ErbB2 overexpression in breast tumors results in increased metastasis and angiogenesis and reduced survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, we did a spontaneous metastasis assay using MDA-MB-435 human breast cancer cells stably transfected with constitutively active ErbB2 kinase (V659E), a kinase-dead mutant of ErbB2 (K753M), or vector control (neo). Mice injected with V659E had increased metastasis incidence and tumor microvessel density than mice injected with K753M or control. Increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro. V659E produced increased vascular endothelial growth factor (VEGF) through increased VEGF protein synthesis. This was mediated through signaling events involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase/Akt, mammalian target of rapamycin (mTOR), and p70S6K. The V659E xenografts also had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with controls. To validate the clinical relevance of these findings, we examined 155 human breast tumor samples. Human tumors that overexpressed ErbB2, which have been previously shown to have higher VEGF expression, showed significantly higher p70S6K phosphorylation as well. Increased VEGF expression also significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Our findings show that ErbB2 increases VEGF protein production by activating p70S6K in cell lines, xenografts, and in human cancers and suggest that these signaling molecules may serve as targets for antiangiogenic and antimetastatic therapies. (Cancer Res 2006; 66(4): 2028-37)

Approximately 30% of breast cancer patients have tumors overexpressing ErbB2 (HER2/neu; ref. 1), a receptor tyrosine kinase in the epidermal growth factor receptor family (2). Patients with ErbB2-overexpressing breast tumors have an increased incidence of metastasis and a poorer survival rate when compared with patients whose tumors express ErbB2 at normal levels (1). Activation of ErbB2 is associated with increased receptor tyrosine kinase activity and tyrosine phosphorylation of the receptor (3, 4). We previously showed that transfection of the erbB2 expression vector into the ErbB2 low-expressing MDA-MB-435 human breast cancer cells led to increased ErbB2 protein levels similar to that of endogenous ErbB2 in the SKBR3 human breast cancer cell line (5) and increased their intrinsic metastatic potential (6).

Angiogenesis is a key component of cancer metastasis (7). ErbB2-overexpressing breast tumors tend to be more angiogenic than other breast tumors (8). Angiogenesis is tightly controlled under normal physiologic conditions; however, in pathologic diseases such as cancer, the fine balance between proangiogenic and antiangiogenic factors is disrupted (9). One of the most potent inducers of angiogenesis is vascular endothelial growth factor (VEGF), which induces endothelial cell proliferation and migration (10). VEGF expression in human breast cancers is correlated with increased microvessel density (11) and reduced survival (12). ErbB2 has been implicated in the regulation of VEGF (13). In human breast tumors, overexpression of ErbB2 is correlated with increased VEGF expression (14) and the transcription factor hypoxia-inducible factor-1α has been postulated to mediate the ErbB2 up-regulation of VEGF (15).

P70S6 kinase (p70S6K) is a serine-threonine kinase that regulates protein translation (16) by directly phosphorylating ribosomal protein S6 (rpS6; ref. 17), a subunit of the 40S protein complex involved in the translation of mRNAs that contain an oligopyrimidine tract at their transcriptional start site (18). P70S6K lies downstream from the mammalian target of rapamycin (mTOR; ref. 19). The mTOR/p70S6K pathway may be directly activated by Akt (20) or indirectly activated by Akt through the phosphorylation and inactivation of the tuberous sclerosis complex tumor suppressors (21). mTOR/p70S6K can also be activated by phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent kinase-1 (22) and extracellular signal-regulated kinase (ERK; refs. 23, 24). Although activation of p70S6K involves multiple phosphorylations on its serine and threonine residues, phosphorylation of threonine 389 (T389) by mTOR is critical for p70S6K activation and serves as a marker for mTOR activity (25, 26). Deregulation of the mTOR/p70S6K pathway may play an important role in cancer development as well as in many other diseases (27, 28). Although ErbB2 has been shown to activate PI3K/Akt and ERK, which are upstream activators of mTOR/p70S6K, the role of mTOR/p70S6K in ErbB2-mediated cancer metastasis and angiogenesis remains elusive.

As described herein, we directly studied the effect of ErbB2 receptor tyrosine kinase and its downstream signaling pathways on metastasis and angiogenesis in human breast cancer cells. We compared the metastatic and angiogenic potentials of metastatic human breast cancer cells (MDA-MB-435; ref. 29) stably transfected with constitutively active ErbB2 kinase (V659E) or kinase-dead ErbB2 (K753M) and vector control (30) in vitro and in vivo. We also investigated the relationship between phosphorylated p70S6K-T389, ErbB2, and VEGF expression in human breast cancer specimens. Our data from cell lines, an animal model, and breast cancer patient samples show that ErbB2 activation leads to the translational up-regulation of VEGF and increased angiogenesis through ERK, PI3K/Akt, mTOR, and p70S6K.

Cells. Low ErbB2-expressing MDA-MB-435 human breast cancer cells were stably transfected to overexpress wild-type ErbB2 (435.eB1), a constitutively active ErbB2 kinase mutant (V659E), or a kinase-dead ErbB2 mutant (K753M). The mutant constructs were provided by Dr. Tadashi Yamamoto (Institute of Medical Science, University of Tokyo, Tokyo, Japan; ref. 31) and were subcloned into the pcDNA3 vector (30, 32). MDA-MB-435 ErbB2 stable transfectants were selected with G418 (Geneticin; Life Technologies, Rockville, MD). The cells were maintained in high-glucose DMEM (DMEM/F12; Life Technologies, Inc., Grand Island, NY) with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). Human umbilical vein endothelial cells (HUVEC) were purchased from the American Type Culture Collection (Manassas, VA) and maintained in F-12K nutrient mixture (Kaighn's modification; Life Technologies) with 10% FBS, 100 μg/mL heparin (Sigma-Aldrich, St. Louis, MO), and 30 μg/mL endothelial cell growth supplement (Sigma-Aldrich). The HUVECs were grown on tissue culture dishes coated with 0.5% gelatin.

Western blot analysis. Cells were treated as indicated with 10 nmol/L rapamycin (Cell Signaling Technology, Beverly, MA), 50 nmol/L wortmannin (Calbiochem, San Diego, CA), or 50 μmol/L PD98059 (Calbiochem) for 2 hours in serum-free, phenol red–free DMEM/F12. Cells were lysed with immunoprecipitation buffer [1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 1 mmol/L EGTA, 0.2 mmol/L sodium vanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.5% NP40 supplemented with a protease inhibitor cocktail from Sigma Aldrich]. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), and probed with antibodies against phosphotyrosine (BD Biosciences PharMingen, San Diego, CA), β-actin (Sigma-Aldrich), ErbB2 (Ab-3; Oncogene Science, Inc., Cambridge, MA), and phospho-p70S6K-T389, p70S6K, phospho-Akt-S473, Akt, phospho-tuberin-T1462, tuberin, phospho-p42/44 mitogen-activated protein (MAP) kinase (MAPK)-T202/Y204, and p42/44 MAPK, all from Cell Signaling Technology.

Severe combined immunodeficient mouse spontaneous metastasis assay. MDA-MB-435, neo, eB1, V659E, or K753M breast cancer cells (1 × 106) suspended in 0.15 mL Matrigel (BD Biosciences PharMingen) were injected into the mammary fat pad of 10-week-old female severe combined immunodeficient (SCID) mice (Harlan, Indianapolis, IN), 15 mice per group. At a diameter of 1 cm, tumors were removed from mice and the mice were humanely killed 42 days later. Their lungs were removed, injected with India ink, and examined for the presence of metastases (33).

SCID mouse tumor microvessel density and immunohistochemistry. Xenograft tumors were collected from the mammary fat pad of SCID mice injected with the ErbB2 transfectants in the spontaneous metastasis assay and frozen in liquid nitrogen or fixed in 10% formalin. Tumor microvessel density was determined on 4 μm tumor sections with a rat antimouse CD31 antibody (BD Biosciences PharMingen). CD31+ structures were counted. Branching structures were counted as single vessels. Microvessel density was expressed as the average of number of microvessels per square millimeter.

Immunohistochemical staining of tumors was done as described previously (34) with antibodies against ErbB2 (Neomarker, Fremont, CA), phospho-p70S6K-T389 and phospho-Akt-S473 (Cell Signaling Technology), and VEGF (R&D Systems, Minneapolis, MN). Briefly, sections were subjected to heat-induced epitope retrieval in 0.01 mol/L citrate buffer (pH 6.0) and blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature. Samples were then incubated with primary antibody for 1 hour at room temperature or overnight at 4°C. Immunodetection was done with the EnVision+ system (DAKO, Carpinteria, CA) followed by 3,3′-diaminobenzidine for color development and hematoxylin for counterstaining. Staining intensity was graded as described in the figure captions. Statistical analyses were done with 2 × 2 contingency tables and Mann-Whitney tests, and P < 0.05 was considered statistically significant.

Conditioned medium. Cells (1 × 106) were plated in 60 mm tissue culture plates, washed with serum-free, phenol red-free DMEM/F12, and incubated in serum-free, phenol red-free DMEM/F12 for 48 hours. The medium was collected and centrifuged at 3,500 rpm for 15 minutes to remove debris.

HUVEC migration assay. Polycarbonate membranes from 24-well, 8-μm-pore Transwell plates (Corning Costar, Corning, NY) were coated with 0.5% gelatin. Six hundred microliters of serum-free conditioned medium from the ErbB2 transfectants were placed into the bottom wells and 1 × 104 HUVECs in serum-free DMEM/F12 were plated onto the upper chamber of each well. The plates were incubated at 37°C for 6 hours. The cells were then fixed with 3% glutaraldehyde for 30 minutes and stained with Giemsa stain/PBS at a ratio of 1:9 (Lab Chem, Pittsburgh, PA). Statistical analyses were done by one-way ANOVA with a Bonferroni correction.

VEGF ELISA. Cells (1 × 106) were plated in a 60 mm tissue culture plate and allowed to attach overnight. The cells were then washed and incubated in serum-free, phenol red-free DMEM/F12 with 10 nmol/L rapamycin, 50 μmol/L LY294002, 50 μmol/L PD98059, or solvent control for 48 hours. Cells treated with LY294002 (Calbiochem) were treated twice at 24-hour intervals. The conditioned medium was collected and ELISA assays were done with the VEGF Quantikine ELISA kit (R&D Systems).

Northern blot analysis. Cells (1.5 × 106) were plated in a 100 mm tissue culture dish. Cells were incubated for 48 hours in serum-free medium. RNA was isolated using TRIzol reagent (Invitrogen). Northern blot analysis (35) was conducted using radiolabeled probes for VEGF165 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Densitometric quantification of the VEGF autoradiograms was measured with the AlphaImager 2000 v4.0 (Alpha Innotech Corporation, San Leandro, CA).

VEGF protein stability assay. Cells (2 × 105) were plated in six-well tissue culture plates. Cells were washed the next day and 2.5 mL of serum-free, phenol red–free DMEM/F12 was added to each well. Twenty-four hours later, cells were treated with 50 μg/mL cycloheximide or solvent (DMSO) and treated again with cycloheximide at 48 hours. The conditioned medium was then centrifuged and the VEGF ELISA was done as described above.

Determination of VEGF protein synthesis with ELISA. Cells (5 × 105) were plated in 60 mm dishes and incubated overnight. Cells were then washed, incubated for 4 hours in serum-free DMEM without methionine (Sigma-Aldrich), and labeled for 48 hours with 500 μCi/mL [35S]methionine in serum-free methionine-free medium. After incubation, the conditioned medium was collected and incubated on a high-binding 96-well plate (Corning Costar) coated with of antihuman VEGF capture antibody (AF-293-NA; R&D Systems; 0.8 μg/mL; 100 μL/well) for 2 hours at room temperature. The 96-well plates were blocked with blocking buffer (1% BSA, 5% sucrose, and 0.05% NaN3) before the samples were added. Following incubation, the wells were washed thrice with PBS and 200 μL of scintillation buffer (MicroScint-PS; Perkin-Elmer, Boston, MA) was added to each well. The β cpm values were measured using the Perkin-Elmer TopCount NXT (Perkin-Elmer).

Determination of VEGF protein synthesis with SDS-PAGE. Cells (5 × 105) were plated in 60 mm dishes and incubated overnight. Cells were then washed and incubated in serum-free DMEM without methionine for 4 hours and then labeled for 4 hours with 30 μL of 500 μCi/mL [35S]methionine in 1 mL of serum-free DMEM without methionine. Cell medium and cell lysates were collected. The medium was immunoprecipitated with 1 μg of rabbit VEGF antibody (Upstate Cell Signaling Solutions, Lake Placid, NY), and the cell lysates were immunoprecipitated with 2 μg of VEGF antibody (Upstate Cell Signaling Solutions) overnight at 4°C. Protein G agarose beads (Roche Diagnostics, Indianapolis, IN) were added to each tube for 2 hours at 4°C. The immunoprecipitates from the medium and lysates were then combined and subjected to SDS-PAGE. Part of the gel was stained with Coomassie blue to assess protein loading amounts, and part was exposed to film.

Human tumor samples. Tumor samples were collected from 155 women with primary breast carcinoma who were diagnosed, treated with surgery, and subsequently treated with cyclophosphamide, methotrexate, and 5-flurouracil at The Cancer Hospital, FuDan University (Shanghai, China), from 1988 to 1991 (36). Immunohistochemistry was done as described for xenograft samples (36). Phospho-mTOR-S2448 was detected with antibody from Cell Signaling Technology and ErbB2 was detected with the DAKO HercepTest (DAKO, Carpinteria, CA) and scored as described for the xenografts. VEGF was quantitated with a SAMBA 4000 image analyzer (Samba Technologies, Melan, France; ref. 14). Statistical significance was calculated with Fisher's exact test. Disease-free survival was determined by the Kaplan-Meier method and metastatic frequency was determined by Fisher's exact test.

ErbB2 enhances metastasis and angiogenesis. To assess the involvement of ErbB2 kinase activity in ErbB2-mediated metastasis and angiogenesis, we used the MDA-MB-435 (435) metastatic human breast cancer cells (29) stably transfected with expression vectors of wild-type ErbB2 (eB1), constitutively active ErbB2 kinase (V659E), kinase-dead ErbB2 (K753M), or vector control (30, 32). ErbB2 tyrosine phosphorylation levels (indicating ErbB2 kinase activity) were elevated in the eB1 and the V659E transfectants, whereas it was barely detectable in the K753M and the parental 435 cell lines (Fig. 1A). Because the ErbB2 protein levels in these transfectants are similar (30, 32) as shown by Western blot (Fig. 1A), and by fluorescence-activated cell sorting analysis for membrane ErbB2 (data not shown), differences in biological activities between these transfectants will unlikely be due to ErbB2 protein levels, but rather to ErbB2 kinase activities. To assess the role of ErbB2 kinase activity in metastasis in vivo, we did a spontaneous metastasis assay by injecting the panel of erbB2 transfectants into the mammary fat pad of SCID mice. The incidence of breast cancer metastasis to the lungs was 80% after injection of V659E transfectants and was significantly higher (P = 0.01) than the incidence of metastases in the neo control (23%; Fig. 1B). The mice injected with eB1 transfectants had a 47% incidence of metastasis, whereas the K753M kinase-dead ErbB2 transfectants had a metastatic incidence of only 27%, and were not significantly different from the neo controls (Fig. 1B), indicating that ErbB2 kinase activity is responsible for ErbB2-mediated metastasis.

Figure 1.

ErbB2 increases metastasis and angiogenesis in vivo and in vitro. A, ErbB2 expression and tyrosine phosphorylation levels of MDA-MB-435 and ErbB2 transfectants. Western blotting on total cell lysates from MDA-MB-435 and ErbB2 transfectants with anti-ErbB2 and anti-phosphotyrosine antibodies. β-actin serves as a protein loading marker. B, ErbB2 activation promotes metastasis in a spontaneous metastasis mouse model. Cells (1 × 106) were injected into the mammary fat pad of SCID mice, 15 mice per group. Lung metastases were examined at the end of the assay and the results were analyzed with Fisher's exact test. A P value <0.05, between mice injected with V659E and neo transfectants, was considered statistically significant. C, ErbB2 activation increases angiogenesis in the spontaneous metastasis mouse model. Tumor microvessel density (MVD) was determined in the 435, V659E, and K753M xenografts using anti-CD31 antibody. Three tumor sections were analyzed per mouse. The three most vascular sections per slide, observed under ×200 magnification, were selected for analysis in an area of 1.95 mm2. CD31+ structures were counted. Branching structures were counted as single vessels. Microvessel density was expressed as the average of number of microvessels per square millimeter. Columns, average value; bars, SD. P < 0.001, one-way ANOVA. D, ErbB2 activation promotes HUVEC cell migration. Serum-free conditioned medium was collected from the ErbB2 transfectants and used as a chemoattractant for HUVECs in Corning Costar Transwells for 6 hours. HUVECs were fixed, stained, and migrated cells were counted in six fields under ×200 magnification and averaged per well for three wells. *, P < 0.05; **, P < 0.01, one way ANOVA. Negative control (no conditioned medium) was serum-free DMEM/F12 medium. E, ErbB2 increases VEGF production. Serum-free conditioned medium from neo, V659E, and K753M transfectants were examined for VEGF production by ELISA. **, P < 0.01; ***, P < 0.001, one way ANOVA.

Figure 1.

ErbB2 increases metastasis and angiogenesis in vivo and in vitro. A, ErbB2 expression and tyrosine phosphorylation levels of MDA-MB-435 and ErbB2 transfectants. Western blotting on total cell lysates from MDA-MB-435 and ErbB2 transfectants with anti-ErbB2 and anti-phosphotyrosine antibodies. β-actin serves as a protein loading marker. B, ErbB2 activation promotes metastasis in a spontaneous metastasis mouse model. Cells (1 × 106) were injected into the mammary fat pad of SCID mice, 15 mice per group. Lung metastases were examined at the end of the assay and the results were analyzed with Fisher's exact test. A P value <0.05, between mice injected with V659E and neo transfectants, was considered statistically significant. C, ErbB2 activation increases angiogenesis in the spontaneous metastasis mouse model. Tumor microvessel density (MVD) was determined in the 435, V659E, and K753M xenografts using anti-CD31 antibody. Three tumor sections were analyzed per mouse. The three most vascular sections per slide, observed under ×200 magnification, were selected for analysis in an area of 1.95 mm2. CD31+ structures were counted. Branching structures were counted as single vessels. Microvessel density was expressed as the average of number of microvessels per square millimeter. Columns, average value; bars, SD. P < 0.001, one-way ANOVA. D, ErbB2 activation promotes HUVEC cell migration. Serum-free conditioned medium was collected from the ErbB2 transfectants and used as a chemoattractant for HUVECs in Corning Costar Transwells for 6 hours. HUVECs were fixed, stained, and migrated cells were counted in six fields under ×200 magnification and averaged per well for three wells. *, P < 0.05; **, P < 0.01, one way ANOVA. Negative control (no conditioned medium) was serum-free DMEM/F12 medium. E, ErbB2 increases VEGF production. Serum-free conditioned medium from neo, V659E, and K753M transfectants were examined for VEGF production by ELISA. **, P < 0.01; ***, P < 0.001, one way ANOVA.

Close modal

To determine whether ErbB2-mediated angiogenesis contributed to the ErbB2-mediated metastasis, we stained the mammary fat pad tumor xenografts with antibodies against CD31, a marker of blood vessels. The V659E xenografts had a significant increase in microvessel density when compared with K753M xenografts or the control xenografts (P < 0.001; Fig. 1C), indicating a connection between ErbB2-induced angiogenesis and metastasis in vivo.

We next investigated whether ErbB2 kinase activity regulates angiogenic properties in vitro, which could contribute to the angiogenic activity in vivo. We compared the effects of conditioned medium from the panel of ErbB2 transfectants on endothelial cell migration. We found significant increases in endothelial cell migration (P < 0.01) when HUVECs were stimulated with conditioned medium from the V659E transfectants compared with that from the K753M transfectants (Fig. 1D). These data suggest that the conditioned medium from the V659E transfectants contained angiogenic factors that induced endothelial cell migration, which may contribute to increased angiogenesis in vivo.

Because angiogenesis is an imbalance between proangiogenic and antiangiogenic factors (9), we next asked whether VEGF, the key activator of angiogenesis, can be up-regulated by ErbB2 and may be responsible for the increased angiogenesis in vivo and in vitro. We compared VEGF levels in conditioned medium from V659E, K753M, and the control cell line by ELISA. The V659E cells secreted significantly more VEGF than the control (P < 0.01) or K753M cells (P < 0.001; Fig. 1E), indicating that activation of ErbB2 kinase may have led to increased angiogenesis, at least partly, by up-regulating VEGF.

ErbB2 up-regulates VEGF at the protein synthesis level. Because ErbB2 kinase is involved in the up-regulation of VEGF, we next asked by what mechanism ErbB2 up-regulates VEGF. We examined whether ErbB2 up-regulates VEGF mRNA by measuring the steady-state level of VEGF mRNA in the ErbB2 transfectants and control cells because ErbB2 gene-transfected NIH3T3 mouse fibroblast cells up-regulated VEGF mRNA compared with untransfected NIH3T3 cells (37). We were surprised to find that no significant differences in the steady-state VEGF mRNA levels were detected among these human breast cancer cells expressing different levels of ErbB2 (Fig. 2A), suggesting that ErbB2 up-regulation of VEGF in these cells was not primarily at the mRNA level but at the protein level. Because there was no previous report on how ErbB2 up-regulates VEGF at the protein level, we investigated whether ErbB2 reduces VEGF protein degradation or increases VEGF protein translation. We first compared the VEGF protein stability in the V659E transfectants with that in the parental control. We treated the cells with cycloheximide to inhibit new protein synthesis and determined the VEGF levels in the serum-free conditioned medium at various time points for up to 72 hours. As expected, without cycloheximide inhibiting new VEGF synthesis, the level of secreted VEGF was dramatically higher in the V659E transfectants compared with the control cells. However, with cycloheximide inhibiting new VEGF synthesis, there was no significant difference in VEGF protein levels over time, regardless of the ErbB2 status of the cells (Fig. 2B). Therefore, the difference in VEGF protein levels between the V659E transfectants and the control cells was not due to differences in VEGF protein stability (Fig. 1E). We next investigated whether ErbB2 regulates VEGF protein translation. We labeled newly synthesized proteins in the control, V659E, and K753M cells with [35S]methionine and then detected newly synthesized VEGF in serum-free conditioned medium using radiolabeled VEGF ELISA or by immunoprecipitation of VEGF from lysates and conditioned medium followed by autoradiography of 35S-labeled VEGF in an SDS-PAGE gel. The V659E cell line had more newly synthesized VEGF than the parental, vector, and K753M cells (Fig. 2C and D), implicating a role for ErbB2 in up-regulating VEGF protein synthesis.

Figure 2.

ErbB2 up-regulates VEGF by increasing VEGF protein synthesis. A, similar RNA levels among MDA-MB-435, neo, and ErbB2 transfectants. RNA was collected from cells that had been incubated in serum-free, phenol red-free medium for 48 hours and Northern blot was done with a VEGF165 probe. Quantification was done by adding the values of both VEGF mRNA bands and normalizing them to GAPDH. The neo control line was arbitrarily defined as 1.0. B, VEGF is stable in ErbB2-low and ErbB2-active cells. Cells were grown in serum-free, phenol red-free medium and treated with 50 μg/mL cycloheximide (CHX) at 24 and 48 hours. Conditioned medium was collected and assayed for VEGF using ELISA. C, ErbB2 promotes VEGF protein synthesis. Cells were incubated in methionine-free medium for 4 hours, followed by incubation for 48 hours in [35S]methionine-labeled serum-free medium. VEGF was captured from this conditioned medium with 96-well plates coated with VEGF antibody. V659E transfectants had the highest cpm of newly synthesized [35S]methionine-labeled VEGF. Negative, negative control, [35S]methionine-labeled medium that had not been exposed to cells. D, protein synthesis assay using immunoprecipitation of VEGF from both lysates and conditioned medium of V659E and parental cells incubated in serum-free methionine-free medium for 4 hours followed by labeling with [35S]methionine for 4 hours (top). The SDS-PAGE gel was stained with Coomassie blue (bottom) to show protein loading.

Figure 2.

ErbB2 up-regulates VEGF by increasing VEGF protein synthesis. A, similar RNA levels among MDA-MB-435, neo, and ErbB2 transfectants. RNA was collected from cells that had been incubated in serum-free, phenol red-free medium for 48 hours and Northern blot was done with a VEGF165 probe. Quantification was done by adding the values of both VEGF mRNA bands and normalizing them to GAPDH. The neo control line was arbitrarily defined as 1.0. B, VEGF is stable in ErbB2-low and ErbB2-active cells. Cells were grown in serum-free, phenol red-free medium and treated with 50 μg/mL cycloheximide (CHX) at 24 and 48 hours. Conditioned medium was collected and assayed for VEGF using ELISA. C, ErbB2 promotes VEGF protein synthesis. Cells were incubated in methionine-free medium for 4 hours, followed by incubation for 48 hours in [35S]methionine-labeled serum-free medium. VEGF was captured from this conditioned medium with 96-well plates coated with VEGF antibody. V659E transfectants had the highest cpm of newly synthesized [35S]methionine-labeled VEGF. Negative, negative control, [35S]methionine-labeled medium that had not been exposed to cells. D, protein synthesis assay using immunoprecipitation of VEGF from both lysates and conditioned medium of V659E and parental cells incubated in serum-free methionine-free medium for 4 hours followed by labeling with [35S]methionine for 4 hours (top). The SDS-PAGE gel was stained with Coomassie blue (bottom) to show protein loading.

Close modal

ErbB2-mediated up-regulation of VEGF involves activation of mTOR and p70S6K signaling. To uncover the molecular mechanisms for increased VEGF translation by ErbB2, we focused on signaling events that converge on p70S6K, a known stimulator of protein synthesis. First, we asked whether ErbB2 overexpression could lead to increased p70S6K-T389 phosphorylation (p70S6K-T389-P), which indicates p70S6K activity (25). Western blot with antibody for T389 phosphorylated p70S6K showed that V659E transfectants possessed increased p70S6K-T389-P compared with control and K753M transfectants (Fig. 3A). Conversely, p70S6K-T389-P was reduced when ErbB2 was inhibited by RNA interference in the BT474 breast cancer cell line that overexpresses endogenous ErbB2 (data not shown). Together, these data indicated that overexpression of activated ErbB2 leads to increased p70S6K-T389-P, an indication of increased mTOR activity (25, 26).

Figure 3.

Activation of P70S6K by ErbB2 is involved in ErbB2-mediated up-regulation of VEGF. A, ErbB2 activation leads to increased p70S6K phosphorylation. Western blot of cell lysates with antibodies recognizing phosphorylated p70S6K-T389. B, rapamycin inhibits p70S6K phosphorylation in MDA-MB-435, V659E, and K753M cells. Western blot of cell lysates from 435, V659E, and K753M cells treated for 2 hours with 10 nmol/L rapamycin or methanol as solvent control in serum-free medium. C, wortmannin reduced p70S6K phosphorylation in V659E cells. Western blot of cell lysates from 435, V659E, and K753M cells treated for 2 hours with 50 nmol/L wortmannin or DMSO as solvent control in serum-free medium. D, PD98059 reduced p70S6K phosphorylation in MDA-MB-435 and K753M cells but increased p70S6K phosphorylation in V659E cells. Western blot analysis of MDA-MB-435, V659E, and K753M cells treated with 50 μmol/L of the MEK inhibitor PD98059. E, rapamycin and PD98059 reduced VEGF production in V659E, MDA-MB-435, and K753M cells. VEGF ELISA of conditioned medium from 435, V659E, and K753M cells treated with 10 nmol/L rapamycin (RP) or 50 μmol/L PD98059 (PD) for 48 hours or with 50 μmol/L LY294002 (LY) twice in 48 hours. LY294002 was used in place of wortmannin because wortmannin has a very short half-life in tissue culture (∼30 minutes; ref. 50). DMSO (DM) was administered as vehicle control. Conditioned medium from three independent tissue culture plates was assayed for each treatment group. One-way ANOVA was done to look for statistically significant differences. Within each cell type (435, V659E, and K753M), the inhibitor-treated cells were compared with the DMSO-treated control group using the Bonferroni method for multiple comparisons. **, P < 0.01, statistically significant.

Figure 3.

Activation of P70S6K by ErbB2 is involved in ErbB2-mediated up-regulation of VEGF. A, ErbB2 activation leads to increased p70S6K phosphorylation. Western blot of cell lysates with antibodies recognizing phosphorylated p70S6K-T389. B, rapamycin inhibits p70S6K phosphorylation in MDA-MB-435, V659E, and K753M cells. Western blot of cell lysates from 435, V659E, and K753M cells treated for 2 hours with 10 nmol/L rapamycin or methanol as solvent control in serum-free medium. C, wortmannin reduced p70S6K phosphorylation in V659E cells. Western blot of cell lysates from 435, V659E, and K753M cells treated for 2 hours with 50 nmol/L wortmannin or DMSO as solvent control in serum-free medium. D, PD98059 reduced p70S6K phosphorylation in MDA-MB-435 and K753M cells but increased p70S6K phosphorylation in V659E cells. Western blot analysis of MDA-MB-435, V659E, and K753M cells treated with 50 μmol/L of the MEK inhibitor PD98059. E, rapamycin and PD98059 reduced VEGF production in V659E, MDA-MB-435, and K753M cells. VEGF ELISA of conditioned medium from 435, V659E, and K753M cells treated with 10 nmol/L rapamycin (RP) or 50 μmol/L PD98059 (PD) for 48 hours or with 50 μmol/L LY294002 (LY) twice in 48 hours. LY294002 was used in place of wortmannin because wortmannin has a very short half-life in tissue culture (∼30 minutes; ref. 50). DMSO (DM) was administered as vehicle control. Conditioned medium from three independent tissue culture plates was assayed for each treatment group. One-way ANOVA was done to look for statistically significant differences. Within each cell type (435, V659E, and K753M), the inhibitor-treated cells were compared with the DMSO-treated control group using the Bonferroni method for multiple comparisons. **, P < 0.01, statistically significant.

Close modal

To investigate whether ErbB2 regulates VEGF through p70S6K-T389-P, we inhibited the p70S6K upstream kinase mTOR with a specific inhibitor, rapamycin. Rapamycin effectively reduced p70S6K-T389-P but did not notably inhibit phosphorylations of Akt, tuberin, and ERK (Fig. 3B; data not shown). Rapamycin also significantly reduced VEGF levels in the conditioned medium from all three cell lines under serum-free conditions (Fig. 3E). Thus, ErbB2-mediated activation of mTOR and p70S6K contributes to VEGF up-regulation in these human breast cancer cells.

Both Akt and ERK are up-regulated by ErbB2 (38) and both are known upstream stimulators of mTOR/p70S6K (20, 21, 23, 24). Akt activates p70S6K by inhibiting tuberin through phosphorylation on T1462 that allows mTOR activation (see Fig. 6; ref. 21). ERK is known to activate mTOR through inhibition of tuberin in embryonic kidney cells (39) and induce p70S6K activation (23). Therefore, we examined whether Akt and ERK may contribute to ErbB2-mediated activation of p70S6K by comparing between the low ErbB2-expressing MDA-MB-435, the kinase-active ErbB2 (V659E), and the kinase-dead ErbB2 (K753M) transfectants for the activating phosphorylations on Akt-S473, ERK-T202/Y204, and inactivating phosphorylation on tuberin-T1462. As expected, Akt-S473, tuberin-T1462, and ERK-T202/Y204 were highly phosphorylated in V659E cells compared with both the parental breast cancer cells and the K753M transfectants (Fig. 3B,, C, and D). Interestingly, rapamycin-treated V659E cells showed increased Akt-S473 phosphorylation, which was not seen in the parental or K753M cells, suggesting feedback inhibition of Akt from mTOR/p70S6K in V659E cells (Fig. 3B; data not shown). This may partially be responsible for the less effective VEGF inhibition by rapamycin in V659E cells (35% inhibition in V659E compared with ∼50% inhibition in 435 and K753M; Fig. 3E). Activated Akt in V659E cells may allow VEGF translation activity through downstream signaling events other than p70S6K (40). This ErbB2-dependent feedback loop is consistent the findings in a recent report that showed a PI3K-dependent increase in AKT activation following rapamycin treatment of lung cancer cell lines (41).

To further explore p70S6K regulation by PI3K/Akt in V659E cells versus that in the parental cells and kinase-dead K753M transfectants, cells were treated with 50 nmol/L wortmannin to inhibit PI3K. Wortmannin dramatically inhibited Akt-S473, p70S6K-T389, and tuberin-T1462 phosphorylations in V659E cells almost to the basal levels of the untreated parental and K753M cells (Fig. 3C). Wortmannin also inhibited p70S6K-T389-P in the parental or kinase-dead cells but the magnitude of the decrease was less than in the kinase-active cells (Fig. 3C). However, surprisingly, the PI3K inhibitor LY294002 did not inhibit VEGF production in V659E cells (Fig. 3E) despite effective inhibition of Akt and p70S6K phosphorylation (data not shown) and effective inhibition of VEGF production in the 435 and K753M cells (Fig. 3E). Thus, ErbB2-activated cells must have an additional PI3K/Akt-independent means of up-regulating VEGF production and pathways other than PI3K/Akt may play a substantial role in regulating VEGF production in ErbB2-activated breast cancer cells.

We next examined the role of ERK in p70S6K activation in these breast cancer cells by treating cells with 50 μmol/L PD98059 to inhibit MAP/ERK kinase (MEK), the activating kinase for ERK (42). In parental cells and ErbB2 kinase-dead cells (K753M), MEK inhibition abolished ERK-T202/Y204 phosphorylation that corresponded to inhibition of p70S6K-T389-P (Fig. 3D). In V659E cells, 50 μmol/L PD98059 reduced ERK-T202/Y204 phosphorylation but did not abolish ERK-T202/Y204 phosphorylation as in the 435 parental and K753M kinase-dead cells. To our surprise, compared with untreated V659E cells, PD98059-treated V659E cells had an increased p70S6K-T389-P, which paralleled with increased phosphorylations on Akt-S473 and tuberin-T1462 (Fig. 3D). Similar up-regulation in phosphorylation was also observed with a dominant negative mutant of ERK (data not shown). This suggests a possible negative regulation of Akt/tuberin/p70S6K by the highly activated ERK pathway in V659E cells (Figs. 3D and 6). Interestingly, ERK inhibition by PD98059 was able to reduce VEGF production in all three cell lines (Fig. 3E), indicating that ERK also contributes to VEGF production independent of Akt/mTOR/p70S6K, particularly in the V659E cell line (Fig. 3E). Taken together, the data indicate that ERK, Akt, and mTOR/p70S6K act as downstream mediators of ErbB2 and play major, albeit complex, roles in the regulation of VEGF production.

Activation of Akt/mTOR/p70S6K protein synthesis pathway correlates with ErbB2 overexpression and VEGF up-regulation in vivo. We next examined the mouse mammary fat pad tumor xenografts from the spontaneous metastasis assay (Fig. 1B and C) to validate the in vitro findings in vivo and investigate the role of the Akt/mTOR/p70S6K signaling pathway in regulating VEGF in vivo. Xenografts were stained with antibodies against ErbB2, phosphorylated Akt-S473, phosphorylated p70S6K-T389, and VEGF. Compared with the xenografts from mice injected with the ErbB2 low-expressing MDA-MB-435 cells, the V659E xenografts had strong ErbB2 staining, as expected, and also had significantly higher levels of phosphorylated Akt-S473 (P < 0.05), phosphorylated p70S6K-T389 (P < 0.001), and increased VEGF (P < 0.05; Fig. 4A and B), which correlated with their increased angiogenesis (Fig. 1C). The above data confirms the importance of PI3K/Akt and mTOR/p70S6K signaling in ErbB2-mediated VEGF up-regulation and angiogenesis in vivo.

Figure 4.

Significantly stronger Akt-S473-P, p70S6K-T389-P, and VEGF levels in ErbB2-activated V659E xenografts than in ErbB2 low-expressing xenografts (MDA-MB-435). A, xenografts of V659E cells had strong membrane staining of ErbB2 and more intense staining of phosphorylated p70S6K-T389, phosphorylated Akt-S473, and VEGF than the parental 435 cell line. Representative results of staining with indicated antibodies. B, immunostaining and statistical analysis of phosphorylated Akt and p70S6K, and VEGF levels between the MDA-MB-435 and V659E xenografts. Analysis of immunohistochemistry was graded on a 0 to 2 intensity scale: membrane ErbB2 (0, negative: no staining or faint incomplete membrane staining; 1, weak positive: weak to moderate complete membrane staining in 10% of tumor cells; 2, strong positive: complete and strong membrane staining in more that 10% of tumor cells.) Phospho-p70S6K-T389 (0, <10% of cells with weak staining; 1, tumors with >10% cells having weak staining or <20% cells with strong staining; 2, tumors with >20% of cells with strong staining). Akt-S473-P and VEGF (0, no staining; 1, weak staining or strong staining is <50% of cells; 2, strong staining in >50% of cells). Statistical analyses were done with Mann-Whitney tests, and P < 0.05 was considered statistically significant.

Figure 4.

Significantly stronger Akt-S473-P, p70S6K-T389-P, and VEGF levels in ErbB2-activated V659E xenografts than in ErbB2 low-expressing xenografts (MDA-MB-435). A, xenografts of V659E cells had strong membrane staining of ErbB2 and more intense staining of phosphorylated p70S6K-T389, phosphorylated Akt-S473, and VEGF than the parental 435 cell line. Representative results of staining with indicated antibodies. B, immunostaining and statistical analysis of phosphorylated Akt and p70S6K, and VEGF levels between the MDA-MB-435 and V659E xenografts. Analysis of immunohistochemistry was graded on a 0 to 2 intensity scale: membrane ErbB2 (0, negative: no staining or faint incomplete membrane staining; 1, weak positive: weak to moderate complete membrane staining in 10% of tumor cells; 2, strong positive: complete and strong membrane staining in more that 10% of tumor cells.) Phospho-p70S6K-T389 (0, <10% of cells with weak staining; 1, tumors with >10% cells having weak staining or <20% cells with strong staining; 2, tumors with >20% of cells with strong staining). Akt-S473-P and VEGF (0, no staining; 1, weak staining or strong staining is <50% of cells; 2, strong staining in >50% of cells). Statistical analyses were done with Mann-Whitney tests, and P < 0.05 was considered statistically significant.

Close modal

The most critical question is whether Akt/mTOR/p70S6K signaling is important for VEGF up-regulation in ErbB2-overexpressing breast tumors from patients. To address this clinically important issue, we immunohistochemically detected phosphorylated p70S6K-T389 in 155 primary breast tumor samples that had also been stained for ErbB2, phosphorylated Akt-S473, phosphorylated mTOR-S2448 (a PI3K/Akt phosphorylation site), and VEGF. Out of the 155 breast tumors, 46% had “strong” p70S6K-T389-P staining (ranked as 2 on a 0-2 scale; data not shown). ErbB2 overexpression was previously correlated with VEGF expression in these breast cancers (14) and phosphorylated Akt and phosphorylated mTOR correlated with p70S6K-T389-P (36). Here, we further investigated whether ErbB2 overexpression correlated with p70S6K activation that contributes to increased VEGF in human breast cancers. Indeed, we found that ErbB2 overexpression significantly correlated with p70S6K-T389-P (P < 0.01; Fig. 5A and B) and p70S6K-T389-P correlated with increased VEGF (P < 0.05). Additionally, Akt-S473-P and mTOR-S2448-P significantly correlated with VEGF expression in these same human breast tumor samples (P < 0.05 and P < 0.01, respectively; Fig. 5A). Notably, we detected a trend for reduced disease-free survival in these patients whose tumors had high p70S6K-T389-P levels, although it did not reach statistical significance (Fig. 5C; P = 0.06). However, combination of ErbB2 overexpression and high p70S6K-T389-P levels in patients' tumors is significantly associated with reduced disease-free survival of patients compared with those whose tumors had low levels of both ErbB2 and p70S6K-T389-P (P < 0.01; Fig. 5D). Because most patients who die from breast cancer die from metastases, we compared the patients' incidence of metastases with their tumor p70S6K-T389-P levels. We found that patients whose tumors had high levels of p70S6K-T389-P were more likely to develop metastases (P < 0.05; Fig. 5E), indicating that p70S6K-T389-P may predict patient's metastatic incidence. Together, our data clearly showed the importance of the mTOR/p70S6K protein translation signals in ErbB2-mediated VEGF up-regulation and metastasis in these breast cancer patients.

Figure 5.

ErbB2 overexpression correlates with p70S6K-T389 phosphorylation levels, and higher p70S6K-T389-P levels in patients' tumors correlate with decreased disease-free survival and increased metastasis. A, 155 human breast tumor samples were collected from FuDan University in Shanghai, China. Tumor samples were stained with antibodies to ErbB2, p70S6K-T389-P, Akt-S473-P, mTOR-S2448-P, and VEGF. The staining results were graded and scored as in Fig 4. ErbB2 was detected with the DAKO Hercep Test and scored similarly to the xenografts. VEGF was quantitated with a SAMBA 4000 image analyzer. VEGF expression was analyzed in nine random fields and a numerical value was given based on the percentage of immunostained surfaces and the mean absorbance, which measured the staining intensity. P < 0.05, as calculated with Fisher's exact test, was considered statistically significant. B, immunohistochemistry representing the correlation of ErbB2 overexpression with high p70S6K-T389-P levels in patient tumors. C, the Kaplan-Meier method was used to determine differences in disease-free survival between patients whose tumors expressed low levels of phosphorylated p70S6K-T389 (0, low level; 1, intermediate level) compared with patients whose tumors expressed high levels of phosphorylated p70S6K (2, high level). E/N, event (disease recurrence)/number of total subjects. D, the Kaplan-Meier method was used to determine whether ErbB2 overexpression combined with high p70S6K-T389-P levels could predict patient disease-free survival after surgery. Patients with both high levels of ErbB2 and p70S6K (line 4) had highly significantly reduced survival compared with patients with both low levels of ErbB2 and p70S6K-T389-P (line 1; P < 0.01). High ErbB2 levels alone (line 3) also predicted worse survival than low levels of both ErbB2 and p70S6K-T389-P (P < 0.05). P values were calculated with Fisher's exact test. E, high levels of p70S6K-T389 phosphorylation in patient tumors correlated with increased incidence of metastasis. Fisher's exact test was used to determine if there was a correlation between p70S6K-T389 levels and the incidence of metastasis after surgery in these 155 patients. P < 0.05 was considered statistically significant.

Figure 5.

ErbB2 overexpression correlates with p70S6K-T389 phosphorylation levels, and higher p70S6K-T389-P levels in patients' tumors correlate with decreased disease-free survival and increased metastasis. A, 155 human breast tumor samples were collected from FuDan University in Shanghai, China. Tumor samples were stained with antibodies to ErbB2, p70S6K-T389-P, Akt-S473-P, mTOR-S2448-P, and VEGF. The staining results were graded and scored as in Fig 4. ErbB2 was detected with the DAKO Hercep Test and scored similarly to the xenografts. VEGF was quantitated with a SAMBA 4000 image analyzer. VEGF expression was analyzed in nine random fields and a numerical value was given based on the percentage of immunostained surfaces and the mean absorbance, which measured the staining intensity. P < 0.05, as calculated with Fisher's exact test, was considered statistically significant. B, immunohistochemistry representing the correlation of ErbB2 overexpression with high p70S6K-T389-P levels in patient tumors. C, the Kaplan-Meier method was used to determine differences in disease-free survival between patients whose tumors expressed low levels of phosphorylated p70S6K-T389 (0, low level; 1, intermediate level) compared with patients whose tumors expressed high levels of phosphorylated p70S6K (2, high level). E/N, event (disease recurrence)/number of total subjects. D, the Kaplan-Meier method was used to determine whether ErbB2 overexpression combined with high p70S6K-T389-P levels could predict patient disease-free survival after surgery. Patients with both high levels of ErbB2 and p70S6K (line 4) had highly significantly reduced survival compared with patients with both low levels of ErbB2 and p70S6K-T389-P (line 1; P < 0.01). High ErbB2 levels alone (line 3) also predicted worse survival than low levels of both ErbB2 and p70S6K-T389-P (P < 0.05). P values were calculated with Fisher's exact test. E, high levels of p70S6K-T389 phosphorylation in patient tumors correlated with increased incidence of metastasis. Fisher's exact test was used to determine if there was a correlation between p70S6K-T389 levels and the incidence of metastasis after surgery in these 155 patients. P < 0.05 was considered statistically significant.

Close modal

Here, we report data from cultured human breast cancer cell lines, a mouse spontaneous metastasis model, and tumors from breast cancer patients that show enhanced angiogenesis by the ErbB2 kinase through increased protein translation of VEGF that is mainly due to activation of mTOR/p70S6K. To study ErbB2 downstream signaling pathways that contribute to ErbB2-mediated up-regulation of VEGF, we have generated a panel of isogenic stable ErbB2 transfectants in the MDA-MB-435 human breast cancer cell line that expresses ErbB2 at levels similar to or lower than that in ErbB2-overexpressing breast tumors from patients (Fig. 1A; ref. 5). Thus, differences in signaling and biological properties can be mostly attributed to differences in ErbB2 kinase activity and findings should be relevant to breast cancer patients. Indeed, when these ErbB2 transfectants were injected into the mammary fat pad of SCID mice, the tumor biology mimics the cancer biology observed in tumors of breast cancer patients. The V659E transfectant expressing the kinase constitutively active ErbB2 mutant had a similar trend in promoting angiogenesis and metastasis as the eB1 transfectant expressing the wild-type ErbB2. Because the V659E transfectant had more marked biological effects that provided a bigger window for monitoring the downstream signaling events, we used the V659E transfectant in most of our experiments. Our data from these experimental systems provided tangible links in the biological phenotypes between in vitro tissue culture of the ErbB2 transfectants of breast cancer cells, to the mouse mammary fat pad xenograft model, and finally to ErbB2 overexpressing human breast tumors.

ErbB2 has been linked to the transcriptional up-regulation of VEGF in mouse NIH3T3 fibroblast cells that overexpress ErbB2 (37) and in human breast cancer cells treated with heregulin (13); thus, we were surprised to discover that ErbB2 overexpression and activation per se did not alter the mRNA levels of VEGF compared with ErbB2-low-expressing parental cells or the kinase-dead transfectants under serum-free conditions. Instead, ErbB2 increased the protein translation of VEGF. This is the first report that ErbB2 up-regulates VEGF at the protein translation level. We have found that the ErbB2-overexpressing cells with increased VEGF translation had an elevated inhibitory phosphorylation on T1462 of tuberin, consistent with a previous report that VEGF protein translation is up-regulated in tuberous sclerosis complex-2 (tuberin) null mouse embryo fibroblasts (43). The elevated inhibitory phosphorylation on tuburin-T1462 provides a link between ErbB2-mediated PI3K/Akt activation (indicated by S473 phosphorylation) and mTOR/p70S6K activation (indicated by p70S6K-T389 phosphorylation) that contributes to increased VEGF translation. The strong correlation between ErbB2 overexpression and p70S6K-T389 phosphorylation in patient samples support the notion that ErbB2 can exert its oncogenic function through up-regulation of protein translation.

Our data indicated that ErbB2 low-expressing MDA-MB-435 parental cells and kinase-dead ErbB2 transfectants depended on both ERK and PI3K/Akt to activate mTOR and p70S6K to promote VEGF translation (Fig. 6, left). Both pathways are more highly activated in ErbB2-overexpressing V659E cells than in the parental cells and up-regulation of VEGF translation through mTOR and p70S6K in V659E cells may to come from the superactivations of both pathways (Fig. 6, right). The data also suggest a novel pathway that regulates VEGF translation, independent of mTOR/p70S6K in the erbB2-overepressing V659E cells. ERK can stimulate rp-S6 phosphorylation independently of p70S6K (44) and therefore may be a major mediator of VEGF translation in these cells as well. Clearly, the role of these p70S6K-independent pathways in VEGF regulation in the erbB2-activated breast cancer cells is an important area for future investigation.

Figure 6.

Signaling networks leading to VEGF translation in ErbB2 low- versus high-expressing breast cancer cells. Left, in the ErbB2 low-expressing MDA-MB-435 breast cancer cells, the activation of mTOR/p70S6K contributes to VEGF protein expression and inhibition of mTOR/p70S6K with rapamycin-decreased p70S6K-T389 and VEGF secretion. Right, in the ErbB2 kinase-active V659E cells, both PI3K/Akt and MEK/ERK signaling pathways were more highly activated than in the MDA-MB-435 cells and can contribute to the activation of mTOR/p70S6K. Inhibition of mTOR/p70S6K with rapamycin in V659E cells decreased p70S6K-T389-P and partially inhibited VEGF protein secretion. When mTOR/p70S6K was inhibited by rapamycin, Akt phosphorylation levels increased, suggesting a feedback inhibition of Akt from mTOR/p70S6K. Inhibition of PI3K with wortmannin effectively decreased Akt-S473-P, tuberin-T1462-P, and p70S6K-T389-P, whereas inhibition of MEK/ERK by PD98059 led to increased p70S6K-T389-P, tuberin-T1462-P, and Akt-S473-P. These data indicated that the highly activated ERK may inhibit Akt/mTOR/p70S6K.

Figure 6.

Signaling networks leading to VEGF translation in ErbB2 low- versus high-expressing breast cancer cells. Left, in the ErbB2 low-expressing MDA-MB-435 breast cancer cells, the activation of mTOR/p70S6K contributes to VEGF protein expression and inhibition of mTOR/p70S6K with rapamycin-decreased p70S6K-T389 and VEGF secretion. Right, in the ErbB2 kinase-active V659E cells, both PI3K/Akt and MEK/ERK signaling pathways were more highly activated than in the MDA-MB-435 cells and can contribute to the activation of mTOR/p70S6K. Inhibition of mTOR/p70S6K with rapamycin in V659E cells decreased p70S6K-T389-P and partially inhibited VEGF protein secretion. When mTOR/p70S6K was inhibited by rapamycin, Akt phosphorylation levels increased, suggesting a feedback inhibition of Akt from mTOR/p70S6K. Inhibition of PI3K with wortmannin effectively decreased Akt-S473-P, tuberin-T1462-P, and p70S6K-T389-P, whereas inhibition of MEK/ERK by PD98059 led to increased p70S6K-T389-P, tuberin-T1462-P, and Akt-S473-P. These data indicated that the highly activated ERK may inhibit Akt/mTOR/p70S6K.

Close modal

Previously, it had been suggested that there may be a negative regulatory loop of Akt activity (41, 45, 46). Recently, Akt has been discovered to be phosphorylated on S473 by the mTOR/rictor complex (47). Now, we found that inhibition of mTOR/p70S6K by rapamycin resulted in an increased Akt-S473 phosphorylation in V659E cells but not in the parental cells (Fig. 3C; data not shown). This suggested that a feedback inhibition of the highly activated Akt from mTOR/p70S6K functions primarily in the V659E cells (Fig. 6, right). Furthermore, ERK inhibition in V659E cells by PD98059 increased p70S6K-T389-P compared with untreated V659E cells, which paralleled with increased phosphorylations on Akt-S473 and tuberin-T1462 (Fig. 3D). This suggested a possible negative regulation of Akt-mTOR/p70S6K by the highly activated ERK pathway in V659E cells (Figs. 3D and 6, right). This negative regulation of Akt-mTOR/p70S6K by the highly activated ERK has never been reported previously and was not seen in the parental or kinase-dead ErbB2 cells, suggesting that it may come from ErbB2-related signals, or specifically, the superactivated ERK resulting from constitutively activated ErbB2.

The complexity of the signaling involved in ErbB2-mediated VEGF translation is manifested by the findings that in ErbB2-overexpressing cells, rapamycin, LY294002, and PD98059 did not reduce VEGF levels in conditioned medium to a level similar to that seen in low ErbB2 expression or kinase-dead ErbB2 (Fig. 3E). This indicated that there are other as yet undefined ErbB2-mediated activating signals for VEGF protein synthesis. Future studies are required to reveal these other signaling pathways.

Blocking protein synthesis is a strategy currently under intensive clinical investigation for cancer treatment. CCI-779, RAD001, and AP23573, rapamycin analogues, are all at various stages of clinical trials (48). The phase II studies on CCI-779 in breast and renal cancers have recently been completed and is expected to proceed to phase III clinical trials (48). The antiangiogenic properties of mTOR inhibitors have also been shown in endothelial cells (49). Our data showed that rapamycin has antiangiogenic effects in breast cancer cells in vitro as a consequence of inhibiting the ability of ErbB2 to up-regulate VEGF protein translation. This further supports the notion that targeting ErbB2-mediated VEGF protein synthesis by inhibiting the Akt/mTOR/p70S6K pathway may have potential for inhibiting angiogenesis. On the other hand, our studies show that inhibiting mTOR with rapamycin in our breast cancer cell lines overexpressing ErbB2 results in increased Akt phosphorylation, which may affect other components of the Akt-related signaling networks. Thus, the use of rapamycin analogues needs to be more carefully evaluated. Future studies along this line will help to develop tailored cancer therapies specifically targeting deregulated signaling networks from ErbB2 activation in breast cancer cells.

Note: X. Zhou and W. Yang are currently in Shanghai Cancer Hospital, Fudan University, Shanghai, China.

Grant support: Cancer Center Core grant P30-CA 16672; NIH grants 2RO1-CA60448, 1RO1-CA109570, and PO1-CA099031 project 4 (D. Yu); and U.S. Army Research and Material Command grant DAMD 17-02-1-0462 (D. Yu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Pu Liu and Dawn R. Christianson for technical assistance, Dr. Juri Gelovani for use of the Perkin-Elmer TopCount NXT, Don Norwood for editorial assistance, and Kristine R. Broglio for statistical assistance.

1
Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science
1989
;
244
:
707
–12.
2
Yamamoto T, Ikawa S, Akiyama T, et al. Similarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor.
Nature
1986
;
319
:
230
–4.
3
Bargmann CI, Weinberg RA. Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene.
Proc Natl Acad Sci U S A
1988
;
85
:
5394
–8.
4
Stern DF, Kamps MP, Cao H. Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo.
Mol Cell Biol
1988
;
8
:
3969
–73.
5
Yu D, Liu B, Tan M, Li J, Wang SS, Hung MC. Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to Taxol via mdr-1-independent mechanisms.
Oncogene
1996
;
13
:
1359
–65.
6
Tan M, Yao J, Yu D. Overexpression of the c-erbB-2 gene enhanced intrinsic metastasis potential in human breast cancer cells without increasing their transformation abilities.
Cancer Res
1997
;
57
:
1199
–205.
7
Folkman J. Role of angiogenesis in tumor growth and metastasis.
Semin Oncol
2002
;
29
:
15
–8.
8
Blackwell KL, Dewhirst MW, Liotcheva V, et al. HER-2 gene amplification correlates with higher levels of angiogenesis and lower levels of hypoxia in primary breast tumors.
Clin Cancer Res
2004
;
10
:
4083
–8.
9
Iruela-Arispe ML, Dvorak HF. Angiogenesis: a dynamic balance of stimulators and inhibitors.
Thromb Haemost
1997
;
78
:
672
–7.
10
Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor.
Endocr Rev
1997
;
18
:
4
–25.
11
Toi M, Inada K, Suzuki H, Tominaga T. Tumor angiogenesis in breast cancer: its importance as a prognostic indicator and the association with vascular endothelial growth factor expression.
Breast Cancer Res Treat
1995
;
36
:
193
–204.
12
Gasparini G, Toi M, Gion M, et al. Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma.
J Natl Cancer Inst
1997
;
89
:
139
–47.
13
Yen L, You XL, Al Moustafa AE, et al. Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis.
Oncogene
2000
;
19
:
3460
–9.
14
Yang W, Klos K, Yang Y, Smith TL, Shi D, Yu D. ErbB2 overexpression correlates with increased expression of vascular endothelial growth factors A, C, and D in human breast carcinoma.
Cancer
2002
;
94
:
2855
–61.
15
Yen L, Benlimame N, Nie ZR, et al. Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers.
Mol Biol Cell
2002
;
13
:
4029
–44.
16
Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G. Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k.
EMBO J
1997
;
16
:
3693
–704.
17
von Manteuffel SR, Dennis PB, Pullen N, Gingras AC, Sonenberg N, Thomas G. The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k.
Mol Cell Biol
1997
;
17
:
5426
–36.
18
Meyuhas O. Synthesis of the translational apparatus is regulated at the translational level.
Eur J Biochem
2000
;
267
:
6321
–30.
19
Chung J, Kuo CJ, Crabtree GR, Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.
Cell
1992
;
69
:
1227
–36.
20
Sekulic A, Hudson CC, Homme JL, et al. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.
Cancer Res
2000
;
60
:
3504
–13.
21
Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.
Nat Cell Biol
2002
;
4
:
648
–57.
22
Pullen N, Dennis PB, Andjelkovic M, et al. Phosphorylation and activation of p70s6k by PDK1.
Science
1998
;
279
:
707
–10.
23
Lehman JA, Gomez-Cambronero J. Molecular crosstalk between p70S6k and MAPK cell signaling pathways.
Biochem Biophys Res Commun
2002
;
293
:
463
–9.
24
Kelleher RJ, III, Govindarajan A, Jung HY, Kang H, Tonegawa S. Translational control by MAPK signaling in long-term synaptic plasticity and memory.
Cell
2004
;
116
:
467
–79.
25
Pearson RB, Dennis PB, Han JW, et al. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.
EMBO J
1995
;
14
:
5279
–87.
26
Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB, Thomas G. Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site.
J Biol Chem
2002
;
277
:
20104
–12.
27
Holland EC, Sonenberg N, Pandolfi PP, Thomas G. Signaling control of mRNA translation in cancer pathogenesis.
Oncogene
2004
;
23
:
3138
–44.
28
Hay N, Sonenberg N. Upstream and downstream of mTOR.
Genes Dev
2004
;
18
:
1926
–45.
29
Sellappan S, Grijalva R, Zhou X, et al. Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line.
Cancer Res
2004
;
64
:
3479
–85.
30
Tan M, Li P, Klos KS, et al. ErbB2 promotes Src synthesis and stability: novel mechanisms of Src activation that confer breast cancer metastasis.
Cancer Res
2005
;
65
:
1858
–67.
31
Akiyama T, Matsuda S, Namba Y, Saito T, Toyoshima K, Yamamoto T. The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain.
Mol Cell Biol
1991
;
11
:
833
–42.
32
Tan M, Jing T, Lan KH, et al. Phosphorylation on tyrosine-15 of p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance to Taxol-induced apoptosis.
Mol Cell
2002
;
9
:
993
–1004.
33
Wexler H. Accurate identification of experimental pulmonary metastases.
J Natl Cancer Inst
1966
;
36
:
641
–5.
34
Lee S, Yang W, Lan KH, et al. Enhanced sensitization to Taxol-induced apoptosis by herceptin pretreatment in ErbB2-overexpressing breast cancer cells.
Cancer Res
2002
;
62
:
5703
–10.
35
Ausubel FM. Current protocols in molecular biology, p. 2 v. (loose-leaf). Brooklyn (New York): Greene Publishing Associates; J. Wiley; 1987.
36
Zhou X, Tan M, Stone Hawthorne V, et al. Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers.
Clin Cancer Res
2004
;
10
:
6779
–88.
37
Laughner E, Taghavi P, Chiles K, Mahon PC, Semenza GL. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1α (HIF-1α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.
Mol Cell Biol
2001
;
21
:
3995
–4004.
38
Zhou BP, Hung MC. Dysregulation of cellular signaling by HER2/neu in breast cancer.
Semin Oncol
2003
;
30
:
38
–48.
39
Tee AR, Anjum R, Blenis J. Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin.
J Biol Chem
2003
;
278
:
37288
–96.
40
Dufner A, Andjelkovic M, Burgering BM, Hemmings BA, Thomas G. Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation.
Mol Cell Biol
1999
;
19
:
4525
–34.
41
Sun SY, Rosenberg LM, Wang X, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition.
Cancer Res
2005
;
65
:
7052
–8.
42
Sebolt-Leopold JS. Development of anticancer drugs targeting the MAP kinase pathway.
Oncogene
2000
;
19
:
6594
–9.
43
Brugarolas JB, Vazquez F, Reddy A, Sellers WR, Kaelin WG, Jr. TSC2 regulates VEGF through mTOR-dependent and -independent pathways.
Cancer Cell
2003
;
4
:
147
–58.
44
Pende M, Um SH, Mieulet V, et al. S6K1(−/−)/S6K2(−/−) mice exhibit perinatal lethality and rapamycin-sensitive 5′-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway.
Mol Cell Biol
2004
;
24
:
3112
–24.
45
Garami A, Zwartkruis FJ, Nobukuni T, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.
Mol Cell
2003
;
11
:
1457
–66.
46
Radimerski T, Montagne J, Hemmings-Mieszczak M, Thomas G. Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling.
Genes Dev
2002
;
16
:
2627
–32.
47
Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
Science
2005
;
307
:
1098
–101.
48
Chan S. Targeting the mammalian target of rapamycin (mTOR): a new approach to treating cancer.
Br J Cancer
2004
;
91
:
1420
–4.
49
Sawyers CL. Will mTOR inhibitors make it as cancer drugs?
Cancer Cell
2003
;
4
:
343
–8.
50
Balciunaite E, Kazlauskas A. Early phosphoinositide 3-kinase activity is required for late activation of protein kinase Cϵ in platelet-derived-growth-factor-stimulated cells: evidence for signalling across a large temporal gap.
Biochem J
2001
;
358
:
281
–5.