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Epidermal Growth Factor Promotes Epidermal Growth Factor Receptor Nuclear Accumulation by a Pathway Dependent on Cytoskeleton Integrity in Human Breast Cancer Cells
Archives of Medical Research 40 (2009) 331e338
ORIGINAL ARTICLE
Epidermal Growth Factor Promotes Epidermal Growth Factor Receptor Nuclear Accumulation by a Pathway Dependent on Cytoskeleton Integrity in Human Breast Cancer Cells Pedro Cortes-Reynosa, Teresa Robledo, and Eduardo Perez Salazar Departamento de Biologia Celular, CINVESTAV-IPN, Mexico, D.F., Mexico Received for publication October 20, 2008; accepted May 21, 2009 (ARCMED-D-08-00466).
Background and Aims. The epidermal growth factor receptor (EGFR) is activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events. Emerging evidence indicates a mode of EGF signaling in which growth factor signals are transmitted via EGFR nuclear transport. The aim of this study was to determine whether EGF promotes EGFR nuclear accumulation and the role of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity in breast cancer cells. Methods. MCF-7 cells were treated without or with 100 ng/ml EGF for various times and nuclear extracts were obtained. Nuclear accumulation of EGFR was analyzed by SDS-PAGE followed by Western blotting of nuclear extracts using an anti-EGFR Ab or with a phosphospecific Ab against the Tyr-1068 of EGFR and with anti-Rb Ab as the loading control. DNA binding activity of EGFR was analyzed by EMSA using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS). Results. EGF induces the nuclear accumulation of EGFR, an increase in EGFR phosphorylation at Tyr-1068 and the formation of the complex EGFR-DNA in MCF-7 and MDA-MB-231 breast cancer cells. In addition, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity. Conclusions. This study demonstrates that in breast cancer cells EGF promotes nuclear accumulation of EGFR and is dependent on clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: Breast cancer, nuclear EGFR, EGFR-DNA complex, Cytoskeleton.
Introduction The epidermal growth factor receptor (EGFR) is a member of the ErbB family of tyrosine kinase receptors, which includes EGFR (ErbB1/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors are activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events (1,2). EGF contributes to mammary
Address reprint requests to: Eduardo Perez Salazar, Ph.D, Departamento de Biologia Celular, CINVESTAV-IPN, Av. IPN #2508, 07360 Mexico, D.F., Mexico; Phone: (þ52) (55) 5747-3991; FAX: (þ52) (55) 5747-3393; E-mail: [email protected]
development, and it is a major regulator of breast cancer that become steroid hormone-resistant (3,4). Furthermore, it has been reported that EGFR is expressed in 14e91% of patients with breast cancer, and its expression is significantly associated with the loss of steroid hormone sensitivity. It also correlates with high disease recurrence and decreased patient survival (5e8). EGFR activation induced by binding of EGF promotes the formation of homodimers or heterodimers and the phosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated residues recruit signaling proteins such as Shc, Grb2, phospholipase Cg and Src, with subsequent activation of signal transduction pathways (9,10). However, EGFR also is localized in the
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2009.06.007
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nucleus of cancer cells, human placenta, pregnant uterus and regenerating liver, and it has the potential to associate with the promoter region of cyclin D1, strongly suggesting that EGFR might function as a transcription factor (11e13). Moreover, it has been reported that one juxtamembrane region in the EGFR family members present a putative nuclear localization sequence, which mediates the localization of EGFR in the nucleus (14) and that ErbB-2 receptor translocates directly from the cytoplasmic membrane to the nucleus through endocytosis by using the importin b1 and the nuclear pore protein Nup358 (15) In the present study we report that EGF induces nuclear accumulation of EGFR, nuclear EGFR phosphorylation at Tyr-1068 and an increase in EGFR-DNA complex formation in breast cancer cells. Furthermore, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity.
peroxidase-conjugated, goat Abs to rabbit) (1:5000) for 2 h at 22 C. After washing three times with PBS/0.1% Tween 20, the immunoreactive bands were visualized using ECL detection reagents. Preparation of Nuclear Extracts Nuclear extracts were prepared as described previously (11,16e18). Briefly, cells were lysed with 0.1% Nonidet P40 in Buffer A (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, and 1 mM PMSF). Lysates were pelleted at 12,000 rpm for 30 sec and resuspended in Buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM DTT, and 0.2 mM PMSF). Nuclear fraction was recovered by centrifugation at 12,000 g for 15 min at 4 C. The supernatant was recovered and centrifuged again at 15,000 g for 5 min, and the resulting supernatant was the non-nuclear fraction.
Materials and Methods Materials EGF, colchicine, cytochalasin D, sucrose and filipin III were from Sigma (St. Louis, MO). AG1478 was from Calbiochem (San Diego, CA). EGFR antibody (Ab), retinoblastoma (Rb) Ab and phosphospecific Abs to Tyr-845 and Tyr-1068 of EGFR were from Cell Signaling Technology (Beverly, MA). Paxillin Ab was from BD (San Diego, CA). IkB-a Ab, Src Ab and lamin B1 Ab were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat Ab to rabbit was from Zymed (San Francisco, CA). ECL reagent was from Amersham Pharmacia Biotech (Piscataway, NJ). Cell Culture MCF-7 and MDA-MB-231 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.7 g/L sodium bicarbonate and 5% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 and 95% air at 37 C. For experimental purposes, confluent cultures of these cells were serum-starved for 12 h before treatment with inhibitors and/or EGF.
Electrophoretic Mobility Shift Assay (EMSA) EMSAs were performed as described before (11,16). Briefly, double-stranded oligonucleotide-containing specific binding sites for EGFR (ATRS sequence) (11), 50 -CTTGTTTACGG TCCCCCCATTTTTTACGCTGGTCCT-30 , was used as probe. ATRS oligonucleotide was labeled with (g32P) ATP using T4 polynucleotide kinase. The 32P-labeled oligonucleotide probe (|1 ng) was incubated with 5 mg of nuclear extract in a reaction mixture containing 3 mg of poly (dI-dC), 0.25 M HEPES, pH 7.5, 0.6 M KCl, 50 mM MgCl2, 1 mM EDTA, 7.5 mM DTT, and 9% glycerol for 20 min at 4 C. One hundred-fold excess of unlabeled ATRS probe or an irrelevant oligonucleotide (50 -ACGTGTGATGAAATGCTAGGCGATC-30 ) was used as specific and nonspecific competitors. Samples were fractionated on a 4% polyacrylamide gel in 0.5X Tris borate-EDTA buffer. Gels were dried and analyzed by autoradiography. In supershift assays, nuclear extracts were incubated for 30 min at 4 C with the Ab against EGFR or IgG before addition of the radiolabeled probe.
Results
Western Blotting
EGF Stimulation Induces Nuclear Accumulation of EGFR in MCF-7 Cells
Equal amounts of protein were separated by SDS-PAGE using 8% separating gels followed by transfer to nitrocellulose membranes. After transfer, membranes were blocked using 5% non-fat dried milk in PBS, pH 7.2, and incubated overnight at 4 C with the primary Ab as indicated. The membranes were washed three times with PBS/0.1% Tween 20 and then incubated with secondary Abs (horseradish
Nuclear detection of EGFR has been reported in cancer cells and primary tumors including those of skin, breast, bladder, cervix, adrenocorticord, thyroid and oral cavity (11,12,19e22). To examine whether EGF induces accumulation of endogenous EGFR in the nucleus, confluent cultures of MCF-7 cells were treated with or without 100 ng/mL EGF for various times. Nuclear extracts were
Cytoskeleton Regulates Nuclear Accumulation of EGFR
obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. As shown in Figure 1A (upper panel), in untreated cells a small amount of EGFR was localized in the nucleus and treatment of cells with EGF induced the nuclear accumulation of EGFR. Western blotting with anti-Rb Ab of the same membranes confirmed that similar amounts of nuclear proteins were recovered in the absence or in the presence of EGF (Figure 1A, lower panel). To rule out the possibility that the signal seen in nuclear extracts was due to contamination, nuclear extracts (nuclear fraction) and non-nuclear fraction were analyzed by immunoblotting with anti-paxillin Ab, anti-Rb Ab, anti-IkB-a Ab, anti-Src Ab, anti-E-cadherin Ab and anti-lamin B1 Ab. Our results showed that Rb and lamin B1 were present only in the nuclear fraction, whereas the cytoplasmic proteins paxillin, IkB-a, Src and E-cadherin were absent (Figure 1B). Next, we determined whether EGF induces phosphorylation of nuclear EGFR at specific tyrosine residues such as Tyr-845 and Tyr-1065. MCF-7 cells were treated with or without 100 ng/mL EGF for various times and nuclear extracts were obtained. Nuclear extracts were analyzed by SDS-PAGE followed by Western blotting with a phosphospecific Ab against the Tyr-845 of EGFR [anti-EGFR (P)Y845] to localize EGFR phosphorylated at Tyr-845 and with a phosphospecific Ab against the Tyr-1068 of
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EGFR [anti-EGFR(P)Y1068] to localize EGFR phosphorylated at Tyr-1068. As illustrated in Figure 2A, EGF induced an increase in the nuclear EGFR phosphorylated at Tyr1068. In contrast, in untreated cells and cells stimulated with EGF, nuclear EGFR phosphorylated at Tyr-845 was not detected in the nucleus (results not shown). To examine whether kinase activity of EGFR plays a role in the nuclear accumulation of EGFR induced by EGF, we studied the effect of AG1478 in the nuclear accumulation of EGFR. AG1478 is a potent and selective inhibitor of EGFR kinase activity and it has been used previously to inhibit EGFR kinase activity in breast cancer cells (23,24). MCF-7 cells were treated for 30 min in the absence or presence of 300 nM AG1478 and then stimulated with 100 ng/ mL EGF for another 20 min. Nuclear extracts were obtained and analyzed by Western blotting with anti-EGFR Ab. Our results showed that treatment of MCF-7 cells with AG1478 inhibited EGFR nuclear accumulation in response to EGF (Figure 2B). EGF Treatment Induces DNA Binding Activity of EGFR Because previous studies had already shown that EGFR is a putative transcription factor and is able to bind to DNA (11), we determined whether EGF treatment of MCF-7 cells triggers nuclear translocation with subsequent binding to DNA. MCF-7 cells were treated with 100 ng/mL EGF for
Figure 1. Epidermal growth factor (EGF) induces nuclear accumulation of epidermal growth factor receptor (EGFR) in MCF-7 cells Confluent cultures of MCF-7 cells were treated at 37 C with 100 ng/mL EGF for various times as indicated; nuclear extracts were subsequentally obtained. (A) EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. The graph represents the mean SD of at least three independent experiments and is expressed as the fold stimulation above control (unstimulated) value. Asterisks denote comparisons made to control cultures (unstimulated). *p !0.005, ***p !0.001 by one-way ANOVA. (B) Nuclear fraction and non-nuclear fraction were analyzed by Western blotting with anti-paxillin Ab, anti-Rb Ab, anti-IkB-a, anti-Src Ab, anti-Lamin B1 Ab and anti-E-cadherin Ab. Radiograms shown are representative of at least three independent experiments.
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Figure 2. Role of EGFR kinase activity in the nuclear accumulation of EGFR. (A) Confluent cultures of MCF-7 cells were treated at 37 C with 100 ng/mL EGF for various times as indicated, and subsequently nuclear extracts were obtained. Nuclear EGFR phosphorylation at Tyr-1068 was analyzed by Western blotting of nuclear extracts with anti-EGFR(P)Y1068 Ab or with anti-Rb Ab as the loading control. (B) MCF-7 cells were treated at 37 C for 30 min in the absence () or in the presence (þ) of 300 nM AG1478 and then stimulated without () or with (þ) 100 ng/mL EGF for 20 min and nuclear extracts were obtained. EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. The graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. ***p ! 0.001 by one-way ANOVA. Radiograms shown are representative of at least three independent experiments.
various times and nuclear extracts were obtained. EMSAs were then performed using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS). As illustrated in Figure 3A, EGF induced EGFR-DNA complex formation in a timedependent manner. The specificity of these complexes was demonstrated by inhibition of binding in the presence of a cold competitor.
To further substantiate that EGFR binds to the ATRS sequence, we performed supershift assays using nuclear extracts of MCF-7 cells stimulated with EGF and one Ab against EGFR. MCF-7 cells were treated with 100 ng/ mL EGF for 5 min and nuclear extracts were obtained. Nuclear extracts were treated with 2 mg EGFR Ab for 30 min at 4 C and then EMSAs were performed. Our results showed that pre-treatment with the EGFR Ab inhibited
Figure 3. EGF induces EGFR DNA binding activity in MCF-7 cells. (A) Nuclear extracts were prepared from MCF-7 cells untreated or treated with 100 ng/ mL EGF for various times, as indicated. (B) Nuclear extracts were pre-treated in either the absence () or the presence (þ) of 1 mg Ab to EGFR or control IgG. EGFR DNA binding activity was analyzed by electrophoretic shift mobility assay (EMSA) as described in Methods. Notice that pre-treatment with EGFR Ab inhibited the EGFR-DNA complex formation. Autoradiograms shown are representative of at least three independent experiments.
Cytoskeleton Regulates Nuclear Accumulation of EGFR
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Figure 4. EGF promotes nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells. Confluent cultures of MDA-MB-231 cells were treated at 37 C without or with 100 ng/mL EGF for various times as indicated and nuclear extracts were obtained. (A) EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. (B) Nuclear EGFR phosphorylation at Tyr-1068 was analyzed by Western blotting of nuclear extracts with anti-EGFR(P)Y1068 Ab or with anti-Rb Ab as the loading control. The graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. **p !0.01, ***p !0.001 by one-way ANOVA. Radiograms shown are representative of at least three independent experiments.
the EGFReDNA complex formation. In contrast, treatment with an unspecific Ab (2 mg IgG) did not inhibit the EGFReDNA complex formation (Figure 3B). These results demonstrate that EGFR is forming a complex with the ATRS DNA sequence. EGF Treatment Induces Nuclear Accumulation of EGFR in MDA-MB-231 Breast Cancer Cells In order to substantiate further that EGF induces nuclear accumulation of EGFR in breast cancer cells, we examined whether EGF promotes nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells. MDA-MB231 cells were stimulated for various times with 100 ng/ mL EGF and nuclear extracts were obtained and analyzed by Western blotting with anti-EGFR Ab. In agreement with our previous results, our findings showed that EGF stimulation induced an increase in the nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells (Figure 4A). To determine the phosphorylation status of EGFR in the nucleus, MDA-MB-231 breast cancer cells were stimulated for various times with EGF and nuclear extracts were analyzed by Western blotting with anti-EGFR(P)Y845 and anti-EGFR(P)Y1068 Abs. Our results showed that treatment with EGF induced an increase in the nuclear EGFR phosphorylated at Tyr-1068 in MDA-MB-231 cells (Figure 4B). In contrast, in untreated cells and cells stimulated with EGF, EGFR phosphorylated at Tyr-845 was not detected in the nucleus (results not shown).
EGFR Nuclear Accumulation Requires Endocytosis and Cytoskeletal Integrity It has been reported that one clathrin-mediated endocytosis mechanism mediates ErbB-2 nuclear translocation, whereas endocytosis of EGFR is accompanied by reorganization of microtubule network (15,25). Therefore, we examined the contribution of cytoskeleton and the role of clathrin- and caveolae-mediated endocytosis in the nuclear accumulation of EGFR. We demonstrated that cytochalasin D specifically disrupts the organization of actin fibers, whereas colchicine promotes the disappearance of microtubules in MCF-7 cells (26). Therefore, in order to determine the involvement of cytoskeleton in the nuclear accumulation of EGFR, we determined the effect of colchicine and cytochalasin D in the EGFR nuclear accumulation. Cultures of MCF-7 cells were treated in the absence or presence of 10 mM colchicine for 1 h or 2.4 mM cytochalasin D for 2 h and then stimulated with 100 ng/mL EGF for 20 min. Nuclear extracts were obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. As illustrated in Figure 5A, treatment with colchicine and cytochalasin D inhibited the nuclear accumulation of EGFR. To investigate whether EGFR nuclear accumulation involves an endocytosis mechanism mediated by caveolae microdomains and/or clathrin-coated pits, MCF-7 cells were treated for 45 min in the absence or presence of 5 mg/mL filipin III, a polyene antibiotic that binds to
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Figure 5. Role of cytoskeleton in the nuclear accumulation of EGFR. (A) Confluent cultures of MCF-7 cells were treated for 1 h in the absence () or in the presence (þ) of 10 mM colchicine (Colc) or for 2 h with 2.4 mM cytochalasin D (Cyt D) and then stimulated with 100 ng/mL EGF for a further 20 min. (B) MCF-7 cells were treated for 45 min in the absence () or presence (þ) of 5 mg/mL filipin III or for 1 h in the absence () or presence (þ) 375 mM sucrose and then stimulated with 100 ng/mL EGF for a further 20 min. EGFR nuclear translocation was analyzed by Western blotting of nuclear extracts with antiEGFR Ab or with anti-Rb Ab as the loading control. Graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. **p !0.01, ***p !0.001 by one-way ANOVA. Radiograms are representative of at least three independent experiments.
cholesterol and removes it from the membrane causing reversible caveolae disassembly (27,28), or they were incubated for 1 h with hypertonic medium containing sucrose at a concentration of 375 mM, which interferes with the clathrin pathway (29), followed by stimulation with 100 ng/ mL EGF for 20 min. Nuclear extracts were obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. Our findings showed that treatment with filipin III completely inhibited the nuclear accumulation of EGFR, whereas treatment with sucrose partly inhibited the nuclear accumulation of EGFR (Figure 5B).
Discussion The presence of receptor tyrosine kinases in the nucleus has been reported. In particular, EGFR, ErbB-3/HER-3 and fibroblast growth factor receptor I (FGFR-I) are present in an uncleaved form in the nucleus (11,30e32). However, little is known about the regulation of nuclear EGFR accumulation in breast cancer cells. By using a method to obtain nuclear extracts from breast cancer cells previously described (11,16,33), and two breast cancer cell lines, we show here that stimulation with EGF induces nuclear
accumulation of EGFR and requires EGFR kinase activity. These results are in agreement with previous reports that demonstrate EGFR is localized in the nucleus of breast cancer tumors and correlates with poor survival in patients with breast cancer (11,12). In addition, our results show that nuclear EGFR accumulation decreases at 30 min of treatment. Therefore we propose that degradation of EGFR takes places after 20 min of treatment or that nuclear EGFR is translocated from nucleus to cytosol. However, additional studies are necessary to substantiate this hypothesis. In glioblastoma cell lines, treatment with EGF stimulates global phosphorylation of the EGFR at Tyr-845, Tyr-992, Tyr-1068 and Tyr-1054, whereas treatment with phorbol 12myristate 13-acetate stimulates phosphorylation of EGFR only at Tyr-1068 (9,34). In the present study we used Abs that detect the phosphorylated state of Tyr-845 and Tyr-1068 of EGFR to elucidate whether EGF modulates the phosphorylation of these residues in the nuclear EGFR of breast cancer cells. Our results demonstrate that stimulation of breast cancer cells with EGF induces an increase in the nuclear accumulation of EGFR phosphorylated at Tyr-1068. In contrast, EGF does not induce accumulation of EGFR phosphorylated at Tyr-845. However, EGFR nuclear accumulation kinetics does not coincide with the nuclear accumulation of EGFR phosphorylated at Tyr-1068. Specifically, nuclear accumulation
Cytoskeleton Regulates Nuclear Accumulation of EGFR
of EGFR phosphorylated at Tyr-1068 reaches a maximum at 30 min of treatment when EGFR nuclear accumulation declines toward baseline levels. These results suggest that EGFR phosphorylation at Tyr-1068 does not trigger the nuclear accumulation of EGFR and that EGF promotes phosphorylation of nuclear EGFR at their tyrosine residues to different times of stimulation. In addition, because our findings also show that nuclear accumulation of EGFR is dependent on EGFR kinase activity, we propose that EGFR phosphorylation at their different amino acids plays an important role in the nuclear accumulation of EGFR and in the DNA binding activity. Our findings also suggest that PKC activity may play an important role in the phosphorylation at Tyr1068, promoting changes in the conformation of EGFR. In support of this proposal, it has been reported that phosphorylation of Tyr-992, Tyr-1068 and Tyr-1086, localized between the tripeptides Asp-Glu-Glu and Tyr-Gln-Gln are highly critical in the conformation change of EGFR (35). Furthermore, in agreement with our results, treatment with EGF does not induce phosphorylation of EGFR at Tyr-845, whereas ionizing radiation promotes phosphorylation of EGFR at Tyr-845 in human bronchial carcinoma cells A549 and human squamous carcinoma cells FaDu (36). EGFR and HER2 interact with specific DNA sequences on the promoters of cyclin D1/iNOS and cyclooxygenases2 genes, respectively (11,19). Consequently, we consider the possibility that EGF induces EGFReDNA complex formation. In line with this notion, our results demonstrate that EGF induces an increase in EGFReDNA complex formation. These findings support the hypothesis that EGFR in breast cancer cells mediates the transcription of genes related with the tumorigenesis process. In support of this proposal, a positive correlation has been reported between EGFR and cyclin D1/iNOS in a cohort of breast carcinomas and that nuclear EGFR and E2F1 interact with B-Myb promoter and activate its transcription, leading to accelerated G1/S cell cycle progression (12,19,37). Upon ligand activation of EGFR, there is a rapid decrease in the cell surface number of the receptor. It is mediated by clathrin- or caveolae-mediated endocytosis and dependent on microtubule network where microtubules are considered work as rails along which endosomes are transported (38e42). Furthermore, endocytosis of EGF is accompanied by the reorganization of microtubule network (25). Therefore, we hypothesize that nuclear accumulation of EGFR in response to EGF requires receptor endocytosis and an intact cytoskeleton. Supporting this hypothesis, we demonstrate here that EGFR nuclear accumulation is dependent on clathrin-coated pits and the integrity of caveolae microdomains because treatment with filipin III and hypertonic medium of sucrose inhibits nuclear accumulation of EGFR. In addition, integrity of microtubule network and an intact actin cytoskeleton are also necessary for the nuclear accumulation of EGFR. In line with this notion, it has been reported that EGFRs reside in rafts/caveolae and, following EGF
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stimulation, EGFRs rapidly leave rafts/caveolae followed by clathrin-dependent endocytosis (43e45). Moreover, stimulation of MDA-MB-468 breast cancer cells with EGF promotes co-localization of EGFR with early endosome antigen-1 (EEA-1) (46). In conclusion, our results demonstrate that stimulation of breast cancer cells with EGF induces the nuclear accumulation of EGFR in a fashion dependent on EGFR kinase activity, clathrin-coated pits, caveolae microdomain integrity and cytoskeleton integrity. Our findings also demonstrate that EGF induces an increase in the phosphorylation of EGFR at Tyr-1068 and the EGFReDNA complex formation. These findings support the proposal that nuclear EGFR and cytoskeleton integrity play an important role in breast cancer.
Acknowledgments We thank Nora Ruiz for technical assistance. This work was supported by grants from UC MEXUS-CONACYT (CN-03-70) and CONACYT (43370 and 83802).
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30. Offterdinger M, Schofer C, Weipoltshammer K, et al. c-erbB-3: a nuclear protein in mammary epithelial cells. J Cell Biol 2002;157: 929e939. 31. Maher PA. Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2. J Cell Biol 1996;134:529e536. 32. Stachowiak MK, Maher PA, Joy A, et al. Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells. Mol Biol Cell 1996;7:1299e1317. 33. Richer JK, Lange CA, Manning NG, et al. Convergence of progesterone with growth factor and cytokine signaling in breast cancer. Progesterone receptors regulate signal transducers and activators of transcription expression and activity. J Biol Chem 1998;273: 31317e31326. 34. Amos S, Martin PM, Polar GA, et al. Phorbol 12-myristate 13-acetate induces epidermal growth factor receptor transactivation via protein kinase Cdelta/c-Src pathways in glioblastoma cells. J Biol Chem 2005;280:7729e7738. 35. Bishayee A, Beguinot L, Bishayee S. Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail. Mol Biol Cell 1999;10:525e536. 36. Dittmann K, Mayer C, Kehlbach R, et al. Radiation-induced caveolin1 associated EGFR internalization is linked with nuclear EGFR transport and activation of DNA-PK. Mol Cancer 2008;7:69. 37. Hanada N, Lo HW, Day CP, et al. Co-regulation of B-Myb expression by E2F1 and EGF receptor. Mol Carcinog 2006;45:10e17. 38. Xie J, Qian L, Wang Y, et al. Role of the microtubule cytoskeleton in traffic of EGF through the lacrimal acinar cell endomembrane network. Exp Eye Res 2004;78:1093e1106. 39. Murray JW, Wolkoff AW. Roles of the cytoskeleton and motor proteins in endocytic sorting. Adv Drug Deliv Rev 2003;55: 1385e1403. 40. Sebastian S, Settleman J, Reshkin SJ, et al. The complexity of targeting EGFR signalling in cancer: from expression to turnover. Biochim Biophys Acta 2006;1766:120e139. 41. Orlichenko L, Huang B, Krueger E, et al. Epithelial growth factorinduced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells. J Biol Chem 2006;281:4570e4579. 42. Zhu JX, Goldoni S, Bix G, et al. Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis. J Biol Chem 2005;280:32468e32479. 43. Mineo C, James GL, Smart EJ, et al. Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol Chem 1996;271:11930e11935. 44. Mineo C, Gill GN, Anderson RG. Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem 1999;274: 30636e33064. 45. Pike LJ. Growth factor receptors, lipid rafts and caveolae: an evolving story. Biochim Biophys Acta 2005;1746:260e273. 46. Lo HW, Ali-Seyed M, Wu Y, et al. Nuclear-cytoplasmic transport of EGFR involves receptor endocytosis, importin beta1 and CRM1. J Cell Biochem 2006;98:1570e1583.
ORIGINAL ARTICLE
Epidermal Growth Factor Promotes Epidermal Growth Factor Receptor Nuclear Accumulation by a Pathway Dependent on Cytoskeleton Integrity in Human Breast Cancer Cells Pedro Cortes-Reynosa, Teresa Robledo, and Eduardo Perez Salazar Departamento de Biologia Celular, CINVESTAV-IPN, Mexico, D.F., Mexico Received for publication October 20, 2008; accepted May 21, 2009 (ARCMED-D-08-00466).
Background and Aims. The epidermal growth factor receptor (EGFR) is activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events. Emerging evidence indicates a mode of EGF signaling in which growth factor signals are transmitted via EGFR nuclear transport. The aim of this study was to determine whether EGF promotes EGFR nuclear accumulation and the role of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity in breast cancer cells. Methods. MCF-7 cells were treated without or with 100 ng/ml EGF for various times and nuclear extracts were obtained. Nuclear accumulation of EGFR was analyzed by SDS-PAGE followed by Western blotting of nuclear extracts using an anti-EGFR Ab or with a phosphospecific Ab against the Tyr-1068 of EGFR and with anti-Rb Ab as the loading control. DNA binding activity of EGFR was analyzed by EMSA using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS). Results. EGF induces the nuclear accumulation of EGFR, an increase in EGFR phosphorylation at Tyr-1068 and the formation of the complex EGFR-DNA in MCF-7 and MDA-MB-231 breast cancer cells. In addition, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity. Conclusions. This study demonstrates that in breast cancer cells EGF promotes nuclear accumulation of EGFR and is dependent on clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: Breast cancer, nuclear EGFR, EGFR-DNA complex, Cytoskeleton.
Introduction The epidermal growth factor receptor (EGFR) is a member of the ErbB family of tyrosine kinase receptors, which includes EGFR (ErbB1/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors are activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events (1,2). EGF contributes to mammary
Address reprint requests to: Eduardo Perez Salazar, Ph.D, Departamento de Biologia Celular, CINVESTAV-IPN, Av. IPN #2508, 07360 Mexico, D.F., Mexico; Phone: (þ52) (55) 5747-3991; FAX: (þ52) (55) 5747-3393; E-mail: [email protected]
development, and it is a major regulator of breast cancer that become steroid hormone-resistant (3,4). Furthermore, it has been reported that EGFR is expressed in 14e91% of patients with breast cancer, and its expression is significantly associated with the loss of steroid hormone sensitivity. It also correlates with high disease recurrence and decreased patient survival (5e8). EGFR activation induced by binding of EGF promotes the formation of homodimers or heterodimers and the phosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated residues recruit signaling proteins such as Shc, Grb2, phospholipase Cg and Src, with subsequent activation of signal transduction pathways (9,10). However, EGFR also is localized in the
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2009.06.007
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nucleus of cancer cells, human placenta, pregnant uterus and regenerating liver, and it has the potential to associate with the promoter region of cyclin D1, strongly suggesting that EGFR might function as a transcription factor (11e13). Moreover, it has been reported that one juxtamembrane region in the EGFR family members present a putative nuclear localization sequence, which mediates the localization of EGFR in the nucleus (14) and that ErbB-2 receptor translocates directly from the cytoplasmic membrane to the nucleus through endocytosis by using the importin b1 and the nuclear pore protein Nup358 (15) In the present study we report that EGF induces nuclear accumulation of EGFR, nuclear EGFR phosphorylation at Tyr-1068 and an increase in EGFR-DNA complex formation in breast cancer cells. Furthermore, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity.
peroxidase-conjugated, goat Abs to rabbit) (1:5000) for 2 h at 22 C. After washing three times with PBS/0.1% Tween 20, the immunoreactive bands were visualized using ECL detection reagents. Preparation of Nuclear Extracts Nuclear extracts were prepared as described previously (11,16e18). Briefly, cells were lysed with 0.1% Nonidet P40 in Buffer A (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, and 1 mM PMSF). Lysates were pelleted at 12,000 rpm for 30 sec and resuspended in Buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM DTT, and 0.2 mM PMSF). Nuclear fraction was recovered by centrifugation at 12,000 g for 15 min at 4 C. The supernatant was recovered and centrifuged again at 15,000 g for 5 min, and the resulting supernatant was the non-nuclear fraction.
Materials and Methods Materials EGF, colchicine, cytochalasin D, sucrose and filipin III were from Sigma (St. Louis, MO). AG1478 was from Calbiochem (San Diego, CA). EGFR antibody (Ab), retinoblastoma (Rb) Ab and phosphospecific Abs to Tyr-845 and Tyr-1068 of EGFR were from Cell Signaling Technology (Beverly, MA). Paxillin Ab was from BD (San Diego, CA). IkB-a Ab, Src Ab and lamin B1 Ab were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat Ab to rabbit was from Zymed (San Francisco, CA). ECL reagent was from Amersham Pharmacia Biotech (Piscataway, NJ). Cell Culture MCF-7 and MDA-MB-231 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.7 g/L sodium bicarbonate and 5% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2 and 95% air at 37 C. For experimental purposes, confluent cultures of these cells were serum-starved for 12 h before treatment with inhibitors and/or EGF.
Electrophoretic Mobility Shift Assay (EMSA) EMSAs were performed as described before (11,16). Briefly, double-stranded oligonucleotide-containing specific binding sites for EGFR (ATRS sequence) (11), 50 -CTTGTTTACGG TCCCCCCATTTTTTACGCTGGTCCT-30 , was used as probe. ATRS oligonucleotide was labeled with (g32P) ATP using T4 polynucleotide kinase. The 32P-labeled oligonucleotide probe (|1 ng) was incubated with 5 mg of nuclear extract in a reaction mixture containing 3 mg of poly (dI-dC), 0.25 M HEPES, pH 7.5, 0.6 M KCl, 50 mM MgCl2, 1 mM EDTA, 7.5 mM DTT, and 9% glycerol for 20 min at 4 C. One hundred-fold excess of unlabeled ATRS probe or an irrelevant oligonucleotide (50 -ACGTGTGATGAAATGCTAGGCGATC-30 ) was used as specific and nonspecific competitors. Samples were fractionated on a 4% polyacrylamide gel in 0.5X Tris borate-EDTA buffer. Gels were dried and analyzed by autoradiography. In supershift assays, nuclear extracts were incubated for 30 min at 4 C with the Ab against EGFR or IgG before addition of the radiolabeled probe.
Results
Western Blotting
EGF Stimulation Induces Nuclear Accumulation of EGFR in MCF-7 Cells
Equal amounts of protein were separated by SDS-PAGE using 8% separating gels followed by transfer to nitrocellulose membranes. After transfer, membranes were blocked using 5% non-fat dried milk in PBS, pH 7.2, and incubated overnight at 4 C with the primary Ab as indicated. The membranes were washed three times with PBS/0.1% Tween 20 and then incubated with secondary Abs (horseradish
Nuclear detection of EGFR has been reported in cancer cells and primary tumors including those of skin, breast, bladder, cervix, adrenocorticord, thyroid and oral cavity (11,12,19e22). To examine whether EGF induces accumulation of endogenous EGFR in the nucleus, confluent cultures of MCF-7 cells were treated with or without 100 ng/mL EGF for various times. Nuclear extracts were
Cytoskeleton Regulates Nuclear Accumulation of EGFR
obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. As shown in Figure 1A (upper panel), in untreated cells a small amount of EGFR was localized in the nucleus and treatment of cells with EGF induced the nuclear accumulation of EGFR. Western blotting with anti-Rb Ab of the same membranes confirmed that similar amounts of nuclear proteins were recovered in the absence or in the presence of EGF (Figure 1A, lower panel). To rule out the possibility that the signal seen in nuclear extracts was due to contamination, nuclear extracts (nuclear fraction) and non-nuclear fraction were analyzed by immunoblotting with anti-paxillin Ab, anti-Rb Ab, anti-IkB-a Ab, anti-Src Ab, anti-E-cadherin Ab and anti-lamin B1 Ab. Our results showed that Rb and lamin B1 were present only in the nuclear fraction, whereas the cytoplasmic proteins paxillin, IkB-a, Src and E-cadherin were absent (Figure 1B). Next, we determined whether EGF induces phosphorylation of nuclear EGFR at specific tyrosine residues such as Tyr-845 and Tyr-1065. MCF-7 cells were treated with or without 100 ng/mL EGF for various times and nuclear extracts were obtained. Nuclear extracts were analyzed by SDS-PAGE followed by Western blotting with a phosphospecific Ab against the Tyr-845 of EGFR [anti-EGFR (P)Y845] to localize EGFR phosphorylated at Tyr-845 and with a phosphospecific Ab against the Tyr-1068 of
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EGFR [anti-EGFR(P)Y1068] to localize EGFR phosphorylated at Tyr-1068. As illustrated in Figure 2A, EGF induced an increase in the nuclear EGFR phosphorylated at Tyr1068. In contrast, in untreated cells and cells stimulated with EGF, nuclear EGFR phosphorylated at Tyr-845 was not detected in the nucleus (results not shown). To examine whether kinase activity of EGFR plays a role in the nuclear accumulation of EGFR induced by EGF, we studied the effect of AG1478 in the nuclear accumulation of EGFR. AG1478 is a potent and selective inhibitor of EGFR kinase activity and it has been used previously to inhibit EGFR kinase activity in breast cancer cells (23,24). MCF-7 cells were treated for 30 min in the absence or presence of 300 nM AG1478 and then stimulated with 100 ng/ mL EGF for another 20 min. Nuclear extracts were obtained and analyzed by Western blotting with anti-EGFR Ab. Our results showed that treatment of MCF-7 cells with AG1478 inhibited EGFR nuclear accumulation in response to EGF (Figure 2B). EGF Treatment Induces DNA Binding Activity of EGFR Because previous studies had already shown that EGFR is a putative transcription factor and is able to bind to DNA (11), we determined whether EGF treatment of MCF-7 cells triggers nuclear translocation with subsequent binding to DNA. MCF-7 cells were treated with 100 ng/mL EGF for
Figure 1. Epidermal growth factor (EGF) induces nuclear accumulation of epidermal growth factor receptor (EGFR) in MCF-7 cells Confluent cultures of MCF-7 cells were treated at 37 C with 100 ng/mL EGF for various times as indicated; nuclear extracts were subsequentally obtained. (A) EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. The graph represents the mean SD of at least three independent experiments and is expressed as the fold stimulation above control (unstimulated) value. Asterisks denote comparisons made to control cultures (unstimulated). *p !0.005, ***p !0.001 by one-way ANOVA. (B) Nuclear fraction and non-nuclear fraction were analyzed by Western blotting with anti-paxillin Ab, anti-Rb Ab, anti-IkB-a, anti-Src Ab, anti-Lamin B1 Ab and anti-E-cadherin Ab. Radiograms shown are representative of at least three independent experiments.
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Figure 2. Role of EGFR kinase activity in the nuclear accumulation of EGFR. (A) Confluent cultures of MCF-7 cells were treated at 37 C with 100 ng/mL EGF for various times as indicated, and subsequently nuclear extracts were obtained. Nuclear EGFR phosphorylation at Tyr-1068 was analyzed by Western blotting of nuclear extracts with anti-EGFR(P)Y1068 Ab or with anti-Rb Ab as the loading control. (B) MCF-7 cells were treated at 37 C for 30 min in the absence () or in the presence (þ) of 300 nM AG1478 and then stimulated without () or with (þ) 100 ng/mL EGF for 20 min and nuclear extracts were obtained. EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. The graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. ***p ! 0.001 by one-way ANOVA. Radiograms shown are representative of at least three independent experiments.
various times and nuclear extracts were obtained. EMSAs were then performed using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS). As illustrated in Figure 3A, EGF induced EGFR-DNA complex formation in a timedependent manner. The specificity of these complexes was demonstrated by inhibition of binding in the presence of a cold competitor.
To further substantiate that EGFR binds to the ATRS sequence, we performed supershift assays using nuclear extracts of MCF-7 cells stimulated with EGF and one Ab against EGFR. MCF-7 cells were treated with 100 ng/ mL EGF for 5 min and nuclear extracts were obtained. Nuclear extracts were treated with 2 mg EGFR Ab for 30 min at 4 C and then EMSAs were performed. Our results showed that pre-treatment with the EGFR Ab inhibited
Figure 3. EGF induces EGFR DNA binding activity in MCF-7 cells. (A) Nuclear extracts were prepared from MCF-7 cells untreated or treated with 100 ng/ mL EGF for various times, as indicated. (B) Nuclear extracts were pre-treated in either the absence () or the presence (þ) of 1 mg Ab to EGFR or control IgG. EGFR DNA binding activity was analyzed by electrophoretic shift mobility assay (EMSA) as described in Methods. Notice that pre-treatment with EGFR Ab inhibited the EGFR-DNA complex formation. Autoradiograms shown are representative of at least three independent experiments.
Cytoskeleton Regulates Nuclear Accumulation of EGFR
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Figure 4. EGF promotes nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells. Confluent cultures of MDA-MB-231 cells were treated at 37 C without or with 100 ng/mL EGF for various times as indicated and nuclear extracts were obtained. (A) EGFR nuclear accumulation was analyzed by Western blotting of nuclear extracts with anti-EGFR Ab or with anti-Rb Ab as the loading control. (B) Nuclear EGFR phosphorylation at Tyr-1068 was analyzed by Western blotting of nuclear extracts with anti-EGFR(P)Y1068 Ab or with anti-Rb Ab as the loading control. The graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. **p !0.01, ***p !0.001 by one-way ANOVA. Radiograms shown are representative of at least three independent experiments.
the EGFReDNA complex formation. In contrast, treatment with an unspecific Ab (2 mg IgG) did not inhibit the EGFReDNA complex formation (Figure 3B). These results demonstrate that EGFR is forming a complex with the ATRS DNA sequence. EGF Treatment Induces Nuclear Accumulation of EGFR in MDA-MB-231 Breast Cancer Cells In order to substantiate further that EGF induces nuclear accumulation of EGFR in breast cancer cells, we examined whether EGF promotes nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells. MDA-MB231 cells were stimulated for various times with 100 ng/ mL EGF and nuclear extracts were obtained and analyzed by Western blotting with anti-EGFR Ab. In agreement with our previous results, our findings showed that EGF stimulation induced an increase in the nuclear accumulation of EGFR in MDA-MB-231 breast cancer cells (Figure 4A). To determine the phosphorylation status of EGFR in the nucleus, MDA-MB-231 breast cancer cells were stimulated for various times with EGF and nuclear extracts were analyzed by Western blotting with anti-EGFR(P)Y845 and anti-EGFR(P)Y1068 Abs. Our results showed that treatment with EGF induced an increase in the nuclear EGFR phosphorylated at Tyr-1068 in MDA-MB-231 cells (Figure 4B). In contrast, in untreated cells and cells stimulated with EGF, EGFR phosphorylated at Tyr-845 was not detected in the nucleus (results not shown).
EGFR Nuclear Accumulation Requires Endocytosis and Cytoskeletal Integrity It has been reported that one clathrin-mediated endocytosis mechanism mediates ErbB-2 nuclear translocation, whereas endocytosis of EGFR is accompanied by reorganization of microtubule network (15,25). Therefore, we examined the contribution of cytoskeleton and the role of clathrin- and caveolae-mediated endocytosis in the nuclear accumulation of EGFR. We demonstrated that cytochalasin D specifically disrupts the organization of actin fibers, whereas colchicine promotes the disappearance of microtubules in MCF-7 cells (26). Therefore, in order to determine the involvement of cytoskeleton in the nuclear accumulation of EGFR, we determined the effect of colchicine and cytochalasin D in the EGFR nuclear accumulation. Cultures of MCF-7 cells were treated in the absence or presence of 10 mM colchicine for 1 h or 2.4 mM cytochalasin D for 2 h and then stimulated with 100 ng/mL EGF for 20 min. Nuclear extracts were obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. As illustrated in Figure 5A, treatment with colchicine and cytochalasin D inhibited the nuclear accumulation of EGFR. To investigate whether EGFR nuclear accumulation involves an endocytosis mechanism mediated by caveolae microdomains and/or clathrin-coated pits, MCF-7 cells were treated for 45 min in the absence or presence of 5 mg/mL filipin III, a polyene antibiotic that binds to
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Figure 5. Role of cytoskeleton in the nuclear accumulation of EGFR. (A) Confluent cultures of MCF-7 cells were treated for 1 h in the absence () or in the presence (þ) of 10 mM colchicine (Colc) or for 2 h with 2.4 mM cytochalasin D (Cyt D) and then stimulated with 100 ng/mL EGF for a further 20 min. (B) MCF-7 cells were treated for 45 min in the absence () or presence (þ) of 5 mg/mL filipin III or for 1 h in the absence () or presence (þ) 375 mM sucrose and then stimulated with 100 ng/mL EGF for a further 20 min. EGFR nuclear translocation was analyzed by Western blotting of nuclear extracts with antiEGFR Ab or with anti-Rb Ab as the loading control. Graphs represent the mean SD of at least three independent experiments and are expressed as the fold stimulation above control (unstimulated) value. **p !0.01, ***p !0.001 by one-way ANOVA. Radiograms are representative of at least three independent experiments.
cholesterol and removes it from the membrane causing reversible caveolae disassembly (27,28), or they were incubated for 1 h with hypertonic medium containing sucrose at a concentration of 375 mM, which interferes with the clathrin pathway (29), followed by stimulation with 100 ng/ mL EGF for 20 min. Nuclear extracts were obtained and analyzed by SDS-PAGE followed by Western blotting with anti-EGFR Ab or with anti-Rb Ab as the loading control. Our findings showed that treatment with filipin III completely inhibited the nuclear accumulation of EGFR, whereas treatment with sucrose partly inhibited the nuclear accumulation of EGFR (Figure 5B).
Discussion The presence of receptor tyrosine kinases in the nucleus has been reported. In particular, EGFR, ErbB-3/HER-3 and fibroblast growth factor receptor I (FGFR-I) are present in an uncleaved form in the nucleus (11,30e32). However, little is known about the regulation of nuclear EGFR accumulation in breast cancer cells. By using a method to obtain nuclear extracts from breast cancer cells previously described (11,16,33), and two breast cancer cell lines, we show here that stimulation with EGF induces nuclear
accumulation of EGFR and requires EGFR kinase activity. These results are in agreement with previous reports that demonstrate EGFR is localized in the nucleus of breast cancer tumors and correlates with poor survival in patients with breast cancer (11,12). In addition, our results show that nuclear EGFR accumulation decreases at 30 min of treatment. Therefore we propose that degradation of EGFR takes places after 20 min of treatment or that nuclear EGFR is translocated from nucleus to cytosol. However, additional studies are necessary to substantiate this hypothesis. In glioblastoma cell lines, treatment with EGF stimulates global phosphorylation of the EGFR at Tyr-845, Tyr-992, Tyr-1068 and Tyr-1054, whereas treatment with phorbol 12myristate 13-acetate stimulates phosphorylation of EGFR only at Tyr-1068 (9,34). In the present study we used Abs that detect the phosphorylated state of Tyr-845 and Tyr-1068 of EGFR to elucidate whether EGF modulates the phosphorylation of these residues in the nuclear EGFR of breast cancer cells. Our results demonstrate that stimulation of breast cancer cells with EGF induces an increase in the nuclear accumulation of EGFR phosphorylated at Tyr-1068. In contrast, EGF does not induce accumulation of EGFR phosphorylated at Tyr-845. However, EGFR nuclear accumulation kinetics does not coincide with the nuclear accumulation of EGFR phosphorylated at Tyr-1068. Specifically, nuclear accumulation
Cytoskeleton Regulates Nuclear Accumulation of EGFR
of EGFR phosphorylated at Tyr-1068 reaches a maximum at 30 min of treatment when EGFR nuclear accumulation declines toward baseline levels. These results suggest that EGFR phosphorylation at Tyr-1068 does not trigger the nuclear accumulation of EGFR and that EGF promotes phosphorylation of nuclear EGFR at their tyrosine residues to different times of stimulation. In addition, because our findings also show that nuclear accumulation of EGFR is dependent on EGFR kinase activity, we propose that EGFR phosphorylation at their different amino acids plays an important role in the nuclear accumulation of EGFR and in the DNA binding activity. Our findings also suggest that PKC activity may play an important role in the phosphorylation at Tyr1068, promoting changes in the conformation of EGFR. In support of this proposal, it has been reported that phosphorylation of Tyr-992, Tyr-1068 and Tyr-1086, localized between the tripeptides Asp-Glu-Glu and Tyr-Gln-Gln are highly critical in the conformation change of EGFR (35). Furthermore, in agreement with our results, treatment with EGF does not induce phosphorylation of EGFR at Tyr-845, whereas ionizing radiation promotes phosphorylation of EGFR at Tyr-845 in human bronchial carcinoma cells A549 and human squamous carcinoma cells FaDu (36). EGFR and HER2 interact with specific DNA sequences on the promoters of cyclin D1/iNOS and cyclooxygenases2 genes, respectively (11,19). Consequently, we consider the possibility that EGF induces EGFReDNA complex formation. In line with this notion, our results demonstrate that EGF induces an increase in EGFReDNA complex formation. These findings support the hypothesis that EGFR in breast cancer cells mediates the transcription of genes related with the tumorigenesis process. In support of this proposal, a positive correlation has been reported between EGFR and cyclin D1/iNOS in a cohort of breast carcinomas and that nuclear EGFR and E2F1 interact with B-Myb promoter and activate its transcription, leading to accelerated G1/S cell cycle progression (12,19,37). Upon ligand activation of EGFR, there is a rapid decrease in the cell surface number of the receptor. It is mediated by clathrin- or caveolae-mediated endocytosis and dependent on microtubule network where microtubules are considered work as rails along which endosomes are transported (38e42). Furthermore, endocytosis of EGF is accompanied by the reorganization of microtubule network (25). Therefore, we hypothesize that nuclear accumulation of EGFR in response to EGF requires receptor endocytosis and an intact cytoskeleton. Supporting this hypothesis, we demonstrate here that EGFR nuclear accumulation is dependent on clathrin-coated pits and the integrity of caveolae microdomains because treatment with filipin III and hypertonic medium of sucrose inhibits nuclear accumulation of EGFR. In addition, integrity of microtubule network and an intact actin cytoskeleton are also necessary for the nuclear accumulation of EGFR. In line with this notion, it has been reported that EGFRs reside in rafts/caveolae and, following EGF
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stimulation, EGFRs rapidly leave rafts/caveolae followed by clathrin-dependent endocytosis (43e45). Moreover, stimulation of MDA-MB-468 breast cancer cells with EGF promotes co-localization of EGFR with early endosome antigen-1 (EEA-1) (46). In conclusion, our results demonstrate that stimulation of breast cancer cells with EGF induces the nuclear accumulation of EGFR in a fashion dependent on EGFR kinase activity, clathrin-coated pits, caveolae microdomain integrity and cytoskeleton integrity. Our findings also demonstrate that EGF induces an increase in the phosphorylation of EGFR at Tyr-1068 and the EGFReDNA complex formation. These findings support the proposal that nuclear EGFR and cytoskeleton integrity play an important role in breast cancer.
Acknowledgments We thank Nora Ruiz for technical assistance. This work was supported by grants from UC MEXUS-CONACYT (CN-03-70) and CONACYT (43370 and 83802).
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