with cancer prognosis and tumor pathophysiology (35, 36). However, these remain only markers unless functional analyses are able to establish a causal role for them in cancer. Although in vitro cell culture experiments are valuable in gaining insight into the molecular mechanisms of a particular gene of interest, in vivo animal experiments are needed to generate conclusive data in support of the role and relative importance of the gene in tumorigenesis.

In this study, using the glial-specific RCAS/N-tva somatic cell gene transfer model, we provide definitive evidence that the IGFBP2 gene, which is overexpressed during progression in gliomas (astrocytomas and oligodendrogliomas) and a number of other cancer types (prostate, colorectal, ovarian, adrenocortical, breast, and leukemia), exerts a key oncogenic signal in tumorigenesis and tumor progression in two major forms of glioma. Although IGFBP2 by itself does not result in cancer development, only a single additional oncogenic event (K-Ras or PDGFB) is required for IGFBP2 to lead to cancer development and progression, demonstrating the significance of IGFBP2 in cancer.

IGFBP2 likely exerts its oncogenic affect through several mechanisms. We chose to focus on up-regulation and activation of the Akt pathway because of the link between IGFBP2 and several important signaling pathways. IGFBP2 has been shown to bind and regulate the bioavailability of IGFs, in both positive and negative regulatory roles, and can have downstream effects on the Akt pathway (37). IGFBP2 has also been shown to bind integrins (30), which affect cell adhesion and migration, and to use Akt downstream signaling. Results of the present study confirm the ability of IGFBP2 to significantly up-regulate and activate the Akt pathway. The ex vivo inhibitor studies further demonstrate that IGFBP2-expressing cells depend on the Akt signaling pathway because they are selectively killed by Akt inhibitors.

During the review of this article, Mehrian-Shai et al. (38) reported that IGFBP2 is a candidate biomarker for PTEN status and phosphatidylinositol 3-kinase/Akt pathway activation in GBM and prostate cancer. Their study provides additional confirmative evidence of IGFBP2 overexpression in GBM and prostate cancer and further links IGFBP2 overexpression to PTEN mutation and phosphatidylinositol 3-kinase/Akt activation, which are both frequent events in each of the two cancer types (14, 3941). Our study using PTEN wild-type mice showed that IGFBP2 has oncogenic function in the absence of PTEN mutation, which is consistent with the notion that PTEN may serve as an upstream suppressor for IGFBP2 expression. Thus, it is conceivable that IGFBP2 is downstream of PTEN function but upstream of the phosphatidylinositol 3-kinase/Akt pathway.

Because IGFBP2 is expressed in many different types of human high-grade tumors and is present at only very low levels in normal tissues, the results of this study have extremely broad implications for the development of new cancer prognostics and therapeutics. Elevated serum levels of IGFBP2 have been reported in patients with high-grade tumors, such as GBM and prostate cancer, as well as in colorectal and ovarian carcinomas and acute lymphoblastic leukemia (4245). Thus, serum levels of IGFBP2 may constitute a biomarker for Akt pathway activation. The finding that IGFBP2 tumors depend on Akt signaling may also be clinically relevant for patient treatment stratification; patients with IGFBP2 up-regulation may respond better to Akt pathway interventions. Moreover, as additional IGFBP2 signaling pathways are elucidated, novel drugs that are introduced to target these downstream effectors, as well as IGFBP2, may prove to be effective therapeutic agents.

Materials and Methods

RCAS Constructs.

Human IGFBP2-pcDNA3 was digested with HindIII and EcoR1 restriction enzymes (NEB, Ipswich, MA). The 1.4-kb fragment including the entire coding sequence of IGFBP2 was first subcloned into a Yap vector, then transferred into the RCAS-X vector using NotI and ClaI restriction enzymes (NEB). RCAS-PDGFB encodes full-length human PDGFB with an HA tag (20). RCAS-K-Ras encodes the mutant G12D activated K-Ras (21). RCAS-Akt carries the activated form of Akt, designated Akt-Myr Δ11–60, and was kindly provided by Peter Vogt (The Scripps Research Institute, La Jolla, CA). RCAS-GFP, containing full-length GFP, was used as an infection marker and was kindly provided by Yi Li (Baylor College of Medicine, Houston, TX).

Transfection of DF-1 Cells.

DF-1 immortalized chicken fibroblasts were grown in DMEM with 10% FCS (GIBCO, Carlsbad, CA) in a humidified incubator containing 5% CO2 at 37°C. Plasmid forms of RCAS vectors were transfected into DF-1 cells by using the Nucleofector transfection system (Amaxa, Cologne, Germany). Vectors were allowed to replicate as viral particles in culture for 1 month.

N-tva Transgenic Mice.

The N-tva mouse line expresses tva from the glial-specific nestin promoter; production has been previously described (19). The mice are a mixed genetic background, including contributions from C57B16, 129, Balb/C, and FVB/N, and are wild type except for the tva transgene.

Injection of N-tva Transgenic Mice.

DF-1 cells producing various RCAS virions were trypsinized and pelleted by centrifugation; the pellets were resuspended in ≈30 μl of PBS and placed on ice before injection as previously described (20). If a combination of DF-1 cells was injected, cells were thoroughly mixed in equal parts before centrifugation. Using a 10-μl gas-tight Hamilton syringe, a single intracranial injection of 1 μl of cell suspension (≈104 cells) was made in the right frontal region of newborn mice (postnatal day 1), with the tip of the needle just touching the skull base. All mice were coinjected with RCAS-GFP, which served as an injection marker. Hydrocephalus developed in <10% of GFP-only control injected mice; this was a result of inflammation, not tumor, in all cases (SI Fig. 4). Immunohistochemistry of injection controls for PDGFB (HA tag) and GFP is shown in SI Fig. 5 C–F.

Primary Brain Cell Culture.

Newborn N-tva transgenic mice were killed, and the whole brains were mechanically dissociated into small pieces in sterile PBS, Ca2+- and Mg2+-free (pH 7.4), as previously described (20). Dissociation was followed by digestion with 1 ml of 0.25% Trypsin/1 mM EDTA in HBSS (Hanks's balanced salt solution) (GIBCO) in sterile tubes and incubation in 37°C water bath for 15 min with gentle shaking every 5 min. After incubation, fresh medium was added to terminate digestion and large debris was settled. Supernatant containing primary cells was pelleted, washed once with medium, resuspended in DMEM with 10% FCS, and plated.

Ex Vivo Infection of Primary Brain Cell Culture and Inhibitor Analysis.

The supernatants containing various RCAS virions from DF-1 cell cultures transfected with RCAS vectors were collected by using sterile syringes and filtered through 0.22-μm filters, followed by transferring into 60% confluent primary brain cell cultures that had been plated and grown in DMEM with 10% FCS, as previously described (20). Infections were repeated once per day for 2 weeks. After infection, cells were harvested by trypsin digestion for Western blot analysis or replated for inhibitor experiments. For inhibitor experiments, a total of 5 × 103 cells were plated per well in a 96-well plate for each infected line. After 24 h, Akt inhibitor in DMSO (Akt Inhibitor IX; Calbiochem) was added. Cells were harvested by trypsin digestion at days 2, 4, and 6 and analyzed by Trypan blue assay (GIBCO). The percentage of viable versus nonviable cells was calculated.

Brain Sectioning and Immunohistochemistry.

Mice were killed at 13 weeks of age (or before because of symptoms), and the whole brains were fixed in 4% formaldehyde in PBS for at least 24 h. The brains were coronally sectioned into five slices, and these were embedded in paraffin; 7-μm-thick tissue sections were cut with a Leica (Bannockburn, IL) microtome. The sections were stained with H&E. Immunostaining was performed with LSAB2 kits (DAKO Cytomation, Carpinteria, CA). Briefly, deparaffinized slides were treated with antigen unmasking reagent (DAKO) with heating in a microwave steamer for 14 min, followed by 5% hydrogen peroxide in methanol for 30 min. Sections were then blocked with 5% normal goat serum in TBST (TBS and 0.5% Tween 20) for 1 h at room temperature. Antibodies against Akt1 (1:50), pAkt(Ser473) (1:25), pAkt(Thr308) (1:50), pS6K (1:500), and HA tag (1:500) were purchased from Cell Signaling Technology (Boston, MA) and used at the dilutions listed. Antibodies against IGFBP2 (1:250; Santa Cruz Biotechnology, Santa Cruz, CA), GFP (1:500; GeneTex, San Antonio, TX), and GFAP (1:50; Chemicon, Temecula, CA) were purchased and used at listed dilutions. Primary antibodies were diluted in TBST and incubated with sections at 4°C overnight. After washing with TBST, sections were incubated with biotinylated secondary antibodies (DAKO) at room temperature for 1 h. After washing with TBST, avidin-conjugated peroxidase (DAKO) was added at room temperature for 1 h. Finally, after washing with TBST, sections were developed with DAB (DAKO). After terminating the reaction, sections were counterstained with freshly filtered hematoxylin and mounted. Antibodies against phosphohistone H3 (pHH3) (1:1,000; Upstate Biotechnology, Lake Placid, NY) were purchased, and immunohistochemistry was performed as previously described (46). The pHH3 labeling indices were calculated by the standard method of counting the number of positively stained mitotic figures in one high-power (×400) field in the region of greatest tumor cellularity and mitotic activity.

Western Blot Analysis.

Antibodies against phosphatidylinositol 3-kinase, pAkt(Thr308), pAkt(Ser473), Akt, and pS6K were purchased from Cell Signaling Technology and used at 1:1,000. Antibodies against GFP (BD Bioscience, San Jose, CA), S6K (BD Bioscience), and β-actin (Sigma, St. Louis, MO) were purchased and used at 1:1,000. Western blotting was performed as previously described (47).

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Sadhan Majumder and Howard Colman (M. D. Anderson Cancer Center) for assistance and discussion. This work was supported by National Institutes of Health/National Cancer Institute Grant R01 CA98503 (to W.Z. and G.N.F.). The animal facility is supported by National Cancer Institute Cancer Center Support Grant CA-16672. S.M.D. is supported by a training fellowship from the Keck Center Pharmacoinformatics Training Program of the Gulf Coast Consortia (National Institutes of Health Grant 5 T90 DK070109-02) and by a fellowship granted by the American Legion Auxillary of Texas.

Abbreviations

IGFBP2
insulin-like growth factor binding protein 2
PDGFB
platelet-derived growth factor β
GBM
glioblastoma multiforme.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0703145104/DC1.

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