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. 2016 Apr 3;18(11):1487–1497. doi: 10.1093/neuonc/now053

Insulin-like growth factor binding protein-2 regulates β-catenin signaling pathway in glioma cells and contributes to poor patient prognosis

Shilpa S Patil 1, Priyanka Gokulnath 1, Mohsin Bashir 1, Shivayogi D Shwetha 1, Janhvi Jaiswal 1, Arun H Shastry 1, Arivazhagan Arimappamagan 1, Vani Santosh 1, Paturu Kondaiah 1
PMCID: PMC5063512  PMID: 27044294

Abstract

Background

Upregulation of insulin-like growth factor binding protein 2 (IGFBP-2) is often associated with aggressiveness of glioblastoma (GBM) and contributes to poor prognosis for GBM patients. In view of the regulation of β-catenin by IGFBP-2 in breast cancer and the crucial role of β-catenin pathway in glioma invasion, proliferation and maintenance of glioma stem cells, the mechanism of regulation of β-catenin by IGFBP-2, and its role in GBM prognosis was studied.

Methods

Regulation of the β-catenin pathway was studied by immunocytochemistry, Western blot analysis, luciferase assays, and real-time RT-PCR. The role of IGFBP-2 was studied by subcutaneous tumor xenografts in immunocompromised mice using glioma cells engineered to express IGFBP-2 and its domains. GBM patient tumor tissues (n = 112) were analyzed for expression of IGFBP-2 and β-catenin by immunohistochemistry. Survival analysis was performed employing Cox regression and Kaplan-Meier survival analyses.

Results

IGFBP-2 knockdown in U251, T98G, and U373 or overexpression in LN229 and U87 cells revealed a role for IGFBP-2 in stabilization of β-catenin and regulation of its nuclear functions involving integrin-mediated inactivation of GSK3β. Similar results were obtained upon overexpression of the C-terminal domain of IGFBP-2 but not the N-terminal domain. Subcutaneous xenograft tumors overexpressing either full-length or the C-terminal domain of IGFBP-2 showed larger volume as compared with controls. Coexpression of high levels of IGFBP-2 and β-catenin was associated with worse prognosis (P = .001) in GBM patients.

Conclusion

IGFBP-2 potentiates GBM tumor growth by the activation of the β-catenin pathway through its C-terminal domain, and their coexpression possibly contributes to worse patient prognosis.

Keywords: GBM prognosis, GSK3β, IGFBP-2 C-domain, β-catenin signaling

Glioma accounts for almost 30% of all central nervous system tumors and 80% of primary malignant tumors of the central nervous system. Glioblastoma (GBM) comprises 45% of all gliomas and is associated with the poorest survival.1

Insulin-like growth factor binding protein-2 (IGFBP-2) is conventionally known as the modulator of IGF signaling.2 Binding of IGFBP to IGF regulates either the bioavailability to the receptor or the half-life of IGF in circulation.2 Upregulation of IGFBP-2 is often associated with aggressiveness of GBM and poor patient prognosis.3 IGFBP-2 overexpression in glioma cell lines has been shown to increase migration, invasion, and chemoresistance.4–7 Moreover, a single chain monoclonal antibody, which we developed against IGFBP-2, inhibited invasiveness of GBM cells.8

Although most of the IGF-independent functions of IGFBP-2 are known to occur via integrin signaling,4,6,7 the underlying pathways and exact mechanisms behind their regulation remain unknown. So far in glioma, it has been shown that IGFBP-2 activates NFκB9 and JNK5 pathways via α5β1 integrin signaling, which is responsible for IGFBP-2-mediated survival and invasion.

Previously, we have reported the stabilization of β-catenin by IGFBP-2 in breast cancer cells and its association with lymph node metastasis.10 However, the exact mechanism underlying this regulation and its significance with respect to GBM was unclear. The β-catenin pathway is known to play a crucial role in glioma cell proliferation,11 invasion,12 and maintenance of glioma-initiating cells.13 The expression of β-catenin has been shown to increase from low-grade to high-grade glioma,14 and its higher expression is correlated with poorer patient prognosis.15–17 β-catenin is known to act as a cofactor for transcription factors such as T cell factor 3 (TCF3). Embryonic stem cells, as well as poorly differentiated GBM tissues, often share a common gene signature that includes Nanog, Oct-4, and c-Myc expression, which is contributed by the activity of β-catenin-TCF complex.18 Apart from the canonical Wnt-frizzled pathway, other known upstream regulators of β-catenin activity are EGFR16 and integrins.19 The RGD (arginine-glycine-aspartate) motif within the carboxy terminal domain of IGFBP-2 plays a crucial role in integrin binding and activation of integrin signaling.6 Proteolytic cleavage of IGFBP-2 within its linker region results in N and C-terminal fragments, and these fragments are known to have reduced affinity for IGFs.20 Considering this fact, it was assumed that IGFBP-2 loses its IGF regulatory function once cleaved. The C-terminal fragments of IGFBP-2 purified from human hemofiltrate were found to maintain their disulfide bridging pattern,20 ability to bind to cell membrane, and induce proliferation in rat growth plate chondrocytes.21 These studies led us to speculate that the C-terminal domain of IGFBP-2 may be sufficient to induce integrin-mediated functions of IGFBP-2. To date, a protumorigenic function of the C-terminal domain of IGFBP-2 has not been demonstrated.

In view of the above, the aim of this study was to delineate the pathway involved in IGFBP-2-mediated regulation of β-catenin and to determine if the carboxy terminal domain of IGFBP-2 has any role in β-catenin regulation and tumor growth. The current study evaluates possible cross talk between IGFBP-2-induced integrin signaling and β-catenin signaling in glioma, the 2 very important pathways involved in glioma pathogenesis.

Materials and Methods

Cell Lines, Clones, and Transfection

IGFBP-21–328pcDNAmycHis, IGFBP-21–204pcDNAmycHis, IGFBP-2205–328pcDNAmycHis constructs were generated by inserting PCR-amplified regions of full-length, N and C-terminal domains of IGFBP-2 in pcDNAmycHis3.1(+)A. Primer sequences are listed in Supplementary material. Table S1. U87, LN229, U251, A172, T98G, and U373 glioma cell lines were procured in 2013 from a European collection of cell cultures (ECACC) and have been tested and authenticated by DNA profiling and karyotyping. All transfections were done using Lipofectamine 2000 (Life Technologies, USA) as per the manufacturer's protocols. U87 and LN229 stable transfectants were established by selection using G418 (Invitrogen).

Immunocytochemistry

Cells were allowed to grow on cover-slips, fixed with chilled methanol for 10 minutes, incubated with primary antibody overnight (β-catenin, #C2206, Sigma-Aldrich, 1:50), washed with PBS, and then incubated with 1:1500 dilution of anti-rabbit Alexa Flour 488 secondary antibody for 1 hour; 0.5 mg/mL of propidium iodide was used to stain nuclei.

Nuclear-cytoplasmic Fractionation, Treatments, and Western Blotting

Cytoplasmic extract was collected after lysing cells with 0.5% of Nonidet-40 (NP-40). The nuclear pellet was resuspended in lysis buffer (10 mM Tris-Cl; pH 7.4, 4 mM of EDTA, 30 mM KCl, 1% NP-40, 1 mM DTT, 100 µM of sodium ortho-vanadate, 0.5 mM PMSF, and 1x protease inhibitor cocktail from Calbiochem). For treatments, cells were incubated with 250 ng/mL of Wnt3a (R&D Systems)/20 mM of LiCl/10 µM PP2 (Calbiochem)/10 µM PP3 (Calbiochem)/10 µM RGD/10 µM focal adhesion kinase (FAK) inhibitor (Sigma-Aldrich, USA)/10 µM IGF1R (Calbiochem)/500 ng/mL of IGFBP-2 (# 674-B2, R&D Systems, USA) for 24 hours. Twenty µg of total protein was used for Western blot analysis. The antibodies used in the study were IGF1R, p-GSK3β, GSK3β, p-FAK, FAK, p-β-catenin, p-Akt (Ser 473), Akt (obtained from Cell Signaling Technologies, USA); p-Akt 1/2/3 (Thr308), IGFBP-2 (Santa Cruz, USA); β-catenin and β-Actin (Sigma-Aldrich); p-IGF1R was obtained from Abcam, UK; lamin was purchased from IMGENEX, India. IGFBP-2 (#C-18 sc6001, Santa Cruz) and β-catenin (#C2206, Sigma-Aldrich, USA) antibodies were used for immunohistochemistry. An in-house generated rabbit polyclonal anti IGFBP-2 antibody was utilized for immunohistochemistry on xenograft tissues.

Luciferase Assays

Cells were plated in 24 well dishes and after 18–24 hours were transfected with 500 ng each of IGFBP-2-pcDNAmycHis constructs + 500 ng of Top/flash reporter construct + 10 ng of Renilla luciferase construct (pRL-TK). Thirty-six hours after transfection, treatments were given for 24 hours, and cells were lysed in lysis buffer. Assays were done as per the manufacturer's protocol (Dual-Luciferase Reporter Assay System, Promega, USA).

Semiquantitative and Real-time RT-PCR

Two µg of RNA were reverse transcribed using a cDNA synthesis kit (Applied Biosystems, USA). cDNA equivalent of 20 ng of RNA was used for semiquantitative PCR. Real-time PCR reactions were performed using DyNAmoColorFlash SYBR Green qPCR Kit (Thermo Fisher Scientific India Private Ltd.) in ABI Prism 7900HT sequence detection system (Applied Biosystems, USA). The expression of RPL-35a gene and GAPDH (glyceraldehyde 3 phosphate dehydrogenase) were used as normalizing controls (Primer sequences are provided in Supplementary material. Table S2).

Development of Subcutaneous Xenografts in Nude Mice

Four million cells of respective clonal populations (LN229-Vc/LN229-BP2/LN229-BP2-N/LN229-BP2-C) were injected into 4–6 week old nude mice. All animal experiments were conducted in accordance with institutional guidelines and approval of the Institutional Animal Ethics Committee. Tumor growth was monitored over a period of 7–8 weeks, after which the mice were sacrificed and tumors dissected out, weighed, and sectioned for histopathology and immunohistochemistry (IHC). IHC and characterization are described in supplementary information.

Patient Characteristics and Immunohistochemistry

Following institutional ethical committee approval and informed patient consent, tumor tissue samples of participants with newly diagnosed GBM (n = 136) were obtained from patients who underwent surgery at the two clinical centers. The cohort of GBM participants received standard treatment with adjuvant radio-chemotherapy following surgery. Their survival pattern was noted over a period of 47 months. Overall survival was defined as the duration between surgery and the patient's death due to disease.

Tissue samples were formalin-fixed and paraffin-embedded. Details of the IHC staining pattern for IGFBP-2 (n = 136) were retrieved from our previous study22 and were employed for further analysis. IGFBP-2 staining was mainly cytoplasmic in the tumor cells. Similarly, 112 of 136 cases were evaluated for β-catenin based on tissue availability. In brief, the slides were deparaffinized, subjected to antigen retrieval in Tris-EDTA buffer, incubated with primary antibody (β-catenin, #C2206, Sigma-Aldrich, 1:100) followed by secondary antibody and visualized using betazoid diaminobenzidine (M1U539GL10, MACH-1 polymer detection kit). A visual semiquantitative grading scale was applied to assess the intensity of immunoreactivity for GBM tissues. The staining pattern for β-catenin was both cytoplasmic and nuclear. Only nuclear staining was considered for evaluation. The intensity was scored on a scale of zero to 2+; where zero represented nil, 1+ represented faint, and 2+ represented strong staining. Only 2+ staining was considered for analysis. The extent of staining in all cases was depicted as the labeling index (LI), which was defined as the percentage of 2+ staining tumor cells to the total number of tumor cells counted. The median LI served as a cutoff to distinguish cases with high expression from those with low (and absent) expression for IGFBP-2 and β-catenin, respectively.

Statistical Analysis

For analyzing cell line data, the nonparametric unpaired t test was utilized to calculate the P value of difference between 2 experimental datasets. All experiments with P value ≤ .05 were considered statistically significant. SPSS 15.0 statistical software (SPSS, Inc.) was used for clinical data analysis. Correlation of protein expression between IGFBP-2 and β-catenin was performed using Spearman's rho test. For correlation of protein expression with overall survival in GBMs, univariate Cox regression model was employed. Variables that were associated with P < .10 in univariate analyses were considered for inclusion into the multivariate model. Results were reported using the P values, and the estimated hazard ratio (HR). P < .05 was considered significant, and all exact 2-sided P values were reported. A median cutoff was employed to distinguish tumors with high and low expression of the proteins. Further, for clinical relevance of β-catenin and IGFBP-2, log-rank tests for significance were performed, and Kaplan-Meier curves were generated.

Results

IGFBP-2 Regulated Stabilization of Intracellular β-catenin

To establish the role of IGFBP-2-mediated protumorigenic actions in GBM, LN229 and U87 glioma cell lines stably overexpressing IGFBP-2 (U87-IGFBP-2 and LN229-IGFBP-2) were studied for β-catenin regulation. These cell lines were chosen based on their endogenous IGFBP-2 levels.8 Western blot analysis indicated that IGFBP-2 knockdown in U251, T98G, and U373 cell lines and overexpression in LN229 and U87 cell lines resulted in concomitant decrease or increase in the intracellular β-catenin levels respectively, within 48 hours of transfection (Fig. 1A, Supplementary Material, Fig. S1A). Also, a marked increase in intracellular β-catenin levels was observed upon overexpression of IGFBP-2 (Supplementary Material, Figs S1B and C). However, there was no apparent increase in the β-catenin RNA level indicating stabilization of β-catenin protein by IGFBP-2 (Supplementary Material, Fig. S1D). Interestingly, there was no regulation of canonical Wnt ligands upon IGFBP-2 knockdown/overexpression in glioma cell lines, suggesting that β-catenin regulation by IGFBP-2 might be independent of Wnt regulation (Supplementary Material, Fig. S2A).

Fig. 1.

Fig. 1.

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IGFBP-2-regulated nuclear accumulation and activity of β-catenin involves inactivation of GSK3β (A) Western blots showing regulation of GSK3β, AKT, β-catenin, and p-β-catenin upon knockdown/overexpression of IGFBP-2 within 48 hours of transfection. (B) Western blots showing cytoplasmic as well as nuclear accumulation of β-catenin upon overexpression of IGFBP-2 in LN229 and U87 cell lines (C) Top/flash luciferase assay showing increased β-catenin nuclear activity in IGFBP-2–overexpressing cells (n = 3, P < .05). (D) Western blot showing p-IGF1R and IGF1R expression in LN229 and U87 cell lines upon IGFBP-2 overexpression.

IGFBP-2-regulated Nuclear Accumulation and Activity of β-catenin Involves Inactivation of GSK3β

β-catenin is known for its rapid intracellular turnover by a destruction complex that includes axin, adenomatous polyposis coli (APC), β-catenin, and glycogen synthase kinase (GSK3β).13 GSK3β phosphorylates β-catenin at Ser33/37/Thr41 and primes it for proteasomal degradation.23 Phosphorylation of GSK3β at Ser9 position renders this enzyme inactive, which subsequently leads to stabilization of β-catenin.24 IGFBP-2 knockdown in U251, T98G, and U373 cells resulted in decreased phosphorylation of GSK3β at Ser9 (Fig. 1A, Supplementary Material, Fig. S1A). IGFBP-2 overexpression in LN229 and U87 cell lines consequently resulted in increased levels of phosphorylated GSK3β (Fig. 1A). Therefore, inactivation of GSK3β could be one of the principal mechanisms behind IGFBP-2-mediated stabilization of β-catenin. This IGFBP-2-mediated inactivation of GSK3β resulted in concomitant decrease in the levels of phosphorylated β-catenin (Fig. 1A). AKT is known to stabilize β-catenin by inactivating GSK3β.25 However, only 2 cell lines out of 5 showed IGFBP-2-mediated regulation of AKT at the Thr308 position, indicating some other IGFBP-2-mediated upstream regulator of GSK3β (Fig. 1A, Supplementary Material, Fig. S1A). Cytoplasmic stabilization of β-catenin is often found to be associated with its increased nuclear translocation and transcriptional activity. IGFBP-2 overexpression in LN229 and U87 cells resulted in increased cytoplasmic stabilization and subsequent nuclear accumulation of β-catenin that ultimately reflected in the increased β-catenin-TCF reporter activity (Fig. 1B and C).

IGFBP-2-induced β-catenin Activity Was Integrin-signaling Dependent but IGF1R-signaling Independent

The regulation of β-catenin by IGFBP-2 may involve IGF signaling. Hence, we tested the expression of IGF-I and II upon overexpression or knockdown in glioma cells. All glioma cell lines under study did not show detectable mRNA levels of IGF-I or IGF-II (Supplementary Material, Fig. S2B). In addition, there was no regulation of p-IGF1R upon IGFBP-2 overexpression, which indicated that the regulation of β-catenin by IGFBP-2 could be independent of IGF signaling (Fig. 1D). IGFBP-2 is reported to activate FAK upon activation of integrin signaling.21 Activation of α5β1 integrin signaling results in Src-mediated phosphorylation of FAK at Tyr925 position.26,27 It is reported that α5β1 integrin signaling-mediated inactivation of GSK3β acts as a survival signal for leukemic cells.28 Moreover, integrin-activated-FAK is shown to activate HIF-1alpha via inactivation of GSK3β in GBM.29 These earlier reports suggest that activated FAK could be the upstream regulator of GSK3β. We observed that IGFBP-2 overexpression in the LN229 or U87 cell line resulted in increased levels of p-FAK, whereas inhibition of Src-mediated activation of FAK at Tyr925 using Src antagonist PP2, abrogated IGFBP-2-mediated inactivation of GSK3β and subsequent stabilization of β-catenin (Fig. 2A, Supplementary Material, Fig. S3A). PP3 treatment was used as a negative control in this experiment because PP3 does not inhibit Src-mediated phosphorylation of FAK. Similar results were obtained when these cell lines were directly treated with recombinant IGFBP-2 in the presence or absence of PP2 (Fig. 2B) or FAK inhibitor (Supplementary Material, Fig. S3C). This indicated that stabilization of β-catenin by IGFBP-2 involves FAK activation. On the other hand, IGFBP-2-induced inactivation of GSK3β was not inhibited upon inhibition of IGF-1 receptor signaling, which further confirmed that these effects are IGF1R-signaling independent (Fig. 2B). One hour of treatment with recombinant IGFBP-2 was sufficient to inactivate GSK3β, which indicated that it could be direct regulation via a series of phosphorylation events (Supplementary Material, Fig. S2C). These results are further supported by a β-catenin-TCF responsive luciferase reporter assay. Src inhibitor PP2 and integrin inhibitor RGD peptide inhibited IGFBP-2-induced luciferase activity, while IGF1R inhibitor could not inhibit β-catenin nuclear activity (Fig. 2C). Wnt3a and LiCl treatment or IGFBP-2 overexpression resulted in increased nuclear β-catenin activity, whereas there was no synergistic increase in the nuclear activity of β-catenin when IGFBP-2-overexpressing cells were treated with Wnt3a (Fig. 2C). This indicated that either IGFBP-2 or Wnt3a was independently sufficient to induce the β-catenin pathway in glioma cells and might converge at a common β-catenin regulator, GSK3β. The effect of RGD and IGF1R inhibitor was confirmed by Western blot analysis for their targets p-FAK and p-IGF1R, while the activity of Wnt3a and LiCl was confirmed by analyzing expression levels of p-GSK3β (Supplementary Material, Fig. S3B).

Fig. 2.

Fig. 2.

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IGFBP-2-induced β-catenin activity was integrin-signaling dependent but IGF1R signaling-independent (A) Western blots showing regulation of FAK at Tyr925, GSK3β at Ser9 and total β-catenin in IGFBP-2 overexpressing LN229 and U87 cell lines in presence or absence of PP2. PP3 was used as a negative control for PP2. (B) Regulation of p-GSK3β when cells were treated with 500 ng/mL of IGFBP-2 in presence of PP2 or IGF1R inhibitor. (C) Top/flash luciferase assay indicating the activity of nuclear β-catenin in IGFBP-2 overexpressing cells when treated with PP2/PP3/RGD/Wnt3a/LiCl/IGF1R inhibitor (n = 3, P < .05).

Carboxy Terminal Domain of IGFBP-2 Was Sufficient to Activate β-catenin Signaling

Most of the IGF binding-independent mechanisms of IGFBP-2 are attributed to the C-terminal RGD motif through which it is known to activate integrin signaling.6 C-terminal fragments of IGFBP-2 purified from human hemofiltrate are known to have less IGF binding affinity but have an intact domain structure attributed to the disulfide bridging pattern of these fragments.20 It was proposed that C-terminal fragments generated after proteolytic cleavage of IGFBP-2 might have a biological role in cancer progression. To investigate this, we cloned and expressed N-terminal (1–165IGFBP-2) and C-terminal domains (166–289IGFBP-2) of IGFBP-2 separately in LN229 and U87 cell lines (Supplementary Material, Fig. S4, S5, S6A), which have low endogenous expression of IGFBP-2. Interestingly, stable overexpression of C-terminal domain of IGFBP-2 was as potent as full-length IGFBP-2 in the stabilization of β-catenin and in inducing β-catenin nuclear accumulation and activity (Fig. 3A–D). This was also reflected in the regulation of β-catenin transcriptional targets Oct-4, Nanog, MMP2, and c-Myc in these cells (Fig. 3E, Supplementary Material, Fig. S7D). This C-domain-induced stabilization of β-catenin was compromised when cells were treated with PP2, which suggested that IGFBP-2 C-terminal domain alone is sufficient to induce integrin-mediated activation of β-catenin (Supplementary Material, Fig. S6B). Moreover the C-terminal domain of IGFBP-2 induced invasion of LN229 and U87 cells (Supplementary Material, Fig. S7A–C).

Fig. 3.

Fig. 3.

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C-terminal domain of IGFBP-2 was sufficient to activate β-catenin signaling. (A) (B) Confocal image indicating stabilization of β-catenin in LN229 and U87 cells stably overexpressing full-length IGFBP-2 (FL) or its N or C-terminal domains (images at 63X magnification, length of the scale bar represents 20 µm). (C) Western blots showing nuclear and cytoplasmic accumulation of β-catenin in LN229 and U87 cells overexpressing full-length IGFBP-2 and its N or C-terminal domains. (D) Top/flash luciferase assay indicating the nuclear activity of β-catenin-TCF in cells expressing full length or N or C-terminal domains of IGFBP-2 (n = 3, P < .05). (E) Real-time PCR analysis showing the regulation of β-catenin-TCF transcriptional targets in U87 cell line (n = 3, P < .05).

C-terminal Domain of IGFBP-2 Showed Faster in vivo Tumor Growth Than N-terminal Domain

To investigate the role of different domains of IGFBP-2 in glioma tumor progression, LN229 cells engineered to overexpress either full length IGFBP-2 (LN229-IGFBP-2/FL) or C (LN229-BP2-C) and N (LN229-BP2-N) terminal domains were subcutaneously injected into immunocompromised mice. Cells transfected with cloning vector (LN229-Vc/V) served as the control. As shown in Fig. 4, subcutaneous xenografts developed from LN229 cells expressing full length and C-domain IGFBP-2 protein progressed faster than cells expressing N-terminal domain or vector control. The tumor sizes developed from cells expressing full length (Fig. 4A and B) or C and N terminal showed (Fig 4C–E; Supplementary Material, Fig. S8A, S8B) different sizes because these data were obtained from different sets of experiments.

Fig. 4.

Fig. 4.

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C-terminal domain of IGFBP-2 showed faster in vivo tumor growth than N-terminal domain (A) (B) Subcutaneous injection of LN229-IGFBP-2 (FL) cells depicting faster tumor growth in nude mice compared with LN229-Vc (Vc) cells (n = 10, P < .05). (C and D) Subcutaneous injection of LN229-BP2-C (C) cells depicting faster tumor growth in nude mice compared with LN229-BP2-N (N) or LN229-Vc (Vc) cells (n = 10, P < .05). (E) Mean xenograft tumor weight of LN229-IGFBP-2 or LN229-BP2-C compared with LN229-Vc or LN229-BP2-N (n = 10, P < .05). (F) Hematoxylin and eosin (H and E) staining showing the tissue morphology, immunohistochemistry showing the IGFBP-2 and β-catenin expression pattern in xenograft tumors obtained from V (original magnification x160), FL (original magnification x32, inset x160), N (×160) and C (×80, inset ×160) groups of nude mice. Length of the scale bar represents 20 µm.

The xenograft tissue morphology was observed by hematoxylin and eosin staining (Fig. 4F). The immunohistochemical analysis for IGFBP-2 predominantly showed cytoplasmic staining in each subgroup with minimal nuclear staining in 2 cases. Higher IGFBP-2 expression was noted in LN229-BP2-C (C) and LN229-BP2 (FL) groups as compared with LN229-BP2-N (N) and LN229-Vc (V) groups. For β-catenin, both nuclear and cytoplasmic immunopositivity was noted in the samples. While the majority of samples from the V group showed no nuclear staining, the C and FL groups displayed considerable nuclear staining of β-catenin. Variable cytoplasmic staining was also observed in all groups. Of all the groups studied, maximum immunopositivity for both nuclear and cytoplasmic β-catenin staining and maximum cytoplasmic immunopositivity of IGFBP-2 was observed in the FL group (Fig. 4F).

Coexpression of IGFBP-2 and β-catenin Correlated With Poorer Glioblastoma Patient Prognosis

The protein expression pattern of IGFBP-2 and β-catenin was analyzed in the tumor tissues of patients by immunohistochemistry (Fig. 5A). Variable expression of IGFBP-2 for GBM cases (as described in our previous study) was noted,22 with the LI (Labeling Index) ranging from zero to 70% (median LI = 30%). Similarly, the nuclear expression of β-catenin was variable, with median LI being 15%. A positive correlation between IGFBP-2 and β-catenin was noted on Spearman's rho correlation (correlation coefficient = 0.217, P = .021).

Fig. 5.

Fig. 5.

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Coexpression of IGFBP-2 and β-catenin correlated to the worst GBM patient prognosis (A) Immunohistochemistry showing the staining pattern of IGFBP-2 and β-catenin in normal tissue and GBM patient tissues. IGFBP-2 stainings are depicted in normal brain tissue (a), GBM with nil expression (c), moderate expression (e), and high expression (g). Similarly, β-catenin stainings are depicted in normal brain tissue (b), GBM with nil expression (d), moderate expression (f), and high expression (h) (All figures are original x160.) Higher magnification of IGFBP-2 and β-catenin are shown as insets (g and h; original magnification x320). Length of the scale bar represents 20 µm. (B) Kaplan-Meier survival analysis; Prognosis of GBM patients when categorized into IGFBP-2 low and IGFBP-2 high groups on the basis of staining intensity in IHC. (C) Prognosis of GBM patients when categorized into β-catenin low and β-catenin high groups. (D) The IGFBP-2 low, β-catenin low category with a median survival of 21 months, whereas the IGFBP-2 high, β-catenin high category had a median survival of only 12 months. IGFBP-2 and β-catenin coexpression in GBM tissues predicted patient prognosis with statistical significance of P = .001(log-rank test).

The median age of the participants in this cohort was 47 years. The maximum follow-up period was 46 months. On univariate analysis with Cox regression models, the continuous variables such as patient age (HR: 1.022; P = .014) and β-catenin expression (HR: 1.037; P ≤ .001) were noted to influence overall survival significantly. IGFBP-2 expression demonstrated a trend towards significance with respect to overall survival (HR: 1.011; P = .074). Subsequently, in a multivariate Cox model, patient age (HR: 1.021; P = .022) and β-catenin (HR: 1.036; P ≤ .001) were independently associated with poorer prognosis in GBM patients. However, IGFBP-2 (HR: 1.006; P = .425) lost significance, indicating β-catenin to be the stronger indicator of poor prognosis than IGFBP-2 expression.

For the purpose of clinical utility, all cases were dichotomized as high (n = 78) and low (n = 58) expression of IGFBP-2 based on the median LI cutoff of 30% (Supplementary material, Table S3). On Kaplan-Meier survival analysis, the prognostic association of IGFBP-2 trended towards significance (P = .071; Fig. 5B) with a median survival of 13 months in patients with high expression of IGFBP-2 versus 18 months for those with low expression. Similarly, with a median LI cutoff of 15% for nuclear β-catenin, patients with high expression of β-catenin (n = 57) had a significantly worse prognosis with a median survival of 12 months versus 19 months in patients with low expression of β-catenin (n = 55) (P = .002; Fig. 5C).

Further, we evaluated the effect of coexpression of β-catenin and IGFBP-2 and noted that a combinatorial high expression of IGFBP-2 and β-catenin was associated with the worst prognosis (median survival of 12 mo) as opposed to tumors having low expression or lacking both IGFBP-2 and β-catenin, which had the best prognosis (median survival of 21 mo) (Fig. 5D). Those with a high expression of either marker alone had an intermediate prognosis (median survival of 14–16 mo). The statistical significance of these differences in survival has been depicted in Supplementary material, Table S4. These results have been validated using the TCGA dataset, where IGFBP-2 mRNA alone and coexpression of IGFBP-2 and β-catenin mRNA were significantly associated with poor patient prognosis, whereas β-catenin mRNA alone did not show a significant association with patient prognosis (Supplementary Material, Fig. S9A and B, Supplementary material, Table S5 and S6).

Discussion

Despite recent advances in the treatment of GBM, the median survival has been in the range of 13–17 months. Our previous study,22 which corroborated other reports,3,6 suggested overexpression of IGFBP-2 in GBM. However, the role of IGFBP-2 in the pathogenesis of GBM is not clear. Here we demonstrate, for the first time, IGFBP-2 as the upstream regulator of β-catenin signaling in glioma. We observed that IGFBP-2 regulates stabilization and nuclear activity of β-catenin, which was in agreement with our earlier study in breast cancer.10 However, in contrast to our observation in breast cancer, regulation of β-catenin was independent of IGF1R signaling in glioma. One probable reason for this could be that IGFBP-2 did not regulate IGFs in glioma cell lines, which was observed in breast cancer cells. We further delineated this pathway and observed that regulation of β-catenin signaling by IGFBP-2 involves integrin-mediated inactivation of GSK3β. Inactivation of GSK3β is also often found to be associated with misregulation of several other targets such as HIF-1alpha, Snail, Mdm2, IRS1, Myc, PTEN, and VDAC, which may ultimately contribute to tumorigenesis.30

Transcriptional upregulation of Wnt1, Wnt2, or Wnt3a was not involved in the IGFBP-2-mediated regulation of β-catenin, suggesting noncanonical regulation of β-catenin through integrin pathway. The data from this study and the previously published report8 demonstrate a role for secreted IGFBP-2 in β-catenin regulation and protumorigenic actions, respectively. However, we have not examined if there is any role of intracellular IGFBP-2 in this regulation, which is a remote possibility.

Although our emphasis is upon the IGFBP-2-induced nuclear activity of β-catenin, we also observed a marked accumulation of β-catenin in the cytoplasm upon overexpression of IGFBP-2 (Supplementary Material, Fig. S1C). There are reports that discuss the role of cytoplasmic β-catenin in stabilizing oncogenic mRNAs such as Cox2,31 IL6, CA9, and SNAIL2.32 In concordance with this, the protumorigenic role of these molecules has been demonstrated in GBM.33–36

One more novel finding of this study is the demonstration of biological activity of the C-terminal fragments of IGFBP-2 in glioma tumorigenesis. It has been proposed that IGFBP-2 fragments might have important biological function.21 In the background of upregulated IGFBP-2 and increased protease activity in GBM tissues, it is important to study if any such mechanism exists in vivo. Interestingly, C-terminal domain of IGFBP-2, which contains the RGD motif, was sufficient to activate β-catenin signaling and also showed faster in vivo tumor growth. This suggests that C-terminal domain of IGFBP-2 has the protumorigenic role and is probably the mediator of IGFBP-2 associated GBM aggressiveness. This study advocates that under physiological circumstances, cleaved C-terminal fragment of IGFBP-2 might play an important role in the invasiveness of GBM in an IGF-independent manner since IGFBP-2 is known to lose its IGF binding affinity once cleaved. These in vitro and in vivo experiments for the first time elucidate a mechanism of IGFBP-2-induced β-catenin signaling and demonstrate a protumorigenic role for IGFBP-2 C-terminal fragment.

As mentioned before, there was a weak correlation between IGFBP-2 and β-catenin staining on GBM tissues when the Spearman rank correlation coefficient was considered. One probable reason behind this discordance could be the fact that β-catenin stabilization and activity are regulated by multiple upstream regulators such Wnt-frizzled signaling and EGFR16 signaling. Activation of these pathways along with IGFBP-2-integrin activation could be playing an important role in the subsequent regulation of β-catenin signaling. Further, tissues showing the high coexpression of IGFBP-2 and β-catenin had the worst patient prognosis, suggesting that IGFBP-2-integrin interaction might be responsible for activation of multiple arms of signaling such as NFκB9 and JNK5 besides β-catenin signaling, which ultimately determines aggressiveness of tumor and the poorer prognosis of patients.

In conclusion, this study establishes IGFBP-2 as the upstream regulator of the β-catenin pathway in GBM and demonstrates the mechanism behind this regulation (Fig. 6). We observed that IGFBP-2 or its C-terminal domain bind to the cell surface integrins and activate FAK. Activation of FAK in turn leads to the phosphorylation and inactivation of GSK3β. Inactivation of GSK3β results in the stabilization and increased nuclear activity of β-catenin. This IGFBP-2-induced β-catenin activity could be a major contributor in determining IGFBP-2-mediated aggressiveness of GBM. Ability of C-terminal domain of IGFBP-2 to activate β-catenin signaling and tumor growth in vivo makes it an attractive target for therapeutic intervention.

Fig. 6.

Fig. 6.

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A schematic showing the mechanism of IGFBP-2-mediated regulation of β-catenin pathway in glioma cells involving α5β1 integrin, FAK and GSK3β.

Supplementary Material

Supplementary material is available at Neuro-Oncology Journal online (http://neuro-oncology.oxfordjournals.org/).

Funding

Department of Biotechnology, Government of India.

Conflict of interest statement. No conflict reported by any authors.

Supplementary Material

Supplementary Data
supp_18_11_1487__index.html (1.8KB, html)

Acknowledgments

The results published here are in part based upon data generated by The Cancer Genome Atlas (TCGA) pilot project established by the NCI and NHGRI. Information about TCGA and the investigators and institutions that constitute the TCGA research network can be found at http://cancergenome.nih.gov/ (last accessed 30 September 2015).

We acknowledge the help of Kruthika, Priyanka Sehgal for suggestions and Vikas for analysis of TCGA data, confocal facility and real-time PCR facility at the Indian Institute of Science. Financial support for this study was provided by the Department of Biotechnology, Government of India.

SSP is a recipient of a University Grants Commission (India) fellowship. Infrastructure support to MRDG was by the DST-FIST program, Government of India.

References

  • 1.Ostrom QT, Bauchet L, Davis FG et al. The epidemiology of glioma in adults: a “state of the science” review. Neuro Oncol. 2014;16(7):896–913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev. 2002;23(6):824–854. [DOI] [PubMed] [Google Scholar]
  • 3.Lin Y, Jiang T, Zhou K et al. Plasma IGFBP-2 levels predict clinical outcomes of patients with high-grade gliomas. Neuro Oncol. 2009;11(5):468–476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Song SW, Fuller GN, Khan A et al. IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion. Proc Natl Acad Sci USA. 2003;100(24):13970–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mendes KN, Wang GK, Fuller GN et al. JNK mediates insulin-like growth factor binding protein 2/integrin alpha5-dependent glioma cell migration. Int J Oncol. 2010;37(1):143–153. [DOI] [PubMed] [Google Scholar]
  • 6.Wang GK, Hu L, Fuller GN et al. An interaction between insulin-like growth factor-binding protein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility. J Biol Chem. 2006;281(20):14085–14091. [DOI] [PubMed] [Google Scholar]
  • 7.Han S, Li Z, Master LM et al. Exogenous IGFBP-2 promotes proliferation, invasion, and chemoresistance to temozolomide in glioma cells via the integrin β1-ERK pathway. Br J Cancer. 2014;111(7):1400–1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Patil SS, Railkar R, Swain M et al. Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion. J Neurooncol. 2015;123(2):225–235. [DOI] [PubMed] [Google Scholar]
  • 9.Holmes KM, Annala M, Chua CYX et al. Insulin-like growth factor-binding protein 2-driven glioma progression is prevented by blocking a clinically significant integrin, integrin-linked kinase, and NF-κB network. Proc Natl Acad Sci USA 2012;109(9):3475–3480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Sehgal P, Kumar N, Praveen Kumar VR et al. Regulation of protumorigenic pathways by insulin like growth factor binding protein2 and its association along with β-catenin in breast cancer lymph node metastasis. Mol Cancer. 2013;12(1):63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pu P, Zhang Z, Kang C et al. Downregulation of Wnt2 and beta-catenin by siRNA suppresses malignant glioma cell growth. Cancer Gene Ther. 2009;16(4):351–361. [DOI] [PubMed] [Google Scholar]
  • 12.Roth W, Wild-Bode C, Platten M et al. Secreted Frizzled-related proteins inhibit motility and promote growth of human malignant glioma cells. Oncogene. 2000;19(37):4210–4220. [DOI] [PubMed] [Google Scholar]
  • 13.Nager M, Bhardwaj D, Cantí C et al. β-Catenin Signalling in Glioblastoma Multiforme and Glioma-Initiating Cells. Chemother Res Pract. 2012;2012:192362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Schüle R, Dictus C, Campos B et al. Potential canonical wnt pathway activation in high-grade astrocytomas. ScientificWorldJournal. 2012;2012:697313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Rossi M, Magnoni L, Miracco C et al. β-catenin and Gli1 are prognostic markers in glioblastoma. Cancer Biol Ther. 2011;11(8):753–761. [DOI] [PubMed] [Google Scholar]
  • 16.Yang W, Xia Y, Ji H et al. Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature. 2011;480(7375):118–122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kahlert UD, Maciaczyk D, Doostkam S et al. Activation of canonical WNT/β-catenin signaling enhances in vitro motility of glioblastoma cells by activation of ZEB1 and other activators of epithelial-to-mesenchymal transition. Cancer Lett. 2012;325(1):42–53. [DOI] [PubMed] [Google Scholar]
  • 18.Ben-Porath I, Thomson MW, Carey VJ et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40(5):499–507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Burkhalter RJ, Symowicz J, Hudson LG et al. Integrin regulation of beta-catenin signaling in ovarian carcinoma. J Biol Chem. 2011;286(26):23467–23475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Mark S, Kübler B, Höning S et al. Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern. Biochemistry. 2005;44(9):3644–3652. [DOI] [PubMed] [Google Scholar]
  • 21.Kiepe D, Van Der Pas A, Ciarmatori S et al. Defined carboxy-terminal fragments of insulin-like growth factor (IGF) binding protein-2 exert similar mitogenic activity on cultured rat growth plate chondrocytes as IGF-I. Endocrinology. 2008;149(10):4901–4911. [DOI] [PubMed] [Google Scholar]
  • 22.Santosh V, Arivazhagan A, Sreekanthreddy P et al. Grade-specific expression of insulin-like growth factor-binding proteins-2, −3, and −5 in astrocytomas: IGFBP-3 emerges as a strong predictor of survival in patients with newly diagnosed glioblastoma. Cancer Epidemiol Biomarkers Prev. 2010;19(6):1399–1408. [DOI] [PubMed] [Google Scholar]
  • 23.Hagen T, Vidal-puig A. Characterisation of the phosphorylation of beta-catenin at the GSK-3 priming site Ser45. Biochem Biophys Res Commun. 2002;294:324–328. [DOI] [PubMed] [Google Scholar]
  • 24.Fang X, Yu SX, Lu Y et al. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc Natl Acad Sci USA. 2000;97(22):11960–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Atkins RJ, Dimou J, Paradiso L et al. Regulation of glycogen synthase kinase-3 beta (GSK-3β) by the Akt pathway in gliomas. J Clin Neurosci. 2012;19(11):1558–1563. [DOI] [PubMed] [Google Scholar]
  • 26.Focal SI, Hauck CR, Hsia DA et al. v-Src SH3-enhanced interaction with Focal Adhesion Kinase at beta 1 Integrin-containing Invadopodia Promotes Cell Invasion. J Biol Chem. 2002;277(15):12487–12490. [DOI] [PubMed] [Google Scholar]
  • 27.Fu PTCCY, Chang CTYCL, Lin S. migration and invasion by suppressing Src-mediated signaling pathways. Mol Cell Biochem. 2014;387:101–111. [DOI] [PubMed] [Google Scholar]
  • 28.De Toni-Costes F, Despeaux M, Bertrand J et al. A New alpha5beta1 integrin-dependent survival pathway through GSK3beta activation in leukemic cells. PLoS One. 2010;5(3):e9807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Skuli N, Monferran S, Delmas C et al. Alphavbeta3/alphavbeta5 integrins-FAK-RhoB: a novel pathway for hypoxia regulation in glioblastoma. Cancer Res. 2009;69(8):3308–3316. [DOI] [PubMed] [Google Scholar]
  • 30.Sutherland C. What Are the bona fide GSK3 Substrates? Int J Alzheimers Dis. 2011;2011:505607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kim I, Kwak H, Lee HK et al. β-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3′-UTR and interacts with HuR in colon cancer cells. Nucleic Acids Res. 2012;40(14):6863–6872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.D'Uva G, Bertoni S, Lauriola M et al. Beta-catenin/HuR post-transcriptional machinery governs cancer stem cell features in response to hypoxia. PLoS One. 2013;8(11):e80742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Akasaki Y, Liu G, Chung NHC et al. Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma. J Immunol. 2004;173(7):4352–4359. [DOI] [PubMed] [Google Scholar]
  • 34.McFarland BC, Hong SW, Rajbhandari R et al. NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma. PLoS One. 2013;8(11):e78728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Said HM, Hagemann C, Staab A et al. Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1alpha in human glioma in vitro and in vivo. Radiother Oncol. 2007;83(3):398–405. [DOI] [PubMed] [Google Scholar]
  • 36.Kubelt C, Hattermann K, Sebens S et al. Epithelial-to-mesenchymal transition in paired human primary and recurrent glioblastomas. Int J Oncol. 2015;46(6):2515–2525. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_18_11_1487__index.html (1.8KB, html)
supp_now053_now053supp.doc (76.5KB, doc)
supp_now053_now053supp_fig1.tif (2.6MB, tif)
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