Mol Cancer Ther. 2006;5:1571-1576
© 2006 American Association for Cancer Research
Research Articles: Therapeutics
Constitutively active receptor tyrosine kinases as oncogenes in preclinical models for cancer therapeutics
Kristen A. Kellar1,
Matthew V. Lorenzi1,
Ching Ping Ho1,
Dan You1,
Mei-Li Wen1,
Rolf P. Ryseck1,
Simone Oppenheimer1,
Brian E. Fink2,
Gregory D. Vite2,
Bruce R. Rowley1,
Chiang Yu1,
David K. Bol1,
Francis Y. Lee1 and
Tai W. Wong1
1 Oncology Drug Discovery and 2 Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
Requests for reprints: Tai W. Wong, Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ. Phone: 609-252-4187; Fax: 609-252-6171. E-mail: tai.wong{at}bms.com
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Abstract
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Receptor tyrosine kinases (RTK) remain an area of therapeutic interest because of their role in epithelial tumors, and experimental models specific to these targets are highly desirable. Chimeric receptors were prepared by in-frame fusion of the CD8 extracellular sequence with the cytoplasmic sequences of RTKs. A CD8HER2 fusion protein was shown to form disulfide-mediated homodimers and to transform fibroblasts and epithelial cells. CD8RTK fusion proteins transform rat kidney epithelial cells and impart phenotypes that may reflect signaling specificity inherent in the native receptors. Transgenic expression of CD8HER2 and CD8Met in mice resulted in the formation of salivary and mammary gland tumors. The transgenic tumors allow the derivation of allograft tumors and cell lines that are sensitive to inhibition by small molecule kinase inhibitors. This approach provides excellent cell and tumor models for the characterization of signaling properties of diverse RTKs and for the evaluation of rationally designed antagonists targeting these kinases. [Mol Cancer Ther 2006;5(6):1571–6]
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Introduction
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Of the >500 protein kinases that are known to be encoded by the human genome, the 
60 receptor tyrosine kinases (RTK) represent a class that have been most extensively studied because of their importance to the regulation of biological processes (1). Without exception, every RTK that has been characterized has been shown to function in regulating organogenesis and/or tissue homeostasis of adult animals. Not surprisingly, a number of these receptor kinases have been shown to be deregulated in human diseases ranging from diabetes to cancer (2–5). In fact, understanding the role of RTKs in cancer pathobiology has led to the successful development of therapeutic agents that specifically target the biological activity of RTKs, such as the epidermal growth factor receptor, HER2, KIT, platelet-derived growth factor receptor, and the ligand of the vascular endothelial growth factor receptors (6–9). It is, therefore, reasonable to anticipate that the RTKs will continue to be a fruitful area of research for understanding cancer biology and disease intervention.
Elucidation of the biological function of RTKs has often been challenging because of the complexity in receptor and ligand expression. These kinases can be classified into families according to structural relatedness. Each receptor family, such as the human epidermal growth factor receptor (HER) family, may consist of multiple related receptors with distinct ligand-binding and signaling specificity (10). The coexpression of some of these receptors and the ability to form heterodimers may further complicate attempts to define the role of each of these receptors in a biological context.
The design of animal models for characterizing the biological function of RTKs has relied on the overexpression of receptors or their ligands in transgenic animals (11–15). This approach has imposed significant constraints in the interpretation of the data because of the complexity in temporal regulation of ligand or receptor expression, as well as any feedback regulation that may be inherent. For many of these receptors, the availability of a constitutively active receptor offers many advantages in studying the function of the receptor kinase in development or oncogenesis. The extracellular sequence of the human T-cell antigen CD8 is known to form intermolecular disulfide bonds (16). We previously showed that a fusion protein between the extracellular sequence of the CD8 
chain and the cytoplasmic sequence of the human insulin-like growth factor-1 receptor is constitutively activated and drives tumor formation when expressed in transgenic mice (17). We report here that the use of CD8 fusion proteins can be broadly adapted to provide cell and tumor models for characterizing the biological activity of RTKs and for the optimization of kinase inhibitors designed to target these receptors.
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Materials and Methods
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Antibodies and Western Analyses
Antibodies that are specific for HER1 and HER2 were prepared by immunizing New Zealand White rabbits with the peptides CYLRVAPQSSEFIGA (codons Y1196–A1210 of HER1) and CFDNLYYWDQDPPER (codons F1217–R1230 of HER2). A cysteine residue was added to the NH2 termini of the peptides to facilitate chemical coupling to a carrier protein. Coupling of the peptides to Imject Activated Ovalbumin was carried out as recommended by the supplier (Pierce Chemicals, Rockford, IL), and New Zealand white rabbits were immunized by standard procedures. A monoclonal antibody to the human CD8 chain (AHS0802) was obtained from Biosource International (Camarillo, CA). An antibody specific for the COOH terminus of human Met receptor kinase (C-12) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine (PY20) antibody was from BD Biosciences (San Jose, CA). Immunoprecipitation and Western blot analyses were done as described previously (17).
Preparation of CD8RTK Expression Plasmids
A plasmid that encodes the extracellular and transmembrane sequences of human CD8 chain (codons 1–218) in pcDNA3.1+ (Invitrogen, San Diego, CA) was used to prepare in-frame fusions with sequences that encode the cytoplasmic sequences of a number of RTKs. The receptor kinase sequences that were included in the fusion proteins are codons 681 to 1210 of HER1 (National Center for Biotechnology Information accession no. P00533), codons 682 to 1255 of HER2 (accession no. P04626), codons 965 to 1390 of Met (accession no. P08581), and codons 442 to 790 of TrkA (accession no. AAA36770). The HER2 cytoplasmic sequence was also subcloned into the vector pFLAG-CMV2 (Sigma Chemical, St. Louis, MO) to yield an expression plasmid with a FLAG epitope tag added to the NH2 terminus. A full-length HER2 expression plasmid was also prepared in pcDNA3.1+. The CD8HER2 and CD8Met sequences were amplified and transferred into a vector under the control of mouse mammary tumor virus long terminal repeat promoter (11). All cDNA plasmids were verified by sequence determination.
Transfection and Characterization of Stable Transformants
RK3E or NIH3T3 cells were transfected with expression plasmids encoding CD8RTK fusion proteins or activated H-Ras (18, 19). Transfected cells were selected in culture medium containing G418 (0.5 mg/mL), and clones that survived the selection were pooled for further characterization. Growth rates of RK3E and transfectants were determined by plating 25,000 cells per well in 12-well tissue culture plates. Cells were harvested by trypsinization, and cell numbers were determined from triplicate cultures using a Coulter Z2 Counter (Fullerton, CA). For soft agar assays, cells were plated at 1,000 or 10,000 per well in six-well plates. Cells were suspended in 3.6% bactoagar that was placed on top of a bottom layer of 6% agar. After 2 weeks, colonies were stained with crystal violet and counted using a Sorcerer colony counter (Optomax, Hollis, NH). For collagen growth assays, 25,000 cells were resuspended in 1 mL of 3% collagen I (Trevigen, Gaithersburg, MD) that had been neutralized to pH 7. The suspension was put into a 12-well tissue culture plate, and the collagen matrix was allowed to gel at 37°C for 30 minutes. To the collagen layer was added 1 mL of DMEM supplemented with 10% fetal bovine serum. The cultures were allowed to grow for 10 to 14 days and were fed every 3 days.
CD8RTK Transgenic Animals and Allografts
Plasmid DNA encoding CD8HER2 and CD8Met from the mouse mammary tumor virus 1 promoter were microinjected into pronuclei from B6D2 mice. Manipulation of the injected embryos and subsequent breeding in ICR mice were as described (20). Transgene expression was monitored using PCR assays with primers specific for the fusion gene products. Animal care conformed to guidelines of the institutional Animal Care and Use Committee in a facility certified by the Association for Assessment and Accreditation of Laboratory Animals. Salivary gland tumors that emerged in founder animals were surgically removed, and tumor fragments were implanted s.c. into the ventral thoracic regions of female athymic BALB/c nu/nu mice (Harlan Sprague-Dawley, Indianapolis, IN). The transplanted tumors were allowed to grow to 500 to 1,000 mm3 and were then passaged serially in athymic mice at intervals of 2 to 3 weeks. A CD8HER2 salivary gland tumor was subjected to enzymatic dissociation, and a stable cell line (Sal2) was isolated as described before (17). Sal2 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. For efficacy evaluation, groups of eight mice each were randomized to receive either vehicle only or the small molecule kinase inhibitor BMS-462043. BMS-462043 was prepared in a vehicle of 40% propylene glycol/10% Tween 80/50% water and was given by oral gavage once daily for 10 days. Tumor sizes were recorded and converted to tumor weight using the formula: tumor weight = (length x width2) / 2.
Characterization of CD8RTK Fusion Proteins
COS7 cells were transfected with an expression plasmid encoding CD8HER2, and cell lysates were prepared after 48 hours. Lysates were prepared by scraping cells into a lysis buffer that contained 1% Triton X-100, 40 mmol/L Tris-HCl (pH 7.7), 1 mmol/L EDTA, 10% glycerol, 0.15 mol/L NaCl, 1 mmol/L sodium vanadate, 40 µmol/L ammonium molybdate, and 1% Complete protease inhibitors. HER2 polypeptides were immunoprecipitated using an anti-peptide antibody specific for the cytoplasmic sequence of HER2 and were fractionated by gel electrophoresis using sample buffer with or without 2-mercaptoethanol. Tyrosine phosphorylation of the polypeptides was analyzed by Western blotting with anti-phosphotyrosine antibody (PY20). Sal2 cells were treated with porcine epidermal growth factor (Life Technologies, Gaithersburg, MD) at 100 ng/mL for 5 minutes before lysis. Cell lysates were immunoprecipitated with an anti-peptide antibody specific for HER1. Tumor lysates were prepared by homogenizing tumors in lysis buffer and were clarified by centrifugation. CD8HER2 and CD8Met polypeptides were immunoprecipitated using either an antibody to the CD8 extracellular sequence or an antibody to the cytoplasmic sequence of Met. Immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine antibodies (PY20).
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Results
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Expression of CD8HER2 Homodimers Transforms Cells In vitro
The signaling property of a CD8HER2 fusion protein was compared with that of full-length HER2 or the cytoplasmic sequence alone. Expression plasmids encoding these sequences were transfected into COS7 cells, and the protein products were analyzed by immunoprecipitation with an antibody to the HER2 cytoplasmic sequence followed by Western blotting. When fractionated by gel electrophoresis in the presence of a reducing agent, the CD8HER2 fusion protein migrated as a heterogeneous mixture of polypeptides of 100 kDa, that are phosphorylated to an extent at least comparable to that in the full-length receptor (Fig. 1A, lane 7
). The corresponding cytoplasmic sequence of HER2 expressed as a fusion with an eight-amino-acid FLAG peptide epitope showed only a minor extent of tyrosine phosphorylation, although the protein was expressed to a high level (Fig. 1A, lane 6). When fractionated under nonreducing condition, the CD8HER2 polypeptide migrated as a high molecular weight entity (Fig. 1A, lane 3). The use of nonreducing electrophoretic condition in lanes 1 to 4 resulted in the appearance of the immunoglobulin heavy chains as a smear. Treatment with 2-mercaptoethanol had little effect on the electrophoretic mobility of full-length HER2 or its cytoplasmic sequence alone. These observations confirm that the CD8 extracellular sequence promotes disulfide-mediated oligomerization of the fusion protein, resulting in activation of the kinase activity. The activated state of the CD8HER2 protein was verified by its ability to transform cultured fibroblasts. NIH3T3 cells transfected with a plasmid encoding only the CD8 extracellular sequence retain a flat morphology as seen in untransfected cells. By contrast, stable clones that express the CD8HER2 fusion protein assume a highly transformed morphology and form colonies in soft agar (Fig. 1B).
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Figure 1. Constitutive oligomerization and activation of CD8HER2. A, COS7 cells were transfected with expression plasmids encoding a FLAG-tagged HER2 cytoplasmic sequence (lanes 2 and 6), CD8HER2 fusion protein (lanes 3 and 7), or full-length (FL) HER2 (lanes 4 and 8). Cell lysates were immunoprecipitated with antibodies to the cytoplasmic sequence of HER2 and analyzed by Western blotting with anti-phosphotyrosine antibodies (top). The filter was stripped and reprobed with antibodies to the cytoplasmic sequence of HER2 (bottom). B, CD8HER2 transforms NIH3T3 cells. Representative morphology of NIH3T3 cells that were not transfected or transfected with the CD8 vector (top) and stable clones expressing CD8HER2 (bottom left). Right, colonies of the CD8HER2 transfectants in soft agar.
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CD8RTKs Transform Epithelial Cells
The transforming activity of the CD8HER2 fusion protein in epithelial cells was evaluated by transfecting the rat kidney epithelial cell line RK3E. These cells had previously been shown to be highly susceptible to transformation by a number of oncogenes and may be suitable for studying the biological function of RTKs that are typically expressed in cells of epithelial origin (18). RK3E cells expressing a truncated CD8 protein without a fusion partner are morphologically indistinguishable from untransfected cells; they are flat in appearance and have well-defined cell-cell adhesions that are characteristic of untransformed epithelial cells (Fig. 2A
). By contrast, RK3E cells transfected with a CD8HER2 expression plasmid grew in large clusters that are loosely adherent to the substratum. Expression of a CD8 fusion protein containing the cytoplasmic sequence of the Met RTK resulted in a highly polarized morphology that is characteristic of motile cells. Many of these cells take on an elongated shape, with abundant fillopodia clearly visible under the light microscope. Expression of the CD8HER2 and CD8Met fusion proteins affects not only the morphology of the RK3E transfectants but also their growth rate. Compared with parental cells, RK3E cells expressing CD8HER2 or CD8Met grew at a more rapid rate and to a higher density, reflecting their ability to overcome contact inhibition (Fig. 2B). Because many RTKs have morphogenic potential, the RK3E transfectants were examined for their ability to grow in collagen gel. Parental RK3E or cells transfected with a truncated CD8 expression plasmid are not capable of anchorage-independent growth and grew poorly in collagen (Fig. 2C). CD8HER2 expression in the RK3E cells enabled them to form large colonies that, with prolonged incubation, resemble cysts (Fig. 2C; data not shown). RKE cells expressing CD8Met developed a more complex morphology with branch-like extensions (Fig. 2C). Thus, whereas both of these CD8RTK proteins transform epithelial cells, the transformed cells have distinct characteristics that may reflect a difference in receptor signaling. The general applicability of the CD8 fusion approach was further assessed in a soft agar transformation assay using cytoplasmic sequences from a subset of diverse RTKs. All of the CD8RTKs tested were able to support soft agar growth of RK3E transfectants. Although there is some heterogeneity in the size of the colonies, the CD8RTKs were comparable with mutant Ras in their oncogenic potency as measured by the number of colonies formed in soft agar (Fig. 3
).
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Figure 2. CD8HER2 and CD8Met transform rat kidney epithelial cells. A, morphology of RK3E parental cells and cells transfected with CD8HER2 and CD8Met expression plasmids. B, growth rate of parental RK3E cells and transfectants expressing CD8HER2 and CD8Met. Points, averages of triplicate determinations. C, colony growth of RK3E transfectants in type I collagen. Colonies were photographed after 14 d in culture. Two representative colonies of CD8Met transfectants to illustrate their distinct morphology.
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Figure 3. Soft agar clonogenicity of RK3E cells transfected with CD8RTK expression plasmids. Pooled transfectants were cultured in soft agar, and representative cultures were photographed after 14 d (top). Quantitated colony formation (bottom).
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CD8HER2 and CD8Met Expression in Transgenic Mice
The transforming properties of the CD8HER2 and CD8Met fusion proteins were also exploited to develop tumor models for further characterization of mechanism of tumorigenesis and for efficacy evaluation of kinase inhibitors targeting these receptors. The CD8HER2 and CD8Met coding sequences were placed under transcriptional control of a mouse mammary tumor virus promoter and the DNA was microinjected into mouse pronuclei. Founder mice developed tumors in the salivary and mammary glands with comparable latency (Table 1
). Both salivary gland and breast tumors expressed the CD8HER2 fusion protein (Fig. 4A
). A stable cell line (Sal2) was derived from one of the salivary gland tumors and was found to have significant expression of HER1. However, the HER1 receptor was not activated, as shown by the lack of receptor phosphorylation in the absence of added epidermal growth factor (Fig. 4B). This confirms that there is no activating interaction between the CD8HER2 and endogenous HER1. As expected, added epidermal growth factor was able to elicit a robust tyrosine phosphorylation of endogenous HER1. Transgenic mice expressing CD8Met gave rise to salivary and mammary gland tumors with incidence and latency similar to those in the CD8HER2 animals (Table 1). A 75-kDa CD8Met fusion protein was detected in a salivary tumor lysate by immunoprecipitation with either anti-CD8 or anti-Met antibody (Fig. 4C). When immunoprecipitated with an anti-CD8 antibody, the CD8Met fusion protein was recovered in a complex consisting of multiple phosphorylated polypeptides. Immunoprecipitation with an antibody to the COOH terminus of Met did not result in the coimmunoprecipitation of the additional proteins, suggesting that the COOH terminus epitope overlaps with Met sequence that may be involved in the complex formation, as previously shown for the full-length Met receptor (21).
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Figure 4. Expression of CD8HER2 and CD8Met in transgenic tumors. A, salivary and mammary gland tumor lysates were immunoprecipitated with anti-CD8 antibodies and analyzed by Western blotting with anti-phosphotyrosine antibodies (top). The amounts of lysates used in immunoprecipitation were 50 µg (lanes 1 and 4), 100 µg (lanes 2 and 5), and 200 µg (lanes 3 and 6). The filter was stripped of antibodies and reprobed with anti-HER2 antibodies. B, cultures of Sal2 salivary gland tumor cells were lysed with or without treatment with epidermal growth factor (EGF), and lysates were immunoprecipitated with anti-HER1 antibodies. The immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine and anti-HER1 antibodies. C, lysates (200 µg protein each) were prepared from salivary gland tumors from CD8Met transgenic mice and were immunoprecipitated with anti-CD8 or anti-Met antibodies. The immune complexes were analyzed by Western blotting with anti-phosphotyrosine and anti-Met antibodies. The numbers to the left refer to the locations of molecular weight markers, with their molecular weights given in kDa.
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When implanted in nude mice, the CD8HER2 tumors grew with decreasing latency with each successive passage (Fig. 5A
). The allografts were evaluated for their response to BMS-462043, an inhibitor that inhibits HER1 and HER2 kinases (IC50 = 40 nmol/L for both kinases) and the Sal2 cell line (IC50 = 0.46 µmol/L; compound 9b in ref. 22). BMS-462043, given by oral gavage once daily for 10 days, inhibited tumor growth in a dose-dependent manner (Fig. 5B). At 200 mg/kg/d, the compound gave nearly complete inhibition of tumor growth during the course of treatment. The responsiveness of the tumor to treatment with the kinase inhibitor confirms the dependence of the tumor cell proliferation on CD8HER2 kinase activity.
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Figure 5. Establishment of CD8HER2 allografts and inhibition of tumor growth by a HER1/HER2 kinase inhibitor. A, fragments of CD8HER2 salivary gland tumors were excised from the transgenic animals and implanted s.c. in nude mice. Growth rates of the original implants (p0) and subsequent passages (p1, p4, and p7). B, nude mice implanted with Sal2 tumors were treated with vehicle or BMS-462043 once daily for 10 consecutive days (indicated by open triangles above the x axis).
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Discussion
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The biology of the HER kinases in oncogenic signaling exemplifies the technical challenges to discovering novel kinase inhibitors that selectively target individual RTKs. Many human tumor cell lines express HER1 and HER2; yet, receptor expression is not a reliable indicator of dependence on the receptor(s) for cell proliferation or survival. The potential for receptor heterodimer signaling makes it difficult to ascertain responsiveness to agents that are highly selective for one receptor. These considerations provided a rationale for using chimeric receptors to investigate the functional properties of receptor homodimers. Previous work in this area has relied on the use of "domain swapping" to characterize orphan receptors, for which a ligand was not available for activating the kinase (23). The use of the CD8 extracellular sequence has the advantage that the sequence is sufficient to drive disulfide-mediated dimerization. The sequence is also relatively short and is therefore not likely to impart deleterious conformational influence on the folding of the intracellular sequence to which it is fused. We previously used this approach to show the oncogenic properties of a CD8 fusion protein consisting of the cytoplasmic sequence of the insulin-like growth factor-1 receptor. Here, we have extended our analyses to show that the method can be used to activate RTKs of distinct structural classes. Furthermore, it has been shown here that the chimeric receptors can be used to transform epithelial cells, yielding phenotypes that are distinctive and perhaps representative of the signaling properties of the native receptors. The CD8RTKs are highly potent oncogenes not only in cultured cells but also in transgenic mice, inducing tumors at a high frequency and with short latency. The transgenic tumors have proven to be highly adaptable to transplantation as allograft models and have been shown to be highly sensitive to kinase inhibitors that target the native receptors. These tumor models, together with cell lines that are derived from them, will be excellent tools for lead optimization in drug discovery. The CD8Met transgenic mice are prone to develop lung metastases,3 again providing support for the notion that the chimeric receptor is a reasonable surrogate of the full length receptor in its biological function (24).
The ability of the CD8RTK fusion proteins to transform RK3E cells may provide an alternate model for characterization of RTK signaling and optimization of kinase inhibitors. Whereas parental RK3E cells are not tumorigenic, RK3E cells transfected with some of the CD8RTK constructs form tumors when implanted in nude mice4 and may provide an alternative route to target-specific tumor models for lead optimization. As illustrated with CD8HER2 and CD8Met, expression of these oncogenes confers robust proliferation and morphogenic phenotypes. Upon stimulation with hepatocyte growth factor, the Met receptor is known to induce branching morphogenesis in epithelial cells grown in semisolid matrices (25, 26). It has been shown here that RK3E cells expressing CD8Met exhibit similar behavior when grown in collagen. By contrast, RK3E cells expressing CD8HER2 and grown in collagen did not exhibit this differentiation-like phenotype, consistent with the apparent differences in signaling complexes associated with the two chimeric receptors. This in vitro model may be an isogenic system for further dissecting the downstream signaling pathways that regulate the morphologic changes. In this report, we have shown the versatility of the use of CD8RTK fusion proteins as an experimental paradigm for studying RTKs. The approach provides cell and tumor models for further characterization of the biology underlying the oncogenic and morphogenic properties of RTKs. It also provides tools for optimization of drug candidates targeting RTKs, including models for both primary tumors and metastases. This approach holds promise for future application in targeting other RTKs that may emerge as potential therapeutic targets.
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Footnotes
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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: The present address for B.R. Rowley is Bayer Pharmaceutical Division, West Haven, CT. The present address for D.K. Bol is Avalon Pharmaceuticals, Gaithersburg, MD.
3 Unpublished observation. 
4 Unpublished observation.
Received 2/ 9/06;
revised 4/ 6/06;
accepted 4/21/06.
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Abstract
Introduction
