Molecular Cancer Research
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Molecular Cancer Research 5, 393-401, April 1, 2007. doi: 10.1158/1541-7786.MCR-06-0303
© 2007 American Association for Cancer Research
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Signaling and Regulation

Growth Stimulation of Non–Small Cell Lung Cancer Cell Lines by Antibody against Epidermal Growth Factor Receptor Promoting Formation of ErbB2/ErbB3 Heterodimers

Mari Maegawa1, Kenji Takeuchi1, Eishi Funakoshi1, Katsumi Kawasaki1, Kazuto Nishio2, Nobuyoshi Shimizu3 and Fumiaki Ito1

1 Department of Biochemistry, Setsunan University; 2 Department of Genome Biology, Kinki University School of Medicine, Osaka, Japan; and 3 Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan

Requests for reprints: Fumiaki Ito, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan. Phone: 81-72-866-3115; Fax: 81-72-866-3117. E-mail: fito{at}pharm.setsunan.ac.jp


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non–small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family–directed therapies for cancer. (Mol Cancer Res 2007;5(4):393–401)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The epidermal growth factor (EGF) receptor (EGFR) is a member of the structurally related ErbB family of receptor tyrosine kinase. The ErbB family includes four members [i.e., EGFR (ErbB1), ErbB2, ErbB3, and ErbB4], all of which can dimerize with each other; in addition to homodimerization, specific ligands also induce heterodimerization of different pairs of the ErbB family members (1, 2). Although structural similarity exists between the family members, important differences are also present. Unlike the rest of the ErbB family, ErbB3 lacks tyrosine kinase activity (3, 4) and ErbB2 has no known ligand (5). EGF and transforming growth factor {alpha} bind directly only to EGFR, whereas neuregulins (also known as heregulins) are specific for ErbB3 and ErbB4 (6-8). Because expression levels of the family members and their ligands vary considerably in various cells, signaling pathways via activation of EGFR family members are complex.

Ligand-induced dimerization of EGFR is required to elevate its tyrosine kinase activity. The activated EGFR autophosphorylates tyrosine residues in its own COOH terminus, after which the receptor recruits and phosphorylates several signaling molecules such as growth factor receptor binding protein 2 (Grb-2), phospholipase C{gamma}, Src homology and collagen protein (Shc), and Grb2-associated binding protein 1 (Gab1; refs. 9, 10). Thus, ligands for EGFR are able to activate a variety of signaling pathways through their association with these signaling molecules. The mitogen-activated protein kinase (MAPK) pathway leading to phosphorylation of extracellular signal–regulated kinase (ERK)-1/2 plays an essential role in cell growth (9), and the phosphatidylinositol 3-kinase pathway is also important for cell growth and cell survival (11, 12). Another important signaling pathway is one for down-regulation of activated receptors. EGF binding is believed to result in localization of EGFR to clathrin-coated pits, from where EGFR is endocytosed. Recruitment of the Grb2/Casitas B-lineage lymphoma (Cbl) complex to the EGFR and subsequent activation of Cbl–dependent ubiquitination are essential for the delivery of EGFRs into these clathrin-coated pits (13).

Alterations resulting in enhanced EGFR expression or function have been documented in a variety of tumors, including non–small cell lung cancer, breast cancer, and gliomas (14-17). These changes can occur due to increased production of ligands such as EGF and transforming growth factor {alpha}, increased gene transcription or amplification of EGFR, and receptor mutations resulting in constitutive activation of the receptor tyrosine kinase (18, 19). A variety of approaches to block the EGFR-mediated signaling pathway are currently undergoing clinical evaluation, including the use of anti-EGFR monoclonal antibodies (mAb), low molecular weight tyrosine kinase inhibitors, and immunoconjugates (20, 21). A series of anti-EGFR mAbs produced showed inhibitory activity toward the binding of EGF to A431 cells (22). As a result of the inhibition of receptor kinase, these anti-EGFR mAbs prevented ligand-induced stimulation of growth in a variety of cells that expressed both EGFR and ligand (20). It was also reported that anti-EGFR mAbs have the capacity to form receptor-containing complexes that result in receptor internalization, an important mechanism for attenuating receptor signaling (23).

It is important to further explore the primary mechanism by which various antibodies against EGFR affect growth of a variety of cells. In this present study, we decided to examine the growth-inhibitory effect of a mouse anti-human EGFR mAb, B4G7, which had previously been prepared against human A431 cells (24). Contrary to our expectations, B4G7 actually exhibited a growth-stimulatory effect in a variety of cells including gefitinib-sensitive and gefitinib-resistant non–small cell lung cancer cell lines, PC-9 and PC-14, respectively. Because EGF showed no stimulatory effect on the growth of these cell lines, we studied the molecular effects of B4G7 mAb and EGF in more detail. Our results indicate that mAb against EGFR increased growth of several cancer cell lines by stimulating the formation of ErbB2/ErbB3 heterodimers. Therefore, it is of importance to consider the status of all ErbB family members in cancer cells, not just the EGFR, for optimizing EGFR-directed cancer therapies.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Growth-Stimulatory Activity of mAb against EGFR
We previously produced a mouse anti-human EGFR mAb against A431 cells and referred to it as B4G7 (24). In this study, we first examined the effect of the B4G7 mAb on the growth of various human cancer cell lines. The mAb exhibited a growth-stimulatory effect on human non–small cell lung cancer cell lines (PC-9, PC-14, and A549) as well as on A431 human epidermoid carcinoma cells, as determined from the results of a colorimetric assay (Fig. 1 ). The growth-stimulatory action of B4G7 was confirmed by counting the number of PC-14 cells in the presence or absence of B4G7 (data not shown). We also examined the mitogenic activity of purified mouse immunoglobulin G (IgG) toward PC-14 cells and found that their growth was not affected by this control IgG (data not shown). These were unexpected results because many research groups have previously reported that anti-EGFR mAbs block the binding of ligand to various EGFR-expressing human cancer cell lines and thereby abolish ligand-dependent cell proliferation (20). Because EGF showed no stimulatory effect on the growth of these cell lines, we next studied the molecular effects of B4G7 mAb and EGF on EGFR.


Figure 1
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  		FIGURE 1. Growth-stimulatory effect of anti-EGFR mAb. Cells were treated or not with 100 ng/mL EGF or 10 µg/mL B4G7 for 2 d and their growth was estimated by means of WST-1 assay as described in Materials and Methods. Columns, mean (n = 6); bars, SD. *, P < 0.01. Similar results were obtained from five independent experiments.

 
B4G7 mAb Affects Neither the Internalization nor the Down-Regulation of EGFR in PC-14 Cells
As shown in Fig. 2A , the majority of EGFRs were localized at the cell surface in the control cells. After EGF stimulation, the distribution of EGFRs was quite distinct: the cell-surface receptors disappeared; and EGFR-containing vesicles appeared in their place, thus indicating the internalization of EGFR on EGF stimulation. In contrast, most of EGFRs still remained on the cell surface after B4G7 treatment. We next did Western blot analysis to assess the down-regulation of EGFRs after stimulation of PC-14 cells with EGF or B4G7 mAb (Fig. 2B). The down-regulation increased as the incubation time with EGF was lengthened, whereas EGFRs were still detected even 12 h after B4G7 treatment. Thus, B4G7 mAb affected neither the internalization nor the down-regulation of EGFRs.


Figure 2
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  		FIGURE 2. B4G7 affects neither the internalization nor the down-regulation of EGFR in PC-14 cells. A. PC-14 cells were treated with 100 ng/mL EGF or 10 µg/mL B4G7 mAb for 15 min. EGFR localization of these cells was determined by immunostaining as described in Materials and Methods. Bar, 10 µm. B. PC-14 cells were treated or not with EGF or B4G7 for the indicated times. Cell lysates were prepared and used for the detection of EGFR as described in Materials and Methods. The blot was reprobed with a ß-actin antibody to show equal loading. Similar results were obtained from four independent experiments. Immunoblot analyses of total cellular lysates, which were prepared by using the Laemmli SDS buffer containing 5% mercaptoethanol, gave similar results.

 
Activation of EGFR, ERK1/2, and Akt after B4G7 Treatment
Next, we assessed EGFR phosphorylation on Tyr1173 and phosphorylation of ERK1/2 and Akt, downstream molecules of EGFR, after stimulation of PC-14 cells with EGF or B4G7 mAb (Fig. 3 ). Following the addition of EGF or B4G7, tyrosine phosphorylation of EGFR was observed with a peak at 10 to 15 min. ERK1/2 and Akt were phosphorylated even in the absence of EGF or B4G7, and this phosphorylation was further augmented in both EGF- and B4G7-treated cells. It thus seems that the growth-stimulatory activity of B4G7 mAb is not simply explained by its activity to stimulate tyrosine phosphorylation of EGFR and subsequent phosphorylation of ERK1/2 and Akt molecules. Figure 3 also shows the effect of AG1478, a specific inhibitor of EGFR tyrosine kinase, on tyrosine phosphorylation of EGFR. This inhibitor suppressed the tyrosine phosphorylation of EGFR in both EGF- and B4G7-treated cells, although a faint band of phosphorylated EGFR band was still visible in EGF-treated cells after AG1478 treatment. On the other hand, the inhibitory activity of the inhibitor against the phosphorylation of ERK1/2 and Akt was not remarkable. This result suggests that activation of the two signaling proteins ERK1/2 and Akt in B4G7-treated cells occurred via a pathway independent of the tyrosine phosphorylation of EGFR.


Figure 3
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  		FIGURE 3. AG1478 inhibits tyrosine phosphorylation of EGFR but not phosphorylation of Akt and ERK1/2. PC-14 cells were pretreated with 200 nmol/L AG1478 for 2 h and then incubated with EGF or B4G7 for the indicated times. Total cell extracts were then prepared and electrophoresed on 7.5% SDS-PAGE for the detection of EGFR phosphorylation or on 12.5% SDS-PAGE for the detection of phosphorylated ERK1/2 and phosphorylated Akt. EGFR, ERK1/2, and Akt and phosphorylation of these molecules were detected by using each corresponding antibody. Similar results were obtained from three independent experiments.

 
Next, we determined the effect of AG1478 on the B4G7-stimulated cell growth (Fig. 4 ). This inhibitor showed no inhibitory activity against the stimulation of PC-14 cell growth, indicating that the growth stimulation by B4G7 mAb seems to have been independent of the activation of EGFR tyrosine kinase.


Figure 4
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  		FIGURE 4. AG1478 shows no inhibitory activity against B4G7-stimulated cell growth. PC-14 cells were pretreated with 200 nmol/L AG1478 for 2 h and then incubated with 100 ng/mL EGF or 10 µg/mL B4G7. After 2 d of incubation, their growth was estimated by means of the WST-1 assay as described in Materials and Methods. Columns, mean (n = 6); bars, SD. *, P < 0.01. Similar results were obtained from three independent experiments.

 
Stimulated Formation of HER2/HER3 Heterodimer by B4G7
EGFR, ErbB2, ErbB3, and ErbB4 are members of the ErbB family of receptors. ErbB receptors signal through a network involving receptor homodimerization and heterodimerization. Thus, we examined whether B4G7 mAb would cause the down-regulation of other members of the ErbB family although it did not down-regulate EGFR. As shown in Fig. 5A , incubation of PC-14 cells with EGF, but not with B4G7 mAb, caused down-regulation of ErbB2. On the other hand, neither EGF nor B4G7 mAb down-regulated ErbB3. Many studies have shown that ligand-activated EGFRs preferentially recruit ErbB2 into a heterodimeric complex in cells that coexpress ErbB2 (25). Thus, it is most likely that EGF down-regulated ErbB2 through the increased formation of EGFR/ErbB2 heterodimer. In fact, when lysates from EGF-treated PC-14 cells were incubated with anti-EGFR antibody, ErbB2 became detectable in the immunocomplex (Fig. 5B). In contrast, it was not detected in lysates from B4G7-treated PC-14 cells.


Figure 5
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  		FIGURE 5. B4G7 stimulates ErbB2/ErbB3 heterodimer formation. A. PC-14 cells were treated or not with EGF or B4G7 for the indicated times. Lysates were prepared from these cells and used for the detection of ErbB2 and ErbB3 as described in Materials and Methods. The blot was reprobed with a ß-actin antibody to show equal loading. B. EGFR was immunoprecipitated from the lysates by using anti-EGFR antibody, and the immunoprecipitates were examined for EGFR and ErbB2 as described in Materials and Methods. C. PC-14 cells were treated or not with EGF or B4G7 for 15 min. The supernatant fractions of cell lysates were immunoprecipitated with anti–c-erbB3 antibody, and proteins eluted from the immunocomplexes were subjected to SDS-PAGE and used for immunoblot analysis of ErbB2 and ErbB3. Similar results were obtained from three independent experiments.

 
ErbB3 has been observed to preferentially heterodimerize with ErbB2 in several cancers, leading to a strong oncogenic signal thought to promote tumor cell proliferation (26). We thus studied whether treatment of PC-14 cells with EGF or B4G7 would affect complex formation between ErbB2 and ErbB3. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7 but not EGF stimulated heterodimeriztion between ErbB2 and ErbB3 (Fig. 5C).

The EGFR extracellular domain (amino acids 1-621) shares 45% amino acid identity with that of ErbB3. Due to this homology, specific B4G7 antibody against EGFR may show some cross-reactivity against ErbB3. We thus determined whether B4G7 binds to ErbB3 as well as to EGFR. Immunocomplexes were prepared from lysates of PC-14 cells using B4G7 and assayed for the presence of ErbB3 by immunoblot analysis. Because ErbB3 was not detected at all in these complexes (data not shown), B4G7 seems to promote formation of ErbB2/ErbB3 heterodimers by its binding to EGFR but not to ErbB3.

B4G7-Stimulated Cell Growth Requires ErbB2/ErbB3 Heterodimer and ErbB3 Tyrosine Phosphorylation
If ErbB2/ErbB3 heterodimers are formed in the presence of B4G7, ErbB3 of B4G7-treated cells could be phosphorylated by ErbB2 tyrosine kinase. Indeed, ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF (Fig. 6A ). Further, this phosphorylation was inhibited by AG825, a specific inhibitor of ErbB2. On the contrary, EGF-induced phosphorylation of ErbB2 at Tyr1248, possibly through heterodimerization of EGFR/ErbB2, was not inhibited by AG825 (Fig. 6B), indicating a selective inhibitory action of this inhibitor against ErbB2 tyrosine kinase. Further analysis of immunocomplexes formed in the presence of anti-EGFR antibody confirmed that ErbB2 phosphorylation by EGFR tyrosine kinase was not inhibited by this inhibitor: tyrosine-phosphorylated ErbB2 was detected only in the immunocomplexes from EGF-treated cells but not in those from B4G7-treated cells, and this phosphorylation was not inhibited by AG825 (data not shown).


Figure 6
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  		FIGURE 6. AG825 inhibits tyrosine phosphorylation of ErbB3 and cell growth in B4G7-treated cells. A. PC-14 cells were pretreated with 0.5 µmol/L AG825 for 2 h and then treated with EGF or B4G7 for 15 min. The cells were then subjected to immunoprecipitation with anti–c-erbB3 antibody and used for the detection of ErbB3 and its tyrosine phosphorylated form by using anti-ErbB3 antibody and anti-phosphotyrosine antibody (PY20). B. PC-14 cells were incubated with EGF for the indicated times after AG825 (0.5 µmol/L) pretreatment. Cell lysates were then prepared and used for immunoblot analysis of ErbB2 and its phosphorylation by using anti–c-ErbB2 antibody and anti–phospho-erbB2 (Tyr1248) antibody, respectively. C. PC-14 cells were incubated with EGF or B4G7 for 2 d in the presence of 0.5 µmol/L AG825. Their growth was estimated by means of the WST-1 assay as described in Materials and Methods. Columns, mean (n = 6); bars, SD. *, P < 0.01. Similar results were obtained from three independent experiments.

 
To examine association of ErbB2 tyrosine kinase with B4G7-induced cell growth, we treated PC-14 cells with AG825 before their stimulation with EGF or B4G7. As shown in Fig. 6C, cell growth increased by B4G7 was suppressed to the control level by this pretreatment. These results show that ErbB2 tyrosine kinase and the ErbB2/ErbB3 dimer function to promote cell growth in B4G7-treated cells.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
We previously produced a mAb against EGFRs by immunizing BALB/c mice with human epidermoid carcinoma A431 cells (24). This mAb, which we named B4G7, inhibited the binding of 125I-EGF to A431 cells and human fibroblasts and specifically precipitated EGFR of A431 cells. Sato et al. (22) also produced mAbs against A431 cells and found that these antibodies were capable of inhibiting both the binding of EGF to its receptor and ligand-induced cell proliferation. In this study, we made the unexpected finding that B4G7 mAb exerted growth-stimulatory activity toward various cancer cell lines. We initially thought that B4G7 stimulated cell growth via signaling pathways originating from EGFR tyrosine phosphorylation because B4G7 stimulated EGFR tyrosine phosphorylation and also phosphorylation of MAPK and Akt, two downstream signaling molecules of EGFR. However, the growth stimulation by B4G7 was independent of EGFR activation itself because the stimulation was not affected by the presence of AG1478, an EGFR tyrosine kinase–specific inhibitor.

EGFR forms homodimers as well as heterodimers with the other ErbB family members, and cooperation between ErbB family members plays pivotal roles in a variety of critical functions. It is thus reasonable to speculate that binding of B4G7 mAb to EGFR affects cross-talk among the ErbB family and the cellular effects mediated by these receptors. In B4G7-treated PC-14 cells, the EGFR/ErbB2 complex was not detected, although its formation was increased in response to EGF (Fig. 5B). Therefore, B4G7 may have blocked the ability of EGFR to heterodimerize with ErbB2 or ErbB3 and thus facilitated enhanced dimerization between ErbB2 and ErbB3. In fact, ErbB2/ErbB3 heterodimers were formed in the presence of B4G7 but not in the presence of EGF (Fig. 5C). In EGF-treated PC-14 cells, major combinations of ErbB family members were EGFR homodimer and EGFR/ErbB2 heterodimer. AG1478 inhibited, but not completely, EGFR phosphorylation, whereas it inhibited phosphorylation of ERK1/2 and Akt to a lesser extent. The limited efficacy of AG1478 could have arisen from unblocked ErbB2 signaling in the form of EGFR/ErbB2.

At least six different ligands are known to bind to EGFR. These ligands include EGF, transforming growth factor {alpha}, amphiregulin, heparin-binding EGF, betacellulin, and epiregulin (1, 20, 27). A second class of ligands, collectively termed neuregulin, bind directly to ErbB3 and/or ErbB4 (6-8). It is known that ErbB2 and ErbB3 dimerize to produce a high-affinity receptor for neuregulin-1. Because production and secretion of neuregulin-1 have been reported in many human lung cancer cell lines (28), it is very likely that neuregulin-1 or neuregulin isoform is secreted from PC-14 cells and thereafter activates the ErbB2/ErbB3 heterodimer in an autocrine fashion. This likelihood is supported by the finding that ErbB3 phosphorylation was increased on B4G7 addition and abrogated in the presence of AG825, which selectively inhibits the ErbB2 kinase activity.

The physiologic role of ErbB2, in the context of ErbB ligand signaling, is to serve as a coreceptor (29, 30). ErbB2 seems to be the preferred partner of the other ligand-bound ErbBs (25, 31). ErbB3 functions as an indispensable ErbB2 dimerization partner and is required for proliferation of ErbB2-overexpressing tumor cells, and neither ErbB1 nor ErbB4 could replace ErbB3 as partner of ErbB2 to drive proliferation. Therefore, it seems that the ErbB2/ErbB3 formed in the presence of B4G7 but not in the presence of EGF transmits effective proliferation signals in PC-14 cells. This notion is supported by our finding that AG825, a specific inhibitor of ErbB2, suppressed B4G7-stimulated cell growth (Fig. 6C). Taken together, these findings indicate that B4G7 transmitted signals for growth stimulation by increasing the formation of ErbB2/ErbB3 (see Fig. 7 ).


Figure 7
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  		FIGURE 7. Schematic representation of how mAb B4G7 stimulates cell growth. Homodimers and heterodimers of EGFR are formed on EGF addition. On the other hand, the ErbB2/ErbB3 heterodimer is formed in the presence of B4G7 mAb because binding of B4G7 mAb to EGFR inhibits the ability of EGFR to form heterodimers with ErbB2 and ErbB3. ErbB2/ErbB3, unlike EGFR dimers, continues to exist on the cell surface and transmits signals for growth stimulation. ErbB2/ErbB3 may be activated in an autocrine fashion.

 
The signals generated by activated growth factor receptors are generally attenuated by the process of receptor internalization, which leads to receptor degradation (32, 33). Neuregulin has been reported to undergo slow endocytosis followed by receptor recycling to the plasma membrane (34). In contrast, most of the EGF-stimulated EGFR molecules are destined to lysosomal degradation. Due to the consequent clearance of EGFR but not ErbB3 molecules from the cell surface, the mitogenic signal evoked by EGF is less potent than the neuregulin signal (29). In PC-14 cells, EGFR and ErbB2 were down-regulated in the presence of EGF, but their levels were unchanged after B4G7 treatment. Therefore, slow endocytosis and receptor recycling of ErbB2 and ErbB3 may explain the capacity of B4G7 to deliver more sustained mitogenic signals than EGF. Another mechanism for the different mitogenic capacity probably involves the recruitment of distinct sets of downstream effectors to each of the activated ErbB family members. One of the downstream effectors is phosphatidylinositol 3-kinase, the activation of which results in the phosphorylation of the 3' position of phosphatidylinositol 4,5-bis-phosphate to yield phosphatidylinositol 3,4,5-tris-phosphate. Phosphatidylinositol 3,4,5-tris-phosphate, in turn, activates several downstream signaling molecules including Akt. ErbB3 effectively couples to the phosphatidylinositol 3-kinase/Akt pathway because it has six tyrosine phosphorylation sites with YXXMs motifs, which serve as excellent binding sites for phosphatidylinositol 3-kinase (35, 36). Because the phosphatidylinositol 3-kinase/Akt pathway originating from ErbB3 is suggested to play an important role in the stimulation of cell growth (37, 38), it is reasonable to speculate that this pathway is linked to the mitogenic superiority of ErbB3. In this study, we determined the phosphorylation time course of Akt in B4G7- and EGF-treated cells, but there was no significant difference between their phosphorylation kinetics.

Neuregulin activates ERK MAPK, a signaling pathway that is critical in the mitogenic effect of neuregulin (39, 40). It has been indicated that sustained, but not transient, activation of ERK induces phosphorylation of immediate early gene products, which leads to their stabilization and activation, resulting in appropriate gene expression, such as that of cyclin D (41, 42). Further, only sustained ERK activation induces and maintains decreased expression levels of antiproliferative genes (43). Thus, the duration and magnitude of ERK activity is a key determinant for the mitogenic response of various cells to EGF (44, 45). The differential mitogenic response of PC-14 cells to EGF and B4G7 may be related to the differential kinetics of ERK activation in B4G7- and EGF-treated cells. However, both EGF and B4G7 mAb stimulated phosphorylation of ERK in a similar time-dependent manner (see Fig. 3). We studied phosphorylation of ERK and Akt in PC-14 cells treated with EGF or B4G7 for a longer period of time, but observed no significant difference in the phosphorylation time course between B4G7- and EGF-treated cells.4 The duration of Akt and MAPK activities may be essential, but not sufficient, for ensuring G1 phase progression of PC-14 cells. A more detailed side-by-side comparison of PC-14 cells treated with EGF or B4G7 should provide a hint for elucidating the signaling pathway for B4G7-induced cell growth.

A variety of approaches to block the EGFR-mediated signaling pathway are undergoing clinical evaluation, including the use of mAbs against EGFR and ErbB2 (46). The study presented here might have important clinical implications because it indicates that mAb against EGFR stimulated the growth of several cancer cell lines by affecting dimerization of EGFR family members other than EGFR. Similarly, ZD1839, a specific EGFR tyrosine kinase inhibitor, has been reported to inhibit the growth of ErbB2-overexpressing breast cancer cells, possibly by sequestration of ErbB2 and ErbB3 receptors in an inactive heterodimer configuration with EGFR (47). Another group also reported that elimination of ErbB2 signaling resulted in an increase in EGFR expression and activation and that its increased activation contributed to sustained cell survival (48). Changes in receptor-receptor interactions between ErbB family members and compensatory changes in the ErbB family after inhibition of one of its members are of potential importance in optimizing current EGFR family–directed therapies for cancer.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Materials
EGF (ultrapure) from mouse submaxillary glands was purchased from Toyobo Co. Ltd. (Osaka, Japan). FCS, phenylmethylsulfonyl fluoride, pepstatin A, p-toluenesulfonyl-L-arginine methyl ester, leupeptin, and aprotinin came from Sigma (St. Louis, MO). AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline] and AG825 [4-hydroxy-3-methoxy-5-(benzothiazolylthiomethyl)benzylidenecyanoacetamide] were purchased from Calbiochem (San Diego, CA). RPMI 1640 and DMEM were from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Antibodies used and their sources were as follows: anti-phosphotyrosine (PY20; BD Transduction Laboratories, San Jose, CA); anti–phospho-EGFR (Tyr1173) and anti–phospho-erbB2 (Tyr1248) (Upstate Biotechnology, Lake Placid, NY); anti-EGFR (1005) and anti-ErbB3 (C-17; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-Akt (Cell Signaling Technology, Inc., Beverly, MA); anti-MAPK (Sigma); anti–phospho-Akt (Tyr473) and anti-ACTIVE MAPK (Promega, Madison, WI); anti–c-ErbB2/c-Neu (Ab-3; Calbiochem); horseradish peroxidase–conjugated swine anti-rabbit immunoglobulin (DAKO, Glostrup, Denmark); and horseradish peroxidase–linked sheep anti-mouse IgG and biotinylated sheep anti-mouse immunoglobulin (GE Healthcare, Piscataway, NJ). A mouse anti-human EGFR mAb (B4G7) was purified from mouse ascites by ammonium sulfate precipitation and protein G column chromatography. All other chemicals were commercial products of reagent grade.

Cell Culture
Human non–small cell lung cancer cell lines PC-9 and PC-14 were obtained from Tokyo Medical University (Tokyo, Japan). Both lines were cultured in RPMI 1640 supplemented with 5% FCS in 5% CO2 at 37°C in a fully humidified atmosphere. Human adenocarcinoma A549 and epidermoid carcinoma A431 were cultured in DMEM supplemented with 5% FCS. Exponentially growing cells were used in all experiments.

Growth Stimulation Assay
Cells were seeded at a density of 2 x 103 per well into a 96-well microtiter plate and cultured for 2 days in the presence of 5% FCS. They were then treated with 100 ng/mL EGF or 10 µg/mL B4G7 mAb. After incubation for 48 h, growth stimulation was quantified by a colorimetric assay with the WST-1 reagent according to the manufacturer's instruction (Dojindo Laboratories, Kumamoto, Japan).

Preparation of Cellular Lysates and Immunoblotting
PC-14 cells were seeded at a density of 1.2 x 105 per 35-mm-diameter dish and cultured for 2 days. They were then treated or not with 100 ng/mL EGF or 10 µg/mL B4G7 mAb for indicated times at 37°C. When the effects of AG1478 or AG825 were assayed, these inhibitors were added 2 h before the addition of EGF or B4G7. The cells were then washed with ice-cold PBS and subsequently lysed by incubating in hypotonic buffer [10 mmol/L Tris-HCl (pH 7.8), containing 10 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 0.3% NP40]. The lysates were incubated on ice for 10 min and clarified by centrifugation at 1,500 x g for 5 min at 4°C. Total proteins (10 µg/mL) from the supernatant fractions were resolved by SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membranes were sequentially incubated, first with primary antibody for 2 h and then with horseradish peroxidase–conjugated anti-rabbit IgG antibody (1:1,000) or anti-mouse IgG antibody (1:1,000) for 1 h. Finally, the proteins were visualized by use of an enhanced chemiluminescence Western Blotting Detection System (GE Healthcare) and exposed to autoradiography film (Fuji Medical X-ray film RX-U, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Immunostaining of Cells for Confocal Laser Scanning Microscopic Observation
Immunostaining of cells was done as previously described (49). Briefly, PC-14 cells were grown on coverslips for 2 days and then stimulated with 100 ng/mL EGF or 10 µg/mL B4G7 mAb for 15 min. The cells were fixed with methanol for 5 min at –20°C, after which they were washed thrice with 20 mmol/L TBS (pH 7.4) containing 1 mmol/L CaCl2 (TBS-Ca) and incubated with anti-EGFR antibody for 2 h at room temperature. After being washed with TBS-Ca, the cells were incubated with biotinylated sheep anti-mouse immunoglobulin antibody (1:100) for 1 h at room temperature and then with Texas red–labeled streptavidin (GE Healthcare). The stained cells were observed under a confocal laser scanning microscope (MRC1024, Bio-Rad, Hercules, CA).

Immunoprecipitation
PC-14 cells were seeded at 1.2 x 106 per 150-mm dish and incubated in RPMI 1640/5% FCS for 2 days. The cultures were then incubated for 2 h in the presence or absence of 0.5 µmol/L AG825 and treated with either EGF or B4G7 for the indicated times at 37°C. They were lysed in hypotonic buffer and centrifuged at 1,500 x g for 5 min as described above. The supernatant fractions were incubated overnight at 4°C with anti–c-erbB3 (clone 2F12) antibody (LabVision Co., Fremont, CA) or anti-EGFR antibody (B4G7). Immunocomplexes were collected on protein G-sepharose (GE Healthcare). Bound proteins were washed thrice with 10 mmol/L Tris-HCl buffer (pH 7.4) containing 135 mmol/L NaCl, 0.1% NP40, 0.1% Triton X-100, a cocktail of protease inhibitors (0.1 mg/mL phenylmethylsulfonyl fluoride, 2 µg/mL leupeptin, 1 µg/mL pepstatin A, 0.1 µg/mL p-toluenesulfonyl-L-arginine methyl ester), 1 mmol/L sodium orthovanadate, 2 mmol/L EGTA, 5 mmol/L EDTA, 50 mmol/L sodium fluoride, and 30 mmol/L Na4P2O7 and once with TBS and eluted in Laemmli sample buffer containing 2-mercaptoethanol. Eluted proteins were subjected to SDS-PAGE and immunoblotted as described above.

Protein Assay
Protein content was assayed by using a Coomassie Plus Protein Assay reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science; fund for "Research for the Future" Program from the Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science, and Technology; and funding from the Fugaku Trust for Medical Research.

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: M. Maegawa and K. Takeuchi contributed equally to this work.

4 Kenji Takenchi and Fumiaki Ito, unpublished data. Back

Received 9/18/06; revised 1/16/07; accepted 2/ 6/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159–67.[CrossRef][Medline]
  2. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–37.[CrossRef][Medline]
  3. Sliwkowski MX, Schaefer G, Akita RW, et al. Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin. J Biol Chem 1994;269:14661–5.[Abstract/Free Full Text]
  4. Guy PM, Platko JV, Cantley LC, Cerione RA, Carraway KL III. Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc Natl Acad Sci U S A 1994;91:8132–6.[Abstract/Free Full Text]
  5. Klapper LN, Glathe S, Vaisman N, et al. The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci U S A 1999;96:4995–5000.[Abstract/Free Full Text]
  6. Tzahar E, Levkowitz G, Karunagaran D, et al. ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms. J Biol Chem 1994;269:25226–33.[Abstract/Free Full Text]
  7. Lupu R, Colomer R, Kannan B, Lippman ME. Characterization of a growth factor that binds exclusively to the erbB-2 receptor and induces cellular responses. Proc Natl Acad Sci U S A 1992;89:2287–91.[Abstract/Free Full Text]
  8. Holmes WE, Sliwkowski MX, Akita RW, et al. Identification of heregulin, a specific activator of p185erbB2. Science 1992;256:1205–10.[Abstract/Free Full Text]
  9. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–25.[CrossRef][Medline]
  10. Ruff-Jamison S, McGlade J, Pawson T, Chen K, Cohen S. Epidermal growth factor stimulates the tyrosine phosphorylation of SHC in the mouse. J Biol Chem 1993;268:7610–2.[Abstract/Free Full Text]
  11. Marte BM, Downward J. PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem Sci 1997;22:355–8.[CrossRef][Medline]
  12. Takeuchi K, Ito F. Suppression of Adriamycin-induced apoptosis by sustained activation of the phosphatidylinositol-3'-OH kinase-Akt pathway. J Biol Chem 2004;279:892–900.[Abstract/Free Full Text]
  13. Stang E, Blystad FD, Kazazic M, et al. Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits. Mol Biol Cell 2004;15:3591–604.[Abstract/Free Full Text]
  14. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.[Abstract/Free Full Text]
  15. Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002;7(Suppl 4):31–9.[Medline]
  16. Humphrey PA, Gangarosa LM, Wong AJ, et al. Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function. Biochem Biophys Res Commun 1991;178:1413–20.[CrossRef][Medline]
  17. Wong AJ, Bigner SH, Bigner DD, Kinzler KW, Hamilton SR, Vogelstein B. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci U S A 1987;84:6899–903.[Abstract/Free Full Text]
  18. Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003;284:31–53.[CrossRef][Medline]
  19. Chu CT, Everiss KD, Wikstrand CJ, Batra SK, Kung HJ, Bigner DD. Receptor dimerization is not a factor in the signalling activity of a transforming variant epidermal growth factor receptor (EGFRvIII). Biochem J 1997;324:855–61.[Medline]
  20. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000;19:6550–65.[CrossRef][Medline]
  21. Herbst RS, Onn A, Mendelsohn J. The role of growth factor signaling in malignancy. In: Frank DA, editor. Signal transduction in cancer. Boston/Dordrecht/London: Kluwer Academic Publishers; 2003. p. 19–72.
  22. Sato JD, Kawamoto T, Le AD, Mendelsohn J, Polikoff J, Sato GH. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med 1983;1:511–29.[Medline]
  23. Fan Z, Lu Y, Wu X, Mendelsohn J. Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J Biol Chem 1994;269:27595–602.[Abstract/Free Full Text]
  24. Behzadian MA, Shimizu N. Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor. Cell Struct Funct 1985;10:219–32.[Medline]
  25. Graus-Porta D, Beerli RR, Daly JM, Hynes NE. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 1997;16:1647–55.[CrossRef][Medline]
  26. Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF III, Hynes NE. The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci U S A 2003;100:8933–8.[Abstract/Free Full Text]
  27. Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 1997;410:83–6.[CrossRef][Medline]
  28. Gollamudi M, Nethery D, Liu J, Kern JA. Autocrine activation of ErbB2/ErbB3 receptor complex by NRG-1 in non-small cell lung cancer cell lines. Lung Cancer 2004;43:135–43.[CrossRef][Medline]
  29. Pinkas-Kramarski R, Soussan L, Waterman H, et al. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J 1996;15:2452–67.[Medline]
  30. Riese DJ II, van Raaij TM, Plowman GD, Andrews GC, Stern DF. The cellular response to neuregulins is governed by complex interactions of the erbB receptor family. Mol Cell Biol 1995;15:5770–6.[Abstract]
  31. Tzahar E, Waterman H, Chen X, et al. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 1996;16:5276–87.[Abstract]
  32. Jaramillo ML, Leon Z, Grothe S, Paul-Roc B, Abulrob A, O'Connor McCourt M. Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting. Exp Cell Res 2006;312:2778–90.[Medline]
  33. Wiley HS. Trafficking of the ErbB receptors and its influence on signaling. Exp Cell Res 2003;284:78–88.[CrossRef][Medline]
  34. Waterman H, Sabanai I, Geiger B, Yarden Y. Alternative intracellular routing of ErbB receptors may determine signaling potency. J Biol Chem 1998;273:13819–27.[Abstract/Free Full Text]
  35. Ram TG, Ethier SP. Phosphatidylinositol 3-kinase recruitment by p185erbB-2 and erbB-3 is potently induced by neu differentiation factor/heregulin during mitogenesis and is constitutively elevated in growth factor-independent breast carcinoma cells with c-erbB-2 gene amplification. Cell Growth Differ 1996;7:551–61.[Abstract]
  36. Cantley LC, Auger KR, Carpenter C, et al. Oncogenes and signal transduction. Cell 1991;64:281–302.[CrossRef][Medline]
  37. Engelman JA, Janne PA, Mermel C, et al. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Natl Acad Sci U S A 2005;102:3788–93.[Abstract/Free Full Text]
  38. Kim HH, Sierke SL, Koland JG. Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product. J Biol Chem 1994;269:24747–55.[Abstract/Free Full Text]
  39. Fiddes RJ, Janes PW, Sivertsen SP, Sutherland RL, Musgrove EA, Daly RJ. Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells. Oncogene 1998;16:2803–13.[CrossRef][Medline]
  40. Vijapurkar U, Kim MS, Koland JG. Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling. Exp Cell Res 2003;284:291–302.[Medline]
  41. Murphy LO, Smith S, Chen RH, Fingar DC, Blenis J. Molecular interpretation of ERK signal duration by immediate early gene products. Nat Cell Biol 2002;4:556–64.[Medline]
  42. Weber JD, Raben DM, Phillips PJ, Baldassare JJ. Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem J 1997;326:61–8.[Medline]
  43. Yamamoto T, Ebisuya M, Ashida F, Okamoto K, Yonehara S, Nishida E. Continuous ERK activation down-regulates antiproliferative genes throughout G1 phase to allow cell-cycle progression. Curr Biol 2006;16:1171–82.[CrossRef][Medline]
  44. Meloche S, Seuwen K, Pages G, Pouyssegur J. Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity. Mol Endocrinol 1992;6:845–54.[Abstract]
  45. Torii S, Yamamoto T, Tsuchiya Y, Nishida E. ERK MAP kinase in G cell cycle progression and cancer. Cancer Sci 2006;97:697–702.[CrossRef][Medline]
  46. Carter P, Smith L, Ryan M. Identification and validation of cell surface antigens for antibody targeting in oncology. Endocr Relat Cancer 2004;11:659–87.[Abstract/Free Full Text]
  47. Anido J, Matar P, Albanell J, et al. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells. Clin Cancer Res 2003;9:1274–83.[Abstract/Free Full Text]
  48. Hu YP, Venkateswarlu S, Sergina N, et al. Reorganization of ErbB family and cell survival signaling following knockdown of ErbB2 in colon cancer cells. J Biol Chem 2005;280:27383–92.[Abstract/Free Full Text]
  49. Funakoshi E, Hori T, Haraguchi T, et al. Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome. BMC Cell Biol 2003;4:12.[CrossRef][Medline]




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