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Endocrinology Vol. 147, No. 12 6027-6035
Copyright © 2006 by The Endocrine Society

Insulin Growth Factor-I and Epidermal Growth Factor Receptors Recruit Distinct Upstream Signaling Molecules to Enhance AKT Activation in Mammary Epithelial Cells

Jodie M. Fleming, Gwenaëlle Desury, Tiffany A. Polanco and Wendie S. Cohick

Department of Animal Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901-8520

Address all correspondence and requests for reprints to: Wendie S. Cohick, Ph.D., Rutgers, The State University of New Jersey, 108 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901-8520. E-mail: cohick{at}aesop.rutgers.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5–100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACCUMULATION OF cellular defects that leads to breast cancer consists of the failure of tumor suppressor genes, activation of growth-promoting genes, and mutations in oncogenes. The unrepressed activation of growth-promoting genes is typically stimulated by growth factors, their receptors, and activation of their signal transduction pathways. Thus, it is not surprising that the IGF and epidermal growth factor (EGF) families have emerged as major factors in the etiology of breast cancer, in addition to their role in normal mammary gland physiology (1, 2, 3, 4, 5, 6).

The IGF and EGF ligands are mitogenic for both normal and tumorigenic mammary epithelial cells in vitro (7). The major postnatal effects of the IGF system on cell proliferation are mediated by IGF-I through the type I IGF receptor (IGF-IR) (8). In addition, six IGF binding proteins(1, 2, 3, 4, 5, 6) bind IGF and modulate its ability to regulate cell growth via complex mechanisms that are not completely understood (9). The EGF family is comprised of seven ligands, including EGF and TGF{alpha}, and four receptors (ErbB1 through -4) (6). Of the four receptors, ErbB1, commonly known as the EGFR, is the primary receptor that binds EGF and TGF{alpha} and is expressed in ductal epithelial cells of normal breast tissue (10). Interestingly, each of the four ErbB receptors can undergo homo- or heterodimerization, resulting in different dimeric receptors that display individual specificity and affinity toward the EGF ligands (11, 12).

The IGF and EGF receptors share a common molecular structure of a cytoplasmic tail containing tyrosine residues that, when phosphorylated, provide specific docking sites for the Src homology 2 or phosphotyrosine binding domains of a number of adaptor proteins such as Grb2, Grb10, SHC, Crk, insulin receptor substrate (IRS)-1 and -2 (13, 14). The formation of these scaffolding complexes ultimately leads to the activation of the phosphatidylinositol-3 kinase (PI3K) and MAPK signal transduction pathways. These are evolutionary conserved pathways, involved in the control of many essential cellular processes including cell proliferation, survival, differentiation, apoptosis, motility, and metabolism (15, 16). The fundamental biochemistry of these pathways is now fairly well established; however, the mechanisms by which these pathways simultaneously process specific inputs from IGF-I and EGF to generate diverse biological outputs remain unresolved.

Whereas the IGF-I and EGF receptors are capable of binding similar docking proteins, the specific molecules recruited by each receptor to activate the MAPK and PI3K signaling cascades vary between cell types. A commonly reported mechanism for IGF-IR activation of the PI3K pathway is through the direct association of the IGF-IR with IRS-1; activated IRS-1 then associates with the p85 regulatory subunit of PI3K, resulting in activation of PI3K and recruitment of AKT to the membrane for activation (17, 18). EGFR activation of the PI3K pathway is less well characterized, but may also involve Grb2-stimulated activation of Ras (19). Another proposed mechanism by which the EGFR activates PI3K is through heterodimerization with another EGFR homolog, ErbB3, which has been shown to directly bind the regulatory subunit of PI3K (20). Activation of the EGFR is most often reported to be associated with the recruitment and activation of SHC and Grb2 (14). This association directly leads to activation of Ras and Raf, which are the point of entry for activation of the downstream molecules of the MAPK pathway. IGF-IR has been shown to activate the MAPK pathway in several ways including direct interactions with SHC, which binds Grb2, or indirectly through IRS-1, which also has a binding site, Tyr895, for Grb2 (17, 18). All of the aforementioned adaptor molecules represent the basic framework of the pathways activated by IGF-I and EGF. However, this simplistic view of linear pathways has collapsed as more upstream interactions have been identified between each pathway. It is often generalized that IGF-I and EGF activate the same pathways within the cell; however, few studies have attempted to dissect out the individual molecules used by IGF-I and EGF to activate these pathways or how specificity ensues.

Our laboratory, as well as others, has shown that IGF-I and EGF can act in concert to enhance DNA synthesis in mammary epithelial cell (MECs) (21, 22, 23). Both the PI3K and MAPK signal transduction pathways are required for the increase in cell proliferation, leading to the question of how these two mitogens evoke a greater biological response in combination when they use the same signaling pathways. In the present study, we investigated the mechanisms by which these mitogens act together to increase DNA synthesis. We demonstrate that EGF and IGF-I use distinct upstream components of the PI3K pathway to phosphorylate AKT in the MEC line, MAC-T, resulting in enhanced activation of AKT. This is the first study in MECs to report the specific selection of discrete components of the PI3K pathway, by combined treatment with IGF-I and EGF, leading to enhanced cell signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant human IGF-I, TGF{alpha}, and EGF were obtained from GroPep (North Adelaide, Australia), Intergen Co. (Purchase, NY), and Sigma Aldrich (St. Louis, MO), respectively. Cell culture reagents were from Invitrogen Co. (Carlsbad, CA) with the exception of fetal bovine serum (Gemini Bio-Products; Woodland, CA), and phenol-red-free DMEM (Sigma). Tissue culture plasticware was from Becton Dickinson (Franklin Lakes, NJ). Bovine insulin and D-glucose were from Sigma. Antibodies against phosphorylated forms of MAPK kinase (MEK) 1/2 (Ser217/221), AKT (Ser473), p70S6K (Thr421/Ser 424), and total p70S6K were purchased from Cell Signaling Technology (Beverly, MA). Antibodies that recognize total and phospho-c-Src (Tyr416) were obtained from BioSource International (Camarillo, CA). Antibodies against total AKT, SHC, Gab1, Gab2, Grb2, EGF receptor, IRS-1, the p85{alpha} subunit of PI3K, AKT (Thr308), SHC (Tyr317), and antiphosphotyrosine clone 4G10 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). An antibody that recognizes {alpha}-, ß-, and {gamma}-isoforms of actin was obtained from Calbiochem (La Jolla, CA). Antibody against phospho-ERK 1/2 was from Santa Cruz Biotechnology (Santa Cruz, CA). Nonimmune rabbit, sheep, and mouse IgGs were from Sigma. Inhibitors of the MAPK (PD98059), Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2)], and PI3K (LY294002) pathways were purchased from Calbiochem, resuspended as 10 mM stock solutions in dimethyl sulfoxide, and stored at –20 C. A smart pool of double-stranded small interfering (si) RNA against c-Src as well as mutated siRNA control were from Dharmacon Research Inc. (Lafayette, CO). All siRNAs were dissolved in RNase-free buffer based on the manufacturer’s protocol to a concentration of 20 µM.

Cell culture experiments
The bovine MEC line MAC-T was established from primary bovine MECs by immortalization with the Simian virus 40 large-T antigen (24). Cells were routinely maintained in complete media consisting of phenol red-containing DMEM supplemented with 4.5 g/liter D-glucose, 10% fetal bovine serum, 5 µg/ml bovine insulin, 20 U/ml penicillin, 20 µg/ml streptomycin, and 50 µg/ml gentamicin (complete media) at 37 C in a humidified atmosphere with 5% CO2. For experiments, cells were plated in complete media without insulin or phenol red. For analysis of intracellular signaling molecules, cells were grown to near confluence, rinsed twice with 1x PBS, and incubated in phenol red-free serum-free DMEM with 0.2% BSA and 30 nM Na selenite for 24 h before exposure to treatments. Cell lysates were collected using Upstate’s protocol for immunoprecipitation of proteins with modified radioimmunoprecipitation buffer.

siRNA transfection
Cells were plated at 3 x 104 cells/cm2 in 6-well dishes in DMEM supplemented with 10% fetal bovine serum without antibiotics. The following day, cells were transfected in serum-free DMEM with 100 nM siRNA or a mutated siRNA control using GeneEraser (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. After 24 h, media were aspirated, cells were washed, and media were replaced with DMEM supplemented with 0.2% BSA and 30 nM Na selenite. After an additional 24 h, media were replaced with fresh DMEM without additives ± growth factors for the indicated times.

Immunoprecipitation and Western blotting
Cell lysates were centrifuged at 14,000 x g for 15 min at 4 C, and total protein content of the cytosolic fraction was determined using a protein assay (Bio-Rad Laboratories, Hercules, CA). For immunoprecipitation, equal amounts of protein (500–1500 µg) were incubated with protein-A or -G agarose beads (Upstate) and specified antibodies according to the manufacturer’s recommendations for individual antibodies. Before Western blotting, samples were heated at 100 C for 5 min. For Western blotting, immunoprecipitated samples or equal amounts of protein (20–40 µg) were separated by electrophoresis through either 6 or 10% resolving SDS-PAGE gels under reducing conditions, as previously described (21). Membranes were blocked in 5% nonfat dried milk for 1 h at room temperature, incubated with primary antibody overnight at 4 C, washed, and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (Amersham, Piscataway, NJ) as previously described (21). After washing, peroxidase activity was detected using the enhanced chemiluminescence detection system (ECL Plus; Amersham) according to the manufacturer’s recommendations.

AKT in vitro kinase assay
AKT kinase activity in cell lysates stimulated with IGF-I, EGF, TGF{alpha}, or a combination of growth factors was determined using an AKT kinase assay from Cell Signaling Technologies according to their recommendations. Briefly, cell lysates were immunoprecipitated with immobilized Akt antibody overnight at 4 C. The following day, the pellet was washed and incubated for 30 min in 50 µl kinase buffer supplemented with glycogen synthase kinase (GSK)-3 fusion protein and ATP. The resulting supernatant was analyzed by immunoblotting with phospho-GSK antibody and quantitated by densitometry. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test for post hoc analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of both the IGF-IR and EGFR enhance AKT phosphorylation
We previously reported that IGF-I and TGF{alpha} or EGF each induce rapid phosphorylation of AKT in MAC-T cells (21). Upon further analysis, we observed an enhanced phosphorylation of AKT when cells were treated with IGF-I and TGF{alpha} together (Fig. 1AGo). An increase in phosphorylation was observed at both the Ser473 and Thr308AKT phosphorylation sites at all concentrations tested and was detected as early as 5 min and sustained through 15 min (Fig. 1Go, A and B). In a previous study, we extensively characterized the responses to TGF{alpha} and EGF in our cell model and found no detectable differences in the activation of the EGFR by TGF{alpha}, compared with EGF (21). As shown in Fig. 1CGo, the combination of IGF-I and EGF also enhanced AKT phosphorylation at both sites.


Figure 1
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  		FIG. 1. Simultaneous treatment with IGF-I and TGF{alpha} or EGF enhances AKT phosphorylation. Confluent MAC-T cells were serum starved for 24 h before addition of IGF-I, EGF, and TGF{alpha}. Cell lysates (20 µg total protein) were separated by SDS-PAGE and Western blotted with antibodies specific for phosphorylated AktThr308 antibody, stripped and reprobed with phosphorylated AktSer473 antibody, and then stripped and reprobed with total Akt antibody (A and B). C, Separate immunoblots were analyzed for the two phosphorylation sites. Each immunoblot is representative of at least three experiments.

 
Enhanced AKT phosphorylation results in increased AKT activity and subsequent downstream signaling
To ensure that increased AKT phosphorylation translated into an increase in enzymatic activity, an in vitro AKT kinase assay was used (Fig. 2Go, A and B). Treatment of cells with IGF-I (100 ng/ml) and either TGF-{alpha} or EGF (100 ng/ml) consistently resulted in greater increases in AKT activity relative to either growth factor alone (Fig. 2AGo; P < 0.05). Similar results were obtained when combinations of each growth factor were tested at concentrations of 20 ng/ml (Fig. 2BGo). Furthermore, the enhanced activation of AKT translated into a greater increase in phosphorylation of p70S6K, a downstream AKT target, between 5 and 30 min when IGF-I and TGF{alpha} were combined (Fig. 2CGo). The enhanced phosphorylation was no longer observed at 60 min.


Figure 2
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  		FIG. 2. Simultaneous treatment with IGF-I and TGF{alpha} or EGF enhances both AKT kinase activity and phosphorylation of downstream signaling. Confluent MAC-T cells were serum starved for 24 h before addition of growth factors for 5 min (A and B) or indicated times (C). Cell lysates were immunoprecipitated with immobilized Akt antibody and analyzed for Akt kinase activity using a GSK-3 fusion protein as substrate as described in Materials and Methods. Immunoblots were probed with phospho-GSK antibody and quantitated by densitometry. A, Panel above the graph shows a representative immunoblot. Bars, mean ± SE of three separate experiments. *, Significant difference (P < 0.05) from IGF-I, EGF, or TGF{alpha} alone. B, Representative immunoblot of two AKT kinase assays from cells treated with lower doses of growth factors for 5 min. C, Cell lysates (20 µg total protein) were separated by SDS-PAGE and Western blotted with antibodies specific for phosphorylated p85/70S6K Thr421/Ser424 antibody and then stripped and reprobed with total p70 S6 antibody. Immunoblots are representative of three experiments.

 
Activation of the IGF-IR, but not EGFR, promotes the interaction between IRS-1 and the p85{alpha} subunit of PI3K
The enhanced AKT activation suggested that IGF-IR and EGFR use unique molecules within the same signal transduction cascade to exert their effects. To test this hypothesis, we identified the individual signaling molecules upstream of AKT that are activated by each growth factor. We previously reported that IGF-I activates the IGF-IR followed by activation of IRS-1 in MAC-T cells (21). To bridge the connection between IRS-1 and AKT activation after IGF-I treatment, we investigated the involvement of PI3K. PI3K is a heterodimer of two subunits, the regulatory p85 subunit and the catalytic p110 subunit. Activation of PI3K produces lipid second messengers that bind to AKT, stimulating its translocation to the plasma membrane, whereupon activation occurs (25). Coimmunoprecipitation experiments demonstrated that stimulation with IGF-I resulted in a rapid association of IRS-1 with the p85{alpha} subunit of PI3K (Fig. 3Go). Similar to the tyrosine phosphorylation of IRS-1, association of p85{alpha} subunit with IRS-1 was detectable as early as 1 min and was sustained through 15 min after exposure to IGF-I (data not shown). In contrast, association of p85{alpha} with IRS-1 was not detectable after stimulation with TGF{alpha} or EGF (Fig. 3Go).


Figure 3
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  		FIG. 3. Activation of IGF-IR, but not EGFR, promotes an interaction between IRS-1 and p85{alpha}. Confluent MAC-T cells were serum starved for 24 h before addition of IGF-I (100 ng/ml), EGF (100 ng/ml), or TGF{alpha} (100 ng/ml) for 5 min. Cell lysates (500 µg) were immunoprecipitated with an anti-IRS-1 antibody. Controls included cell lysates immunoprecipitated with a nonimmune rabbit IgG or lysis buffer incubated with an anti-IRS-1 antibody (Ab). Proteins were resolved by SDS-PAGE and immunoblotted with phospho-tyrosine and p85{alpha} antibodies. The membrane was stripped and reprobed with a total IRS-1 antibody. The figure shows representative immunoblots of two separate experiments. Similar results were obtained with 100 ng/ml IGF-I and 20 ng/ml TGF{alpha} or 20 ng/ml IGF-I and 20 ng/ml TGF{alpha}.

 
Phosphorylation of Src416 is required by EGFR, but not IGF-IR, to activate the PI3K pathway
In contrast to the activation of IRS-1 by IGF-I, treatment with TGF{alpha} or EGF had no effect on IRS-1 or -2 activation (21). Intrigued by the synergistic activation of AKT by two growth factors that potentially activate the same signaling cascades, we investigated other known substrates of the EGFR to identify the molecules responsible for the activation of AKT. We first attempted to coimmunoprecipitate the EGFR with p85{alpha}; however, direct interactions between the two molecules were not detectable. The nonreceptor tyrosine kinase Src has been shown to associate with activated EGFRs, become transiently activated, and phosphorylate downstream targets in a variety of normal and cancer cells (26). Therefore, Src was analyzed to determine whether it was involved in the ability of EGF to activate AKT.

As shown in Fig. 4Go, EGF-induced phosphorylation of Src between 2 and 5 min. In contrast, IGF-I did not induce Src phosphorylation. Similar results were obtained with TGF{alpha} (data not shown). We next used PP2, a specific inhibitor of Src activation. PP2 completely blocked AKT activation in cells treated with EGF. However, no effect was observed in cells treated with IGF-I (Fig. 5AGo, arrows). LY294002, an inhibitor of PI3K that acts on the ATP-binding site of the enzyme, successfully blocked the activation of AKT by either growth factor, supporting the hypothesis that IGF-I and EGF use different molecules upstream of PI3K to activate AKT (Fig. 5AGo). Similar results were obtained when protein levels of Src were decreased with siRNA (Fig. 5BGo). Even though a complete knockdown of endogenous Src protein was not achieved using siRNA, the attained decrease in Src was sufficient to produce a decrease in the levels of phosphorylated AKT in response to EGF, compared with the control siRNA-treated samples. Similar to the results with PP2, knockdown of Src with siRNA did not affect the ability of IGF-I to activate AKT.


Figure 4
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  		FIG. 4. Src is phosphorylated in response to EGF but not IGF-I. Confluent MAC-T cells were serum starved for 24 h before addition of IGF-I (100 ng/ml) or EGF (100 ng/ml) for the indicated periods of time. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phospho-SrcTyr416 antibody. The specific phospho Src band is indicated by the arrow and resides just below an upper nonspecific band that is seen in all cell lysates. Membranes were stripped and reprobed with a total Src antibody. Figure shows representative immunoblots of three separate experiments.

 

Figure 5
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  		FIG. 5. Inhibition or gene knockdown of Src blocks the ability of EGF, but not IGF-I, to phosphorylate AKT. A, Confluent MAC-T cells were serum starved for 24 h. Cells were pretreated for 30 min with 20 µM PD98059, PP2, or LY294002 before the addition of IGF-I (100 ng/ml) or EGF (20 ng/ml) for 5 min. B, Subconfluent cells were transfected in serum-free DMEM with 100 nM siRNA against c-Src or with a mutated siRNA control. After 24 h media were replaced with serum-free media containing 0.2% BSA and 30 nM Na selenite. After 24 h, media were replaced with fresh media without additives ± EGF (100 ng/ml) or IGF-I (100 ng/ml). Cell lysates were collected after 1 min of treatment for analysis. Total cell lysates were resolved by SDS-PAGE and immunoblotted with phospho-AKT and Src antibodies. Membranes were stripped and reprobed with actin antibody. The figure shows representative immunoblots of three separate experiments.

 
Interestingly, inhibition of Src resulted in an enhancement of basal phosphorylation of the MAPK pathway, as shown by the increased phosphorylation of MEK 1/2 and ERK 1/2 in both basal and IGF-I-treated cells (Fig. 6Go). Therefore, it was difficult to determine whether Src affected the ability of EGF to activate the MAPK pathway.


Figure 6
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  		FIG. 6. Inhibition of Src increases basal phosphorylation of the MAPK pathway. Confluent MAC-T cells were serum starved for 24 h. Cells were pretreated for 30 min with PP2 (20 µM) before the addition of IGF-I (100 ng/ml) or EGF (100 ng/ml) for 5 min. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies that recognize the phosphorylated forms of Src, MEK, and ERK. Membranes were stripped and reprobed with an actin antibody. The figure shows representative immunoblots of three separate experiments.

 
EGF, but not IGF-I, promotes the association of EGFR with SHC, SHC with Grb2, and Gab2 with SHC, Grb2, Gab1, and p85{alpha}
Once we determined that the ability of EGF to phosphorylate AKT required Src activation, the EGFR was coimmunoprecipitated with Src, as well as other potential docking proteins, to identify potential molecular interactions involved in the phosphorylation of AKT. Whereas attempts to coimmunoprecipitate the EGFR with Src were unsuccessful, we demonstrated associations between the EGFR and SHC in response to EGF (Fig. 7AGo) and between SHC and Grb2 (Fig. 7BGo). When EGF-stimulated cell lysates were immunoprecipitated with the scaffolding protein Gab2 (Fig. 7CGo), increases in phosphorylated SHC, Gab1, and Gab2 as well as total Grb2 and p85 were detected in the Gab2 complex. Interestingly, whereas EGF increased the amount of Grb2 and p85 that coimmunoprecipitated with Gab1, we could not detect SHC in this complex (Fig. 7DGo). The associations shown in Fig. 7Go were detectable as early as 1 min and were sustained through 5 min. In contrast, SHC did not coimmunoprecipitate with the EGFR in response to IGF-I. Furthermore, IGF-I did not increase the association between SHC and Grb2 or between Gab2 and SHC, Grb2, or p85. In a previous study and in the present one, we analyzed activation of IGF-IR by EGF and EGFR by IGF-I and found no detectable transactivation (21). This analysis was performed because some studies have reported transactivation between the IGF-IR and EGFR in normal MECs and breast cancer cells as well as COS-7 cells (27, 28, 29, 30, 31).


Figure 7
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  		FIG. 7. EGF, but not IGF-I, promotes the association of EGFR with SHC and SHC with Grb2, Gab2, Gab1, and p85{alpha} in MAC-T cells. Confluent MAC-T cells were serum starved for 24 h before addition of IGF-I or EGF for 5 min. Cell lysates (1 mg) were immunoprecipitated with an antibody against EGFR (A), SHC (B), Gab2 (C), and Gab (D) 1. Controls included cell lysates immunoprecipitated (IP) with a nonimmune rabbit IgG or lysis buffer incubated with antibody alone (Ab). Proteins were resolved by SDS-PAGE and immunoblotted with phospho or total SHC, EGFR, Grb2, p85{alpha}, Gab2, Gab1, or phosphor-tyrosine antibodies. Figures are representative immunoblots of at least three separate experiments for each IP.

 
Inhibition of Src blocks EGF-stimulated phosphorylation of EGFR and its association with SHC and disrupts further downstream signaling
Whereas we identified a complex of molecules potentially involved in the activation of AKT in response to EGF, the sensitivity of the pathway to Src activation was not explained. Therefore, we examined the activation of the EGFR and association of SHC when Src was inhibited with PP2. Interestingly, the phosphorylation of the EGFR was decreased on inhibition of Src (Fig. 8AGo). The EGFR antibody recognized a doublet in both the immunoprecipitated samples and cell lysates, running at approximately 160 and 180 kDa. The inhibition of Src appeared to decrease the phosphorylation of the upper 180-kDa band to below detectable levels, whereas the phosphorylation of the lower 160 kDa was substantially decreased yet still detectable. The association of phosphorylated SHC with the EGFR was decreased by PP2 (Fig. 8AGo). In addition, the associations between Gab2 and phosphorylated SHC, Grb2, p85{alpha}, and phosphorylated Gab1 and Gab2 were also decreased (Fig. 8BGo), as were the associations between Gab1 and Grb2 or p85{alpha} (Fig. 8CGo) Activation of the IGF-IR and IRS-1 by IGF-I was not affected by treatment with PP2 (data not shown).


Figure 8
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  		FIG. 8. Inhibition of Src decreases EGF-stimulated phosphorylation of EGF receptor and its association with SHC, and disrupts further downstream signaling. Confluent MAC-T cells were serum starved for 24 h. Cells were pretreated for 30 min with PP2 (20 µM) before the addition of IGF-I or EGF (100 ng/ml) for 1 min (A and C) or 2 min (B) Cell lysates were immunoprecipitated (IP) with antibodies against (A) EGFR, (B) Gab2 and (C) Gab1. Controls included cell lysates immunoprecipitated with a nonimmune rabbit IgG, lysis buffer incubated with appropriate primary antibody (Ab), and EGF-stimulated cell lysates without immunoprecipitation (lysates). Proteins were resolved by SDS-PAGE and sequentially immunoblotted with antibodies against (A) phospho-tyrosine and total SHC, then stripped and reprobed with total EGFR antibody (B) total SHC, Grb2, p85{alpha}, and phospho-tyrosine, then stripped and reprobed with Gab2 (98 kDa) followed by Gab1 (110 kDa) antibodies (C) Grb2, p85{alpha}, and Gab1. Figures are representative immunoblots of three experiments for each individual IP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
AKT regulates a diverse array of cellular functions including proliferation, differentiation, apoptosis, and intermediary metabolism (32). Overactivation of AKT has been associated with numerous cancers, including breast cancer, and has been shown to induce resistance to both tamoxifen and chemotherapy, making it imperative to identify the mechanisms involved in its regulation (33, 34, 35). Overamplification of ErbB2 can be found in 20–30% of breast cancer cases and is associated with a poor prognosis (36). Many of the events mediated by overexpression of ErbB2 are the result of enhanced signaling through AKT (37). In addition, disruption of signaling through AKT was shown to be essential for the cytotoxic effect of trastuzumab (Herceptin) for breast carcinoma cell lines that were formerly sensitive to this agent became resistant after transfection with a constitutively active AKT transgene (38). Furthermore, numerous small molecule inhibitors, monoclonal antibodies, and antisense strategies specifically targeting the IGF-IR are being developed with the objective of blocking IGF-IR signaling, particularly AKT activation, to prevent breast cancer cell proliferation (39). Because AKT contributes to the progression of breast cancer and confers resistance to conventional therapies, the regulation of AKT is emerging as an attractive molecular target in breast cancer treatment (37). However, the distinct upstream signaling events leading to AKT overactivation are not well characterized.

Whereas numerous strategies have focused on blocking the activation of AKT caused by overamplification of EGFR family members, tumor cells may compensate for EGFR inhibition by activating AKT through the IGF-IR, leading to resistance to endocrine and chemotherapies. Combined treatment of IGF-I and EGF has been shown to synergistically and/or additively enhance cell proliferation in bovine MAC-T cells, murine MEC, and primary human breast epithelial or stromal cells (21, 22, 23, 40). However, no prior studies examined the individual intracellular signaling components activated by these two growth factors within one cell type that elicit this effect. In mammary gland organoid cultures, the combination of IGF-I and EGF was shown to stimulate cell cycle progression through the induction of cyclins required for cell cycle progression (23). Specifically, IGF-I and EGF treatment had a synergistic effect on the induction of cyclin D1 as well as A2 and B1. Of note was the observation that IGF-I was essential for EGF to promote the progression past the G1-S restriction point and induce DNA synthesis (23). The current observation that IGF-I and EGF synergistically phosphorylate AKT suggests a role for this signaling molecule because AKT has been shown to regulate proteins that affect cell cyclins in mammary cells. It has been shown that AKT directly phosphorylates p27 at Thr157, leading to cytoplasmic localization of p27 (41, 42). p27 functions as an assembly factor for cyclin D/cdk4 and cyclin D/cdk6 holoenzyme complexes (43, 44). The expression of AKT, p27, and cyclin D1 were up-regulated in rat mammary tumors induced by 7,12-dimethylbenz[{alpha}]anthracene, compared with normal mammary glands (45). In addition, increased expression of p27 and cyclin D1 has been observed in several highly proliferative human breast cancer cell lines and low-grade primary breast tumors (46). Thus, up-regulation of AKT may lead to increased p27 and cyclin D1 expression, allowing for activation of cdk complexes and promotion of cell cycle progression.

Analysis of the upstream molecules required for AKT activation in our cell model system revealed that IGF-I stimulated the association of IRS-1 with p85{alpha}, thereby activating AKT. Whereas this observation was consistent with other reports, the intricate signaling network initiated by EGFR activation was more complex to decipher. Src has been shown to associate with the EGFR and activate downstream molecules, regulate the internalization of the EGFR, and modulate lateral activation of the EGFR by extracellular stimuli other than EGF (47, 48). Additionally, in approximately 70% of mammary tumors, c-Src is cooverexpressed with at least one member of the EGFR family (26). Therefore, we investigated the involvement of Src in the ability of EGF to activate AKT. Although the association of Src and EGFR are typically associated with MAPK activation, studies have shown a direct link between Src and activation of the PI3K pathway. For example, Stover et al. (49) demonstrated that activated Src can phosphorylate EGFR at Tyr 920 and 891, in vitro, and these sites mediate the binding of PI3K and Src itself. In the present study, activation of EGFR, but not IGF-IR, resulted in phosphorylation of Src416. Blocking Src phosphorylation prevented activation of AKT by EGF and had no effect on the ability of IGF-I to phosphorylate AKT. Inhibiting Src activation also decreased phosphorylation of the EGFR. This suggests a bidirectional interaction between EGFR and Src, given that the EGFR can phosphorylate Src, and Src can affect the phosphorylation of the EGFR. There have been previous reports of a bidirectional interaction between the EGFR and Src in breast cancer cell lines and breast tumor tissues (47). For example, in MCF-7 cells, Src was shown to phosphorylate the EGFR on novel sites in vitro and Src associated with the EGFR after it had been phosphorylated by Src (49). Furthermore, the same Src-phosphorylated sites on the EGFR can be phosphorylated in response to EGF and activate Src in an EGF-dependent manner (49). However, the present report is the first time that a bidirectional interaction between the EGFR and Src has been shown in a nontumorigenic MEC line.

Our inability to directly observe an association between Src and the EGFR, or its downstream targets, may have resulted from the presence of unknown intermediate docking proteins involved in the association. Another possibility is that the formation of heterodimers of the EGFR with another member of the EGFR family hindered our ability to detect the protein complex. It is possible that the doublet we detected represents a heterodimerization of the EGFR with either ErbB2 or ErbB3. In rodents, mammary tumors induced by activated ErbB2 exhibited elevated Src, which formed stable complexes with activated ErbB2 (50). In this model, Src could not directly bind to the EGFR. The finding that Src associates with the carboxyl terminal region of the ErbB2 catalytic domain, and that replacement of this domain in EGFR with a catalytic domain of ErbB2 resulted in an EGFR that could bind Src, support the idea that recruitment of Src via ErbB2 to ErbB2/EGFR heterodimers may be a critical step in EGFR transphosphorylation (51). The EGFR has also been demonstrated to heterodimerize with ErbB3, which possesses six PI3K binding sites. Through this dimerization, EGF is able to activate the PI3K pathway (52, 53). Consistent with the importance of this EGFR/ErbB3 heterodimer with association of PI3K, elevated levels of ErbB3 are observed in mammary tumor progression in transgenic mice that overexpress EGFR in the mammary epithelium (54). Future studies will determine whether heterodimerization of the EGFR is influencing signaling in this cell model.

Characteristically, autophosphorylation of the EGFR recruits SHC, thereby causing its activation. The phosphorylated SHC then binds Grb2/mSOS and in so doing activates the MAPK pathway (55). However, results of the present study also implicate SHC in EGF-stimulated activation of the PI3K pathway in MECs. Coimmunoprecipitation studies revealed associations between EGFR and SHC as well as subsequent downstream associations between Gab2 and SHC, Grb2, Gab1, and p85{alpha}. Gab1 and Gab2 belong to a superfamily of docking proteins that contain binding sites for adapter molecules including Grb2, the phosphotyrosine phosphatase SHP2, and p85{alpha} (56). Therefore, a possible scenario is that the EGFR binds SHC, which recruits Grb2 and phosphorylated Gab2 (and/or Gab1), which bind p85, leading to Akt activation. Little is known concerning the role of Gab2 in mammary epithelial cells. Overexpression of Gab2 has been demonstrated in a subset of breast cancer cell lines, and Gab2 was found to be tyrosine phosphorylated in response to EGF or heregulin (57). Recently overexpression of Gab2 was found to increase proliferation of MCF10A MEC in three-dimensional culture (58). Gab1 and Gab2 have been shown to be phosphorylated by receptor tyrosine kinases such as the EGFR as well as intermediate protein tyrosine kinases such as Src (56). In the present study, the ability of EGF to induce tyrosine phosphorylation of Gab1 and Gab2 was eliminated when Src was blocked. Because this corresponded with a decrease in activation of the EGFR, it is unclear which tyrosine kinase was directly responsible for Gab1 and Gab2 activation.

An interesting and unexpected finding was that the inhibition of Src stimulated an increase in basal MAPK activation. Therefore, it was difficult to determine whether inhibition of Src also blocked the ability of EGF to activate MAPK. Because Src activation is classically associated with pathways involved in proliferation and adhesion, if anything, a decrease in MAPK activation would have been anticipated (59). The increase in basal MAPK may be the result of a redistribution of phosphatases. Phosphorylated Src could possibly be involved in the activation of a phosphatase, which functions to dephosphorylate the MAPK pathway. Therefore, inhibition of Src would prevent the activation of the phosphatase, allowing for enhanced MAPK signaling. Another possibility is that the increase in MAPK is acting as a compensatory mechanism by the cell to account for the loss of signaling through the PI3K pathway. Additional experiments are underway to examine the mechanism involved in the increased MAPK activity associated with inhibition of Src.

This is the first report to identify the individual upstream signaling components that are recruited by IGF-I and EGFRs in MECs when both growth factors are present. This specific recruitment of upstream molecules by each receptor may better represent in vivo conditions reflecting substrate availability when several mitogens are acting in concert. These results suggest a potential mechanism by which IGF-I and EGF may synergize to enhance cell proliferation in normal MECs. Furthermore, resistance of breast cancer cells to EGFR inhibition via the chemotherapeutic agent trastuzumab has been shown to occur through enhanced IGF-IR signaling (60). The ability of the IGF-I and EGFRs to recruit different upstream signaling molecules may contribute to this compensatory action of IGF-I. Defining the individual upstream molecules used by these two receptors could result in new therapies to target a cluster of key molecules to prevent signaling compensation by IGF-I or EGF and ultimately inhibit aberrant cell proliferation.


   Footnotes
 
This work was supported by National Research Initiative Competitive Grant 2003-35206-12811 from the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service and Hatch Project NJ06148 from the New Jersey Agricultural Experiment Station at Rutgers, The State University of New Jersey.

Disclosure summary: all authors have nothing to declare.

First Published Online September 21, 2006

Abbreviations: EGF, Epidermal growth factor; EGFR, EGF receptor; GSK, glycogen synthase kinase; IGF-IR, type I IGF receptor; IRS, insulin receptor substrate; MEC, mammary epithelial cell; MEK, MAPK kinase; PI3K, phosphatidylinositol-3 kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; si, small interfering.

Received March 17, 2006.

Accepted for publication September 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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