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Herstatin, an Autoinhibitor of the Epidermal Growth Factor (EGF) Receptor Family, Blocks the Intracranial Growth of Glioblastoma

Departments of ¹ Biochemistry and Molecular Biology, ² Neurology, ³ Cell and Developmental Biology, and ⁴ Neurosurgery, Oregon Health &Science University and ⁵ Veterans Administration Medical Center, Portland, Oregon

Requests for reprints: Gail M. Clinton, Department of Biochemistry and Molecular Biology, L224, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239. Phone: 503-494-5626; Fax: 503-494-5627; E-mail: Clinton{at}ohsu.edu.

	ABSTRACT

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Purpose: Herstatin, an autoinhibitor of the epidermal growth factor (EGF) receptor family, was evaluated for efficacy against human glioblastoma in vitro and in vivo in a rat intracranial model.

Experimental Design: Glioblastoma controlled by EGF receptor (EGFR; U87MG) or by the truncated mutant, EGFR (U87MG/), were transfected with Herstatin and evaluated for in vitro and in vivo growth in nude rat brain. Cells treated with purified Herstatin in vitro were evaluated for growth and signal transduction.

Results: Herstatin expression prevented tumor formation by U87MG and purified Herstatin inhibited their growth in vitro in a dose-responsive fashion, whereas in vivo and in vitro growth of U87MG/ was resistant to Herstatin. Inhibition of U87MG growth correlated with suppressed EGF activation of EGFR and of Akt but not mitogen-activated protein kinase signaling pathways, whereas EGFR activity and intracellular signaling in U87MG/ were unaffected by Herstatin treatment.

Conclusions: Herstatin may have utility against glioblastoma driven by the EGFR but not the mutant EGFR. Blockade of Akt but not the mitogen-activated protein kinase signaling cascade appears to be critical for suppression of intracranial tumor growth.

Key Words: Epidermal Growth Factor Receptor • EGFR • Herstatin • Intracerebral Xenograft Model

	INTRODUCTION

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Activation of the epidermal growth factor (EGF) family of receptor tyrosine kinases by ligands results in dimerization, tyrosine autophosphorylation, and initiation of two major signaling cascades: the PI3K/Akt pathway and the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway (1). Overexpression and/or autocrine activation of normal EGF receptor tyrosine kinases plays an important role in several human carcinomas (2). In addition, an N-terminally truncated mutant, EGFR (EGFRvIII), which is constitutively active in the absence of ligand, is critical in a subgroup of human glioblastoma (3, 4)

The central role of two members of the EGF receptor tyrosine kinase family, EGFR and HER-2, in human cancers places them as attractive targets in anticancer drug development (2). Monoclonal antibodies directed at the receptor extracellular domains and small molecule kinase inhibitors have generated encouraging preclinical and clinical results (2, 5, 6). Nevertheless, there is a critical need for development of novel receptor inhibitors with alternative mechanisms of action.

Herstatin, distinct from kinase inhibitors and monoclonal antibodies, is a naturally occurring product of the HER-2 gene created by alternative mRNA splicing (7). Herstatin functions as a secreted inhibitor that binds to the extracellular domains of EGFR and HER-2 with nanomolar affinity, disabling multiple receptor combinations in response to a variety of ligands (7–10). In this study, we investigated the antitumor activity of Herstatin against glioblastoma because the targets of Herstatin, EGFR (3, 4) and HER-2 (11), play an important role and because current treatment options are toxic and ineffective (12). Herstatin dramatically blocked U87MG glioblastoma tumor formation corresponding to in vitro inhibition of EGFR signaling through Akt but not MAPK pathways, whereas constitutive signaling and tumorigenic growth driven by EGFR were resistant to Herstatin. Because Herstatin is a secreted protein that can spread from the site of production and act outside the cell, it may be an effective therapeutic against intracerebral tumors.

	METHODS

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Cell Culture. U87MG and U87MG/EGFR (henceforth U87 and U87/) human glioblastoma cell lines were a generous gift from Dr. Webster Cavenee, Ludwig Institute for Cancer Research, Univ. of California at San Diego, CA (13). Stable transfections were conducted as described previously (9). Stable U87/Herstatin (Hst) cell lines were selected with 0.05 mg/mL hygromycin B and maintained in DMEM, 10% fetal bovine serum (FBS), and 0.05 mg/mL hygromycin B. Stable U87//Hst cells were selected and maintained in DMEM, 10% FBS, 0.2 mg/mL G418, and 0.1 mg/mL hygromycin B.

Tumor Implantation and Growth. The care and use of the animals was approved by the Institutional Animal Care and Use Committee and was under the supervision of the Oregon Health & Science University Department of Animal Care. Female athymic nude rats (rnu/rnu) (200-220g, n = 16) were anesthetized with i.p. ketamine (60 mg/kg) and diazepam (97.5 mg/kg). Tumor cells (12 µl, 10⁶ cells, 90% viable by trypan blue exclusion) were inoculated at stereotactic coordinates for intracerebral localization in the right caudate putamen (vertical bregma –6.5 mm, –3.1 mm lateral). Cells were inoculated over a 5-minute period to limit backflow along the injection tract. Rats were followed for survival and were sacrificed by barbiturate overdose when condition warranted or at 8 weeks after tumor implantation. Rat brains were fixed by immersion in 10% neutral buffered formalin for vibratome sectioning (100-µm coronal sections).

Herstatin Purification. S2 insect cells transfected with 6xHis tagged-Herstatin in the pMT/BiP expression plasmid (Invitrogen, Carlsbad, CA) were maintained in insect serum-free medium with L-glutamine (JRH Biosciences, Lenexa KS) supplemented with 300 µg/mL hygromycin. Cells were induced in fresh medium with cupric sulfate (100 µM) for 16 hours. Herstatin was purified to 80% to 90% purity by Ni-NT (Qiagen, Valencia CA) affinity chromatography as previously described (10).

In vitro Growth Assays. The different glioblastoma cell lines were plated into 24-well plates overnight in medium with 5% FBS. The cells were then washed with PBS and medium with 0.2% FBS were added. For the Herstatin treatments, triplicate wells were treated with the indicated concentrations of purified Herstatin or with control vehicle (20 mM Tris, 0.3 mol/L NaCl, 0.15 mol/L histidine, pH 8.0) in medium with 0.2% FBS on days 1 and 3. Viable cells were measured in triplicate wells using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (Promega, Madison, WI) as described (10). The results of the assay were quantified by absorbance at 492 nm in a Fluorostar plate reader.

Western Blot Analysis. Western blotting and Herstatin antibodies were as described (7, 8). Polyclonal Akt, phosphoAkt (phosphorylated at S⁴⁷³), MAPK, phospho-MAPK (phosphorylated at T²⁰² and Y²⁰⁴) were from Cell Signaling (Beverly, MA). Polyclonal anti-EGFR was from Santa Cruz Biotechnology (Santa Cruz, CA) and antiphosphotyrosine monoclonal antibody was from Sigma (St. Louis, MO).

Immunohistochemistry. Primary antibodies used for immunostaining were the Herstatin polyclonal (7), Herstatin monoclonal (Upstate, Lake Placid, NY), and an EGFR monoclonal antibody (NeoMarkers, Fremont CA). Secondary antibodies were Alexa 488 goat anti-mouse and Alexa 594 goat anti-rabbit (Molecular Probes, Eugene, OR). Immunofluorescence was visualized with an Olympus confocal inverted laser-scanning microscope (Melville, NY).

Data Analysis. Films exposed to Western blots were scanned and quantitated by imaging densitometry (model GS-700, Bio-Rad, Hercules CA) and standardized to the maximum phosphorylation signal. For tumor volumetrics, sections were stained with hematoxylin and imaged with a Zeiss AxioCam digital camera attached to a Zeiss Axioplan Universal microscope (Thornwood, NY). Every sixth section (10 per brain) was analyzed using Adobe Photoshop and NIH Image software tools. Tumor volume means and SDs were compared with Microsoft Excel software. Survival times (in days) were compared by paired Student t test using Microsoft Excel. Invitro cell growth inhibition was evaluated with Microsoft Excel software.

	RESULTS

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Effect of Herstatin Expression on In vitro Growth and Tumor Formation. We chose the U87 cell line from a human glioblastoma as a model of a highly lethal primary brain tumor for these studies. The U87 parental cells controlled by the full-length EGFR (14, 15) and the U87/ cell line driven by the N-terminally truncated mutant, EGFR (13), were stably transfected with Herstatin to test effects on growth and tumorigenesis. Two clonal cell lines, U87/Hst and U87//Hst, which expressed comparable levels of Herstatin (Fig. 1A), were selected for further studies. The U87/Hst cells proliferated more slowly than the parental U87 cells (Fig. 1B) suggesting that Herstatin expression inhibited their growth. In contrast, Herstatin expression did not appear to affect the growth of the glioma cells that overexpressed EGFR because the U87//Hst and U87/ cell lines grew at a similar rate (Fig. 1B, right).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 1 Effect of Herstatin expression on in vitro growth. A, U87 or U87/ glioblastoma cells mock transfected or transfected with Herstatin were extracted and 20 µg of protein from each were subjected to Western blot (WB) analysis using antibodies against Herstatin. The blot was developed by chemiluminescence and exposed to X-ray film. B, U87, U87/Hst, U87/, and U87//Hst were plated at 20,000 cells/well into 24-well plates overnight in medium with 5% FBS, washed, and serum was reduced to 0.2%. Viable cells were quantified by the MTS assay in triplicate wells after plating overnight to determine the zero time point and at 1, 2, and 3 days in culture. Points, mean of the A492 nm readout from the MTS assay; bars, SE.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 2 Effect of Herstatin expression on intracranial tumorigenic growth. A, survival of nude rats intracranially inoculated with U87 or U87/ glioblastoma cells with and without Herstatin expression was monitored. Each symbol indicates an individual rat that died or was sacrificed on the indicated day. B, histology of parental U87 intracerebral tumor. Rat brain section (100 µm) stained with hematoxylin shows the darkly staining tumor in the injected hemisphere. C, histology of rat brain with U87/Hst cell inoculation. Arrow, subtle linear staining which at higher power can be identified as the needle track by small amount of hemosiderin. Box, area magnified in Fig. 3.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Fig. 3 Immunohistochemistry and histology of U87//Hst and U87/Hst intracerebral tumors. A, EGFR expression (left) and Herstatin expression (right) in U87//Hst tumor section (100 µm). B, histology and immunohistochemistry of U87/Hst tumor from the cortical surface at the injection site (box in Fig. 2C). Separate formalin-fixed brain sections (100 µm) were processed for hematoxylin staining (left) or Herstatin immunohistochemistry (right). Arrowhead, direction of the needle track; arrows, tumor location.

Herstatin Expression in Implanted Cells. Because the size of the tumors and survival time of animals injected with U87/ versus U87//Hst were not significantly different, we examined by immunocytochemistry whether Herstatin expression was retained after intracerebral implantation. Herstatin was readily detected at the cell surface in tumors formed from the U87//Hst cells (Fig. 3A) but not from the U87/ cells (data not shown), showing that differential Herstatin expression was maintained. Abundant amounts of EGFR, localized to the cell surface, were detected in both the U87//Hst (Fig. 3A) and the U87/ (data not shown). Secretion of Herstatin from the U87//Hst tumor cells did not appear to cause toxicity in the normal brain around the tumor margin.

We examined whether Herstatin expression could also be detected in rat brains inoculated with the U87/Hst cells. Whereas there was no apparent staining around the needle track in the caudate nucleus, the cortex at the injection site showed Herstatinstaining that coincided with the histologically identified residual tumor cells (Fig. 3B). This indicated that U87/Hst cells survived and expressed Herstatin but were unable to proliferate. EGFR staining is not shown because levels are low in the parental U87 cells.

Effects of Purified Recombinant Herstatin on In vitro Growth. Herstatin, expressed as a secreted protein in U87 cells, blocked their in vitro as well as their intracranial growth, suggesting that addition of purified Herstatin may also suppress their proliferation. On the other hand, the U87/ cells that were not inhibited by Herstatin expression were predicted to be resistant to exogenous Herstatin. To test this, the effect of Herstatin on in vitro growth was evaluated in cultured U87 cells and, in parallel, in U87/ cells. Figure 4 demonstrates that exogenous Herstatin caused a dose-responsive inhibition of viable U87 cells (P = 0.0002), whereas the U87/ cells were unaffected (P = 0.535). These results showed the efficacy of purified Herstatin as an antiproliferative agent against U87 cells. Moreover, these results supplied further evidence that the EGFR conferred resistance to Herstatin.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 4 Growth of cultured U87 and U87/EGFR cells treated with different concentrations of purified recombinant Herstatin. Triplicate wells (40,000 cells/well) were treated with the indicated concentrations of purified Herstatin or with control vehicle on days 1 and 3, and viability was measured on day 5. The U87 cells treated with 100 and 300 nmol/L Hst were significantly reduced compared to controls (P = 0.04 and 0.0002, respectively). Linear regression analysis of results obtained with 0, 100, and 300 nmol/L Hst revealed dose-responsive inhibition (P = 0.0002). Herstatin had no effect on U87/ cells at any dose (P = 0.535). Dose-responsive inhibition of U87 by purified Herstatin and no effect on U87/ cells was observed in three separate experiments. Points, mean percent inhibition relative to control vehicle–treated cells; bars, SE.

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Fig. 5 Effect of Herstatin on receptor phosphorylation and intracellular signaling. A, U87 cells were starved in serum-free medium for 24 hours and then treated with indicated concentrations of Herstatin for 25 minutes followed by EGF (5 nmol/L) for 25 minutes. Cells were extracted and 20 µg of protein were analyzed as a Western blot using antibodies against phosphotyrosine (p-Tyr, top). The blot was stripped and reprobed with antibodies against the EGFR (bottom). The density of each signal relative (Rel. Density) to the strongest signal was determined by imaging densitometry. B, effect of Herstatin on tyrosine phosphorylation of EGFR in U87/ cells was determined exactly as in A. C, serum-starved U87 cells were pretreated with 200 nmol/L Herstatin for 25 minutes followed by 5 nmol/L EGF for the indicated times. Cell extracts were examined by Western blotting with the indicated antibodies. D, effects of Herstatin on signaling by the EGFR was conducted exactly as in C.

	DISCUSSION

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Several inhibitors of EGFR including monoclonal antibodies and kinase inhibitors have been intensively investigated as potential cancer therapeutics (5, 6). Studies presented here suggest that Herstatin may be a potent inhibitor of malignant glioblastoma that is driven by the EGFR. Herstatin represents a novel addition to the repertoire of antireceptor agents, distinguished by its novel structure and human origin. It is the only naturally occurring (7) inhibitor of the EGF receptor family that exerts its action on the initial events in receptor activation: dimerization and autophosphorylation. Furthermore, Herstatin blocks activation of both HER-2 and the EGFR (8, 9) that are implicated most often in the progression of numerous human cancers.

Herstatin effectively inhibited EGFR tyrosine phosphorylation and the PI3K/Akt pathway, but MAPK activation was unaffected in the U87 cells. This is in agreement with a previous report demonstrating that Herstatin uncouples EGF activation of these two major signaling pathways in murine fibroblasts (9). In both studies, inhibition of Akt correlated with suppression of proliferation indicated by blocked in vitro and in vivo tumorigenic growth of glioblastoma (this study) and inhibition of EGF and transforming growth factor –mediated proliferation of murine fibroblasts (9). Recent studies have showed that EGF activation of Akt in U87MG cells is important in the regulation of vascular endothelial growth factor production, which mediates tumor vascularity (15). Although both the Akt and MAPK pathway have showed involvement in both proliferative and survival signaling, our findings suggest that obstruction of Akt may be sufficient to achieve growth blockade. Herstatin expression may block tumorigenic growth by preferential inhibition of Akt even though MAPK activity is unabated.

Although Herstatin has been found to prevent activation of the full-length EGFR, our results show that EGFR was resistant. Herstatin inhibits full-length EGFR by binding to its extracellular domain and blocking dimerization (8, 9). Herstatin contains a dimerization arm in its subdomain II (19) and may therefore block the dimerization arm of EGFR, preventing formation of productive receptor dimers. Because EGFR is missing subdomains I and II (3, 4), Herstatin binding could be inhibited, or EGFR, which is missing its dimerization arm, may resist Herstatin-mediated inhibition (19, 20). Preliminary observations indicate that Herstatin binds to EGFR,⁶ suggesting that receptor subdomains I and II do not contain the binding site but rather are required for Herstatin-mediated inhibition.

Understanding the molecular profile of cancer cells, which predicts responsiveness to targeted inhibitors, is crucial to maximizing clinical efficacy. Results presented here indicate that Herstatin dramatically inhibits tumor growth of cells driven by the full-length EGFR, whereas cells overexpressing the truncated EGFR are resistant to Herstatin. In a significant proportion of glioblastoma, amplification of the wild-type EGFR occurs and may precede the generation of EGFR (2). The wild-type EGFR is overexpressed in a subset of glioma as well as in many carcinomas to a much greater extent than observed in the U87 cells used in this study. It is expected that Herstatin will also inhibit glioma with amplified wild-type EGFR because our previous studies have shown that Herstatin binds with nanomolar affinity and inhibits EGF signaling and proliferation in cells that overexpress EGFR (8, 9, 21). These studies further point to constitutively active Akt as a predictor of Herstatin insensitivity.

Inhibition of intracranial growth of U87 cells by Herstatin and the growth-inhibitory effects of purified recombinant Herstatin suggests potential as a therapeutic against human glioblastoma that are driven by the EGFR. Current treatments of this disease are largely limited to chemotherapy and radiotherapy, both of which have toxic side effects and minimal efficacy. Receptor-targeted therapeutics are less toxic than most conventional treatments (2, 5, 6). However, the blood-brain barrier presents an obstacle in the treatment of glioblastoma and other brain neoplasms by limiting agent access to the tumor mass and to infiltrating cells far from the main tumor mass (22). Osmotic opening of the blood-brain barrier provides a method to optimize delivery of peptides, such as Herstatin, and even viral vectors to brain tumors (22). Because Herstatin is a secreted protein that can spread to and inhibit adjacent tumor cells, it may be an effective therapeutic transgene for gene therapy of intracerebral tumors. Another delivery option is convection or clysis to enhance the distribution of injected therapeutic in brain parenchyma (22). Future experiments will test the antitumor efficacy of Herstatin protein as well as Herstatin gene expression in rat intracerebral tumor models after intratumor injection with convection in comparison to osmotic blood-brain barrier disruption delivery.

	ACKNOWLEDGMENTS

 
We thank Maryanne McClellan of Reed College for advice and use of the confocal microscope, Kristina Pinkston and Shannan Walker-Rosenfeld for excellent technical assistance, and Webster Cavenee for the U87MG and U87MG/EGFR cell lines.

	FOOTNOTES

 
Grant support: VA merit review grant, National Institute of Neurological Disorders and Stroke grants NS44687 and NS33618 (E.A. Neuwelt), and National Cancer Institute (G.M. Clinton).

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.

Note: G.M. Clinton and Oregon Health & Science University have a significant financial interest in Receptor BioLogix Inc., which may have a commercial interest in results of this research. This potential conflict of interest has been reviewed and is managed by the Oregon Health & Science University Conflict of Interest in Research Committee.

⁶ L. Shamieh and G.M. Clinton, unpublished observations.

Received 4/30/04; revised 9/22/04; accepted 10/ 7/04.

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