The Antitumor and Antiangiogenic Activity of Vascular Endothelial

Cancer Therapy: Preclinical

The Antitumor and Antiangiogenic Activity of Vascular Endothelial Growth Factor Receptor Inhibition Is Potentiated by ErbB1Blockade Patrizia Sini,1, 2 Lorenza Wyder,1Christian Schnell,1 Terence O’Reilly,1Amanda Littlewood,1 Ralph Brandt,3 Nancy E. Hynes,2 andJeanette Wood1

Abstract Purpose: Receptor tyrosine kinases of the ErbB family play important roles in the controlof tumor growth. Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation, enhan- ces vascular permeability, and plays an important role in tumor vascularization.We evaluated the effects of selectiveVEGFreceptor (VEGFR; PTK787/ZK222584) and ErbB (PKI166 and ZD1839) inhibitors on tumor growth and angiogenesis and asked whether additional therapeutic benefit was conferred by combination treatment. Experimental Design: The antitumor activity of each inhibitor alone or in combination was assessed in human cancer models in immunocompromised mice. ErbB receptor expression and activation of downstream signaling pathway was evaluated in both tumor and endothelial cells. Results: Both ErbB inhibitors significantly enhanced the antitumor activity of PTK787/ ZK222584. In vitro, ErbB1inhibition blocked VEGF release by tumor cells and proliferation of both tumor and endothelial cells. In an in vitro angiogenesis assay, epidermal growth factor (EGF) stimulated the release ofVEGF by smooth muscle cells resulting in increased angiogenesis, a response blocked by administration of PTK787/ZK222584. Under basal condition, both ZD1839 and PTK787/ZK222584 blocked sprouting, likely via inhibition of an autocrine ErbB1 loop and VEGFR signaling, respectively, in endothelial cells. In conditions of limiting VEGF, EGF plays an important role in endothelial cell proliferation, survival, and sprouting. Conclusion: We have shown that activation of ErbB1triggers a plethora of effects, including direct effects on tumor and endothelial cells and indirect effects mediated via induction of VEGF release. Simultaneous blockade of ErbB1andVEGFR pathways results in a cooperative antitumor effect, indicating that this combination may represent a valid therapeutic strategy.

The ErbB family of receptor tyrosine kinases has four members: therefore considered valid targets for anticancer therapy. ErbB1 (EGF receptor), ErbB2 (HER-2/neu), ErbB3 (HER3), and With respect to ErbB1, both antagonistic monoclonal anti- ErbB4 (HER4). These receptors play important roles in normal bodies and small-molecule tyrosine kinase inhibitors have development and in cancer biology. In particular, ErbB1 and shown clinical efficacy. Among these, ZD1839 (Iressa, Astra ErbB2 have been implicated in multiple types of human cancer, Zeneca Pharmaceuticals, Macclesfield, United Kingdom; ref. 3) where they control proliferation, survival, and spread of and PKI166 (Novartis Pharma AG, Basel, Switzerland; ref. 4) metastatic cancer cells (1). Alterations in ErbB receptors have are orally active ErbB1 and ErbB1/ErbB2 TK inhibitors, been correlated with more aggressive disease, lower rates of respectively. ZD1839 induced durable responses in patients survival, and poor response to therapy (2). ErbB receptors are with various tumor types (5). Both compounds, in addition to inhibiting tumor growth (6), have also been documented to have antiangiogenic effects (4, 7–9). Angiogenesis, the formation of new vessels from an existing Authors’ Affiliations: 1Novartis Institute for Biomedical Research, Oncology Research, 2Friederich Miescher Institute for Biomedical Research, Basel, vascular network, is required to sustain tumor growth and Switzerland, and 3Vivo Pharm Pty, Ltd.,Therbaton, Australia invasion (10, 11). A link between angiogenesis and ErbB Received 9/24/04; revised 3/22/05; accepted 3/31/05. receptor signaling has been established. Dividing endothelial Grant support: Novartis Research Foundation (N.E. Hynes). cells express functional ErbB1 (12, 13) and undergo apoptosis The costs of publication of this article were defrayed in part by the payment of page upon blockade of its activity (4). ErbB receptor inhibitors are charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. effective in preventing the release of proangiogenic factors, such Note: Supplementary data for this article are available at Clinical Cancer Research as vascular endothelial growth factor (VEGF), from tumor cells Online (http://clincancerres.aacrjournals.org/). (14–16). VEGF exerts potent mitogenic and prosurvival effects N.E. Hynes and J. Wood contributed equally to the article. on endothelial cells and has been shown to be a key factor in Requests for reprints: Patrizia Sini, Novartis Pharma AG, WKL.125.11.08, Klybeckstrasse, CH-4002 Basel, Switzerland. Phone: 41-61-696-1672; Fax: 41- pathologic situations that involve neovascularization and 61-696-6242; E-mail: [email protected]. changes in vascular permeability (17). The angiogenic signal F 2005 American Association for Cancer Research. is transmitted via cognate receptor tyrosine kinases (KDR and

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Flt-1), the VEGF receptors (VEGFR), located on the cell surface were cultured according to manufacturers’ instructions. The DU145 of the host vascular endothelium. Inhibition of VEGFR sig- prostate cancer cell line was obtained from American Type Culture naling prevents tumor angiogenesis thereby starving tumors of Collection (Rockville, MD). Cells were maintained in MEM EBS essential nutrients and oxygen. Various approaches have been (AMIMED, Allschwil, Switzerland) supplemented with 10% heat- inactivated FCS, 1% L-glutamine, 100 IU/mL penicillin, 100 Ag/mL adopted to interfere with the VEGF/VEGFR system including streptomycin. HC11 mouse mammary epithelial cells were grown as antagonistic antibodies (18–20), dominant-negative VEGFR described previously (35). NCI-H596 human adenosquamous lung mutants (21), recombinant soluble VEGFR proteins (22, 23), cancer cells were cultured in RPMI 1640 supplemented with 10% heat- and small-molecule tyrosine kinase inhibitors. Among these, inactivated FCS, 1% L-glutamine, 100 IU/mL penicillin, 100 Ag/mL PTK787/ZK222584 (Vatalanib, Novartis Pharma/Schering AG, streptomycin. Berlin, Germany), a potent inhibitor of both VEGFRs, inhibited Tumor models. BALB/c nu/nu mice were obtained from Iffa Cre´do growth and angiogenesis in experimental carcinoma models (L’Arbresle, France). Tumors were established by s.c. injection of (24, 25) and displayed activity in early clinical trials (26, 27). DU145 or NCI-H596 cancer cells (minimum 1-2 Â 106 cells in 100 AL Although blockade of ErbB receptor signaling would be PBS). Tumors from donor mice were passaged s.c. as fragments three expected to inhibit both tumor growth and angiogenesis, we times before use. HC11 mouse mammary epithelial cells were trans- postulated that combination with a VEGFR inhibitor may fected with oncogenic neuT and implanted as described previously (35). Three to 4 weeks after transplantation, mice showing mammary tumor provide additional therapeutic benefit. This hypothesis is growth of at least 10% during the previous 7 days were selected for supported by in vivo preclinical data we have obtained using efficacy studies. Animals were assigned to balanced groups, based on different agents to block ErbB and VEGFRs (patent no. WO02/ tumor size, so that means and SE were similar in each group (10-11 41882; refs. 28, 29). In addition, prolonged inhibition of animals/group). Tumor growth and body weights were determined tumor growth (30) and vascularization (31, 32) was observed twice per week (35). when agents targeting the VEGF axis were combined with Measurement of vascular endothelial growth factor concentration in antagonistic antibodies directed against ErbB1. plasma and tumor. Repeated plasma samples were taken from several Here we have investigated the molecular mechanisms mice at different time points (days 0, 1, and 8) and collected in underlying the effect of combining selective ErbB and VEGF separation tubes (BD Microtainer PST LH, NJ), left at room temperature j receptor inhibitors on the signaling pathways and biological for 90 seconds and spun at 4 C for 7 minutes at 7,000 rpm. Tumors were homogenized (BIO 101, Fastprep) in 500 AL PBS + 0.1% bovine functions of endothelial and tumor cells. We discuss the serum albumin and centrifuged. The concentration of human VEGF in hypothesis that in the tumor microenvironment, blockade of the supernatants and plasma was measured using a commercially ErbB1 signaling results in direct inhibition of tumor and available ELISA kit (Quantikine ELISA kit, R&D Systems, Inc., endothelial cell proliferation and survival as well as indirect Minneapolis, MN). inhibition of VEGFR signaling via a decrease in VEGF release. Immunofluorescence staining for terminal deoxynucleotidyl transferase–mediated nick-end labeling, CD31/ErbB1 and CD31/ Materials and Methods activated caspase-3. Cryosection sections (8 Am) of DU145 intra- dermal xenografts were fixed with acetone, blocked with 10% goat Antibodies and reagents. PTK787/ZK222584 was developed as a serum, and incubated with 1:100 dilution of polyclonal anti-ErbB1 and joint venture between Novartis Pharma and Schering. For in vitro assays, 1:200 dilution of monoclonal anti-CD31 for 2 hours at room a 10 mmol/L stock solution of PTK787/ZK222584 and ZD1839 was temperature. A biotinylated goat anti-rabbit antibody, diluted 1:500, prepared in DMSO and diluted in the optimal medium. For in vivo was added for 1 hour; the sections were washed with PBS and incubated studies ZD1839, PKI166, and PTK787/ZK222584 were dissolved in with Streptavidin FITC (diluted 1:200, Southern Biotechnology, 0.5% Tween, 5% DMSO in distilled water at the final concentration of 1 Birmingham, AL) and a TRITC-conjugated goat anti-rat antibody to 2 mg/mL. (diluted 1:200, The Jackson Laboratory) for an additional hour. The following antibodies were used: rabbit polyclonal anti-ErbB1 CD31/activated caspase-3 double staining was done using the same (1005) and anti-ErbB3 (C-17; Santa Cruz Biotechnology, Santa Cruz, protocol described for CD31/ErbB1 staining with the following CA), anti-ERK1/2 and anti-phospho-EKR1/2 (Cell Signaling Technol- modifications: the sections were not fixed and an anti-activated ogy, Beverly, MA), rabbit polyclonal anti-ErbB2 antiserum 21N (33), caspase-3 antibody was used (diluted 1:10). Terminal deoxynucleotide monoclonal anti-ErbB1 (120) and (101) (Santa Cruz Biotechnology), transferase–mediated nick-end labeling staining was done using the monoclonal anti-ErbB2 51 (34), monoclonal anti-ErbB3 (Ab-4; Neo- In situ Death kit (Roche AG, Basel, Switzerland). Markers, Fremont, CA), monoclonal anti-phosphotyrosine 4G10 To determine the vessel density, tumor cryosections where stained for (Novartis Pharma), monoclonal anti-CD31 (BD Bioscience, San Diego, CD31 as described above. Pictures encompassing the whole tumor CA), monoclonal anti-activated Caspase-3 (Oncogene Research Prod- where taken at 10Â magnification using a Zeiss Axioplan microscope. ucts, San Diego, CA), biotinylated anti-rabbit secondary antibody The area of the counted regions was measured using the Openlab 3.1.5 (Vector Labs, Peterborough, United Kingdom), and TRITC-conjugated software (Improvision, Lexington, MA). At least three complete tumors anti-rat secondary antibody (The Jackson Laboratory, West Grove, PA). were counted per treated group. Growth factors used were: recombinant human epidermal growth factor Immunoblot analysis. Wherever indicated, treatment of cultures (EGF) and heparin binding-EGF (Sigma, St. Louis, MO), recombinant with growth factor was done after serum starvation and 2 hours of human betacellulin (R&D System, Inc., Minneapolis, MN), recombi- incubation with the compound. Cells were lysed on ice with buffer nant human transforming growth factor-a (Upstate Biotechnology, containing 1% Triton X-100, 50 mmol/L HEPES (pH 7.5), 150 mmol/L Lake Placid, NJ), recombinant human heregulin (NeoMarkers), sodium chloride, 5 mmol/L EGTA, 1.5 mmol/L magnesium chloride, recombinant human basic fibroblast growth factor (bFGF, Life 10% glycerol, 0.1 mmol/L sodium orthovanadate, 2 mmol/L phenyl- Technologies, Inc., Gaithersburg, MD), recombinant human platelet- methylsulfonyl fluoride, 2 mmol/L sodium pyrophosphate, 10 Ag/mL derived growth factor (PDGF, Upstate), and recombinant dog VEGF125 aprotinin, and 10 Ag/mL leupeptin. Immunoprecipitations were done (VEGF, Novartis Pharma). for 2 hours at 4jC and immunocomplexes were resolved by SDS-PAGE Cell cultures. The human umbilical vascular endothelial cells and analyzed by immunoblotting. (HUVEC) and human pulmonary smooth muscle cell (HPASMC), Tumor cell proliferation. The antiproliferative effect of ZD1839 and purchased from Promo Cell (BioConcept AG, Allschwil, Switzerland), PTK787/ZK222584 on EGF-induced proliferation was evaluated via

Clin Cancer Res 2005;11(12) June 15, 2005 4522 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Combined ErbB and VEGFR Inhibition AntitumorActivity bromodeoxyuridine (BrdUrd) incorporation (Biotrak Cell Proliferation replaced and cultures were treated with different concentrations of ELISA System V.2, Amersham, Buckinghamshire, England). Subcon- ZD1839 followed by growth factor stimulation. Twenty-four hours fluent DU145 cells were seeded into 96-well plates at a density of 8 Â later, quantification of the VEGF in the supernatants and simultaneous 3 10 cells per well and incubated at 37jC and 5% CO2 in growth quantification of the number of viable cells (CellTiter 96 AQueous One medium (MEM EBS containing 10% FCS) for 24 hours. The medium Solution Cell Proliferation Assay, Promega Corp., Madison, WI) were was replaced with MEM EBS containing 0.5% FCS. After 24 hours, the carried out. medium was renewed and EGF (50 ng/mL) was added in the presence Endothelial cell proliferation and survival assays. Subconfluent or absence of ZD1839 or PTK787/ZK222584. Twenty-four hours later, HUVECs were seeded at a density of 5 Â 103 cells per well into 96-well

BrdUrd labeling solution was added and BrdUrd incorporation assayed plates coated with 1.5% gelatin and incubated at 37jC and 5% CO2 in according to manufacturers’ instructions. growth medium (PromoCell Nr. C-22110, BioConcept) containing 5% Measurement of vascular endothelial growth factor production by FCS. After 24 hours, the medium was replaced by either basal medium cultured cells. The concentration of human VEGF in the conditioned (PromoCell Nr. C-22210, BioConcept) containing 1.5% FCS or growth medium of DU145 cultures was measured using an ELISA kit medium. After 24 hours, the medium was renewed and ZD1839 or (Quantikine ELISA kit, R&D Systems). Cells were plated at the density vehicle (DMSO) added in the presence or absence of VEGF (20 ng/mL) or of 1 Â 104 into 96-well plates in growing medium. The medium was EGF (100 ng/mL). Cell proliferation was assessed as described above.

Fig. 1. Effect of the combined treatment with PKI166 or ZD1839 and PTK787/ZK222584 on growth of DU145 human prostate tumor xenografts and on plasmaVEGF level. DU145 cells (1 Â10 6) were injected intradermally in the flanks of nude mice.Treatment was started when tumor volume reached f10 0 mm3 (28-32 days after inoculation). A, PKI166, PTK787/ZK222584, or the combination of both compounds (50 mg/kg each) were given once a day for two consecutive weeks. B, ZD1839, PTK787/ZK222584, or the combination of both compounds (50 mg/kg each) was given once a day for four consecutive weeks. Each group consisted of 5 to 10 mice. Statistical significance of differences of mean values between treatment groups, compared with the control group, was determined using ANOVA followed by multiple-comparison Dunnett’s test. *, P < 0.05, the level of significance for all tests. Inhibition of tumor growth (T/C) was calculated as described in Materials and Methods in individual animals. Points, means; bars, FSE. C,VEGF plasma levels were measured 2 hours after the last dose of the compounds, using a specific ELISA kit, as described in Materials and Methods. Points, means expressed as pg/mL of plasma; bars, SE. *, P < 0.05 versus control (day 8) and ZD1839 (day 0); j, P < 0.05 versus control (day 1) and PTK787 (day 0) by two-way ANOVA andTukey test.

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Fig. 2. Immunofluorescence detection of ErbB1in DU145 prostate xenografts.Tumor sections were double stained for ErbB1 (green, A)andCD31/PECAM-1(red, B)and images captured for endothelial cells (CD31, red) were overlapped with images captured for ErbB1;colocalization of the two markers is shown in yellow (CD31/ErbB1, C and D, white arrows).

The effect of ZD1839 on EGF-induced survival of endothelial cells AG, Munich, Germany). Identically treated plates of HPASMC were was determined by the Cell Death Detection ELISA Plus Kit (Boehringer analyzed for human VEGF release as described above. Mannheim, Indianapolis, IN). HUVECs were plated as described above. Statistical analysis. Results are presented as means F SE. After 24 hours, the medium was replaced by basal medium containing Statistical evaluation of any differences between treatment groups EGF (100 ng/mL) in the presence or absence of ZD1839. After 48 hours, used one-way or two-way ANOVA as appropriate, with post hoc cells were lysed and the supernatants assayed for DNA fragments, as Tukey (pairwise comparisons), or Dunnett (comparisons with recommended by the manufacturer. controls) used with the ANOVA tests. In some cases, data was In vitro coculture angiogenesis assay. The effect of ZD1839 and transformed to achieve normal distribution. For all tests, the level of PTK787/ZK222584 on in vitro angiogenesis was assessed as follows. significance was set at P < 0.05. Note that with these small group HPASMCs were seeded on 48-well tissue culture plates coated with 100 sizes, the desired power level of 0.8 was not always attained. All AL of collagen type I (100 Ag/mL in PBS, Sigma) at a density of 2 Â 104 calculations were done with SigmaStat 2.03. cells per well and allowed to attach for 24 hours. The growth medium 3 was then removed and HUVECs (5 Â 10 cells per well) were plated on Results the HPASMCs monolayer. After 24 hours, the medium was replaced with basal medium containing 1.5% FCS in the presence or absence of The in vivo antitumor effect of ErbB and vascular endothelial different growth factors and/or compounds. The medium was renewed growth factor receptor kinase inhibitors. We investigated the at days 1, 3, 5, and 7; at day 9, cells were fixed with 4% PFA for 5 antitumor activity of the ErbB1 inhibitors PKI166 (Fig. 1A) and minutes at room temperature. The wells were washed with PBS and 10% normal goat serum, in PBS/0.1% Triton X-100, was added for ZD1839 (Fig. 1B), either alone or in combination with PTK787/ 30 minutes. Cells were incubated with monoclonal anti-human ZK222584, in nude mice bearing DU145 human prostate cancer CD31 in 3% normal goat serum in PBS/0.1% Triton X-100 for 2 xenografts. Mice were treated daily with individual drugs or hours followed by incubation with biotinylated goat anti-mouse IgG combinations at doses of 50 mg/kg each. Monotherapy with (Southern Biotechnology) in 3% normal goat serum for an PTK787/ZK222584 or PKI166 resulted in a reduction of tumor additional 2 hours. CD31-positive cells were stained using the volume of 41% and 44%, respectively, which was not statis- Vectastain Elite avidin-biotin complex kit (Vector Laboratories, tically significant. A dramatic cooperative effect, 74% tumor Burlingame, CA) according to manufacturer’s instructions and growth inhibition, was observed in mice receiving the combina- detected by 3-3V-diaminobenzidine tetrahydrochloride (Sigma). Cells were imaged at 1.25Â magnification using an inverted microscope tion of both drugs (Fig. 1A). A similar effect was observed when (Carl Zeiss AG, Feldbach, Germany) connected to a digital camera PTK787/ZK222584 was combined with ZD1839, where treat- (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Acquired ment with both inhibitors resulted in a complete suppression of images were analyzed using Definiens Cellenger software (Definiens tumor growth (Fig. 1B). Studies carried out on the NCI-H596

Clin Cancer Res 2005;11(12) June 15, 2005 4524 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Combined ErbB and VEGFR Inhibition AntitumorActivity small cell lung xenograft model and the neuT-driven GeMag 0.05). Coadministration of ZD1839 and PTK787/ZK222584 orthotopically implanted tumor model (28), evaluating the blunted the PTK-induced increase in plasma VEGF (PTK + activity of PTK787/ZK222584 and PKI166, also showed ZD1839; day 1 versus day 0, P > 0.05) and retained the ZD1839 enhanced (possibly synergistic) antitumor effects of combina- pattern of gradual decline in plasma VEGF levels, although this tion treatment (Supplementary Table S1). No or nonsignificant did not reach statistical significance (Fig. 1C). loss of body weight was observed in animals treated with We next examined the expression and localization of ErbB1 combination of agents (data not shown). Analysis of plasma in sections made from DU145 xenografts. Immunofluorescent from treated mice showed that whereas the level of released staining with an ErbB1-specific antiserum of the untreated VEGF remained essentially constant over 8 days in vehicle- (green staining, Fig. 2A) and treated tumors (Supplementary treated, tumor-bearing mice (P < 0.05, day 1 or 8 versus day 0), Fig. S1) revealed similarly high receptor levels in tumor cells. treatment with ZD1839 resulted in a time-dependent decrease Tumor-associated endothelial cells were stained with an anti- in the plasma concentrations of VEGF (ZD1839 treated: day CD31 antibody (red staining, Fig. 2B). Double staining of 8 versus day 0, P < 0.05; Fig. 1C). In contrast, administration of tumor sections with CD31 and ErbB1 antisera revealed that PTK787/ZK222584 led to a rapid increase in VEGF concen- some endothelial cells lining the blood vessels also express trations, as described previously (ref. 27; PTK787/ZK222584 ErbB1 (yellow staining, Fig. 2C, a-c; shown enlarged in Fig. 2D, treated: day 1 versus day 0, P < 0.05), which was higher than that white arrows). Expression of ErbB1 was also observed in the of controls or ZD1839 groups (P < 0.05). This increase in VEGF vessels associated with tumors in the PKI166- and PTK787- levels was largely resolved by day 8 (day 8 versus day 0, P > treated mice, although it was difficult to assess differences in the

Fig. 3. Effect of ZD1839 and PTK787/ ZK222584 on in vitro cultured DU145 cells. A, ErbB receptor expression and the EGF- or heregulin (HRG) ^ induced autophosphorylation was evaluated by immunoblotting. DU145 prostate cancer cells were serum-starved and treated with EGF (50 ng/mL), heregulin (100 ng/mL), or mock treated (À) for 5 minutes. Total cell lysates were immunoprecipitated (IP ) with specific anti-ErbB antibodies and immunoblotted with the indicated antibodies. B, quiescent DU145 cells were treated with EGF (50 ng/mL), heregulin (100 ng/mL), or mock treated (À)for 5 minutes.Total cells lysates were analyzed by SDS-PAGE followed by immunoblotting with anti-P-ERK, anti-ERK, anti-P-AKT, and anti-AKTantibodies. C and D, serum-starved DU145 cells were incubated in the presence or absence of ZD1839 or PTK787/ZK222584 for 1hour and treated with EGF (50 ng/mL) or mock treated (À) for 5 minutes. C, ErbB1immunoprecipitates were immunoblotted with the indicated antibodies. D, total cell lysates were analyzed by immunoblotting with anti-P-ERK and anti-ERK antibodies. E, DU145 cells were incubated with the indicated concentration of ZD1839 or PTK787/ZK222584 in the presence of EGF (50 ng/mL) for 48 hours at 37jC. Proliferation was assessed by using an in vitro cell proliferation BrdUrd ELISA kit as described in Materials and Methods. Columns, averages of quadruplicate determination; bars, SE. *, P < 0.05 versus control (À); j, P < 0.05 versus EGF (ANOVA andTukey test). F, after incubation with the indicated concentration of ZD1839 for 24 hours, conditional medium was collected from DU145 cancer cells plated either in serum-free medium and treated with EGF, heregulin, or mock treated (control)or(G) in FCS 10%.VEGF secretion was measured using a specific ELISA kit, as described in Materials and Methods and has been normalized for the number of producing cells. Bars, SE. *, P < 0.05 versus control; j, P < 0.05 versus EGF (ANOVA and Tukey or Dunnett’s test).

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Fig. 4. Analysis of ErbB receptor activation and proliferation in HUVECs. HUVEC cultures were serum starved and treated with EGF (100 ng/mL), heparin-binding EGF (HB-EGF, 50 ng/mL), BTC (10 ng/mL), heregulin (HRG, 100 ng/mL), or mock treated (À) for15 minutes. A, total cell lysates were immunoprecipitated (IP ) with specific anti-ErbB1and anti-ErbB2 antibodies and immunoblotted with the indicated antibodies. B, total cell lysates were analyzed by SDS-PAGE followed by immunoblotting with anti-P-ERK and anti-ERK antibodies. C, serum-starved HUVEC cultures were incubated in the presence or absence of ZD1839 (0.1 Amol/L) for1hour and treated with EGF (100 ng/mL) or mock treated (À)for 15 minutes. ErbB1immunoprecipitates were immunoblotted with the indicated antibodies. D, quiescent HUVECs were incubated in the presence or absence of ZD1839 (0.1 Amol/L) or PTK787/ ZK222584 (0.1 Amol/L) for 1hour and treated with EGF (100 ng/mL),VEGF (20 ng/mL), or mock treated (À)for 15 minutes.Total cell lysates were analyzed by SDS-PAGE followed by immunoblotting with anti-P-ERK and anti-ERK antibodies. F, HUVECs were incubated with the indicated concentration of ZD1839 in the absence (Control)orpresenceofEGF, VEGF, or FCS for 48 hours at 37jC. G, quiescent HUVECs were stimulated with the indicated concentration of VEGF in the absence (Control)orpresenceofEGFfor 48 hours at 37jC. Proliferation was assessed using an in vitro cell proliferation BrdUrd ELISA kit. Columns, averages of quadruplicate determination; bars, SE. *, P < 0.05 versus control; j, P < 0.05 versus EGF (ANOVA andTukey test, A). *, P < 0.05 versus control FCS1% (two-way ANOVA andTukey test, B). H, quiescent HUVECs were stimulated with the indicated concentration of VEGF, 50 ng/mL of EGF, the combination of the two growth factor or mock treated (À) for15 minutes.Total cell lysates were analyzed by SDS-PAGE followed by immunoblotting with anti-P-ERK and anti-ERK antibodies.

expression occurred upon treatment due to the collapsed and were treated with EGF or the ErbB3 ligand, heregulin, then fragmented appearance of the vessels. cell lysates were prepared and immunoprecipitated receptors In summary, these results suggest that in the tumor setting, were probed with an anti-phosphotyrosine antibody. EGF ErbB-specific inhibitors might have direct inhibitory effects, treatment led to an increase in ErbB1 and ErbB2 phosphor- both on tumor cells and associated endothelial cells. Further- ylation, whereas heregulin induced activation of ErbB2 and more, because the blockade of tumor growth observed upon ErbB3 (Fig. 3A; the slight increase in ErbB1 phosphorylation treatment with both ErbB1 inhibitors (PKI166 and ZD1839) is due to higher levels of immunoprecipitated receptor). was accompanied by an inhibition in release of proangiogenic Treatment with either EGF or heregulin resulted in an factors (Fig. 1C; data not shown), ErbB receptor inhibition is increase in the active, phosphorylated form of extracellular likely to have indirect antitumor effects. In the following signal-regulated kinase 1/2 (ERK1/2) and AKT (Fig. 3B). experiments, we examined the in vitro effect of ErbB receptor Pretreatment of DU145 cells with ZD1839 led to a dose- inhibition on tumor and endothelial cells. ZD1839 was used dependent decrease in EGF-induced ErbB1 phosphorylation due to its superior selectivity for ErbB1 over other tyrosine (Fig. 3C), accompanied by a blockade of EGF-induced kinases, particularly VEGFR-2 and PDGFR-a, in comparison intracellular signaling; 1 Amol/L ZD1839 was sufficient to with PKI166 (36). completely inhibit ERK1/2 phosphorylation (Fig. 3D). In vitro effect of ZD1839 and PTK787/ZK222584 on DU145 EGF treatment of DU145 cells led to a f1.5-fold increase cells. ErbB1, ErbB2, and ErbB3 were detectable in DU145 cells in BrdUrd incorporation; pretreatment of the cells with 0.1 (Fig. 3A). To examine ErbB receptor activity, DU145 cells Amol/L ZD1839 was sufficient to abrogate this effect. Higher

Clin Cancer Res 2005;11(12) June 15, 2005 4526 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Combined ErbB and VEGFR Inhibition AntitumorActivity concentrations of ZD1839 decreased BrdUrd incorporation to PTK787/ZK222584 reduced VEGF but not EGF-stimulated below basal levels, likely due to further inhibition of autocrine proliferation (Supplementary Fig. S2). activated ErbB1 (Fig. 3E; ref. 37). VEGF did not increase Considering that in vivo, tumors are likely to be exposed to proliferation or survival of DU145 cells (data not shown) and ErbB family ligands and VEGF, we also examined in vitro we failed to observe modulation of EGF action upon proliferation of HUVECs in the presence of EGF and VEGF; 5 treatment of the cells with PTK787/ZK222584 (Fig. 3C-E). and 20 ng/mL VEGF stimulated BrdUrd incorporation, whereas The ErbB receptors play a role in the regulation of 2 ng/mL was ineffective. In the absence of VEGF, EGF angiogenesis, via up-regulation of VEGF expression (13, 14, significantly increased BrdUrd uptake and acted in concert 38, 39). Accordingly, we examined the effect of EGF, heregulin, with VEGF (at 2 and 5 ng/mL) to promote BrdUrd uptake; no and ZD1839 on VEGF expression. EGF but not heregulin potentiation of VEGF by EGF was seen at 20 ng/mL VEGF (Fig. induced a significant increase (P = 0.003) in VEGF in the 4G). With suboptimal concentrations of VEGF, coaddition of conditioned medium of DU145 cultures (Fig. 3F) compared EGF also led to an additive increase in ERK1/2 phosphorylation with controls. In the absence of exogenous ligands, ZD1839 (Fig. 4H). Thus, it is possible that in the presence of showed no significant inhibition of VEGF production. However, physiologically relevant levels of VEGF (293 F 66 pg/mg of starting at 10 nmol/L, ZD1839 significantly impaired the effect DU145 tumor, n = 9), EGF might play a significant role in of exogenous EGF (P < 0.03). ZD1839 had a minor but modulating tumor angiogenesis. inconsistent effect on VEGF production in the presence of ErbB1 inhibition induces in vivo apoptosis of tumor cells and heregulin: at 10 (P = 0.003) and 100 (P = 0.045) nmol/L, the associated endothelial cells. The ability of ErbB1 and VEGFR VEGF levels were significantly below those in the presence of inhibitors to induce apoptosis in vivo was examined next. heregulin (or controls), whereas higher and lower ZD1938 Terminal deoxynucleotide transferase–mediated nick-end concentrations had no statistically significant effect. ZD1839 labeling staining revealed high levels of apoptotic tumor cells treatment also resulted in a significant decrease in VEGF released in DU145 xenografts taken from PKI166- treated mice (Fig. 5A). by cells growing in full serum (Fig. 3G), likely resulting from Considering that tumor-associated endothelial cells express blockade of the autocrine activated receptor (37). In summary, ErbB1 (Fig. 2), we assessed the effect of PKI166 treatment on inhibition of ErbB1 signaling in DU145 tumor cells has a direct endothelial cell survival. Tumor sections were costained with negative effect upon tumor cell proliferation and also leads to a CD31 antiserum and an antibody recognizing activated caspase-3. decrease in VEGF production. Both of these activities might This specific early apoptotic marker, stained fewer cells, contribute to the enhanced antitumor activity observed upon facilitating identification of costained (yellow) endothelial addition of ErbB1 inhibitors to PTK787/ZK222584. cells. Tumors taken from vehicle-treated mice lacked endothelial Expression and activation of ErbB receptors in endothelial cells. cells undergoing apoptosis (Fig. 5B, g), whereas tumors from To study the role of ErbB receptors in endothelial cells, we PKI166-treated mice displayed activated caspase-3 staining of employed two models, HUVECs and human dermal microvas- endothelial cells (Fig. 5B, h). Treatment with PTK787/ cular endothelial cells. ErbB1 and ErbB2 (Fig. 4A) but not ZK222584 caused extensive vessel disorganization and a ErbB3 and ErbB4 (data not shown) were detected in HUVECs. significant reduction in vessel density (Fig. 5C) within the Addition of the ErbB1 ligands, EGF, heparin-binding EGF, and tumor. Moreover, colocalization could be observed between betacellulin to HUVECs led to an increase in ErbB1 tyrosine CD31 and caspase-3 staining (Fig. 5B, i), confirming that phosphorylation and ERK1/2 activation (Fig. 4A and B). None inhibition of VEGF signaling promotes endothelial cell apopto- of the ErbB1 ligands induced an increase in ErbB2 phosphor- sis. Concomitant treatment of mice with PTK787/ZK222584 ylation (data not shown). In agreement with the data showing and PKI166 led to extensive tumor necrosis, rendering it that HUVECs do not express heregulin receptors, heregulin impossible to histologically assess their combined effects. treatment had no effect on ERK1/2 activation (Fig. 4B). Inhibition of ErbB1 might induce endothelial cell apoptosis Identical results were observed in human dermal microvascular by directly affecting cell survival or, indirectly, by blocking the endothelial cells (data not shown) suggesting that in endothe- paracrine secretion of survival factors, such as VEGF. Thus, we lial cells, ErbB1 homodimers have a central role in mediating examined whether the in vitro antiproliferative effect of ZD1839 the response to ErbB family ligands. Addition of 0.1 Amol/L on endothelial cells, (Fig. 4F) was accompanied by induction of ZD1839 to HUVECs completely abolished EGF-dependent programmed cell death. Treatment of serum-starved HUVECs ErbB1 activation (Fig. 4C) and ERK1/2 phosphorylation and with EGF resulted in a concentration-dependent increase in resulted in a reproducible slight attenuation of the VEGF- viability, as attested by decreased DNA fragmentation (Fig. 5D). induced ERK1/2 phosphorylation (Fig. 4D). We are currently EGF-induced endothelial cell viability was blocked even at the investigating the specificity of this effect and the possibility of lowest concentration of ZD1839 (0.1 Amol/L) tested. Because VEGF-mediated ErbB1 transactivation. In contrast, 0.1 Amol/L EGF does not induce VEGF release from endothelial cells (data PTK787/ZK222584 inhibited VEGF-induced but had no effect not shown), we consider it likely that the endothelial cell on EGF-induced ERK1/2 activation. apoptosis observed in the presence of the ErbB1 inhibitor in vivo Active ErbB1 cooperates with vascular endothelial growth (Fig. 5B) is, at least in part, due to direct effects of the inhibitor factor to promote endothelial cell proliferation. We next on the endothelial cell compartment. examined the consequences of activation or inhibition of Effect of ZD1839 and PTK787/ZK222584 on endothelial cell ErbB1, on HUVEC proliferation. EGF weakly, and VEGF and sprout formation in a coculture system. The previous results FCS more potently, increased BrdUrd incorporation (all P < suggested that EGF plays a role in proliferation and survival of 0.05, two-way ANOVA; Fig. 4F). Coaddition of ZD1839 (0.01-1 endothelial cells, particularly in limiting VEGF concentrations. Amol/L) significantly blocked the ability of EGF but not VEGF To investigate whether EGF influences other aspects of or serum to stimulate HUVEC proliferation. Treatment with endothelial cell biology, we examined its effects in an in vitro

www.aacrjournals.org 4527 Clin Cancer Res 2005;11 (12) June 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical angiogenesis assay consisting of cocultured endothelial and autocrine loop in HUVECs. We propose that this is smooth muscle cells. HUVECs when cultured on a monolayer biologically relevant because ZD1839 blocks basal HUVECs of HPASMCs proliferate and migrate, resulting in the formation proliferation and survival (Fig. 4F and Fig. 5C) and very likely of capillary-like sprouts. Cocultures were either left untreated or as a consequence, basal sprouting (Fig. 6A, f). Identical results treated with various peptide ligands in the presence or absence were observed in human dermal microvascular endothelial of inhibitors and sprouting was quantified (Fig. 6). Control cells, suggesting that the EGF effect on sprouting is DMSO-treated HUVECs exhibit a basal level of sprouting (Fig. reproducible in endothelial cells expressing functional ErbB1 6A, a), which was quantified in Fig. 6B (total area) and Fig. 6C (data not shown). (total length). Treatment of the cocultures with VEGF (b), EGF Thus, we propose that ErbB1 signaling provides cues (c), bFGF (d), or PDGF (e) resulted in an increase in sprout determining changes in endothelial cell morphology in the number (Fig. 6A), although the image analysis quantification of presence of suboptimal concentrations of VEGF. When ErbB1 is total sprout area and length failed to show the differences blocked, low levels of VEGF released from the HPASMCs fail to apparent by eye (Fig. 6B-C). PTK787/ZK222584 significantly induce sprouting. When VEGF levels are elevated, signals from blocked sprouting in response to VEGF (l) EGF (m), and PDGF theVEGFRaresufficienttodrivemorphologicchanges (o) and partially blocked the effects of bFGF (n; Fig. 6B-C: independently from ErbB1 activity (Fig. 6A, g). VEGF, P = 0.032 and P = 0.042; PDGF, P = 0.002 and P < 0.001; EGF, P = 0.002 and P < 0.001, versus controls within Discussion growth factor group in Fig. 6B and C, respectively). To examine the possibility that VEGF exerts a direct effect upon endothelial Aberrant activation of ErbB receptors has been correlated cells in the coculture assay, whereas EGF, PDGF, and bFGF with a more aggressive cancer phenotype and poorer patient might act by indirectly stimulating HPASMCs to release VEGF, prognosis (41, 42). Experimental evidence indicates that ErbB we used an ELISA assay to measure its release from HPASMCs. inhibitors can induce tumor stasis or regression and, in The basal level of VEGF released from HPASMCs was increased addition, increase the antitumor efficacy of conventional and 2-fold when cultured in the presence of PDGF and EGF and 4- targeted therapies. In particular, there seems a close relation- fold following bFGF treatment (all P < 0.001 versus control; ship between the ErbB and VEGFR signaling pathways in Supplementary Fig. S3). PTK787/ZK222584 had no effect upon progression of solid tumors. The present study was undertaken bFGF- and PDGF-induced VEGF release, suggesting that to assess the effect of selective ErbB1 inhibitors (PKI166 and PTK787/ZK222584’s ability to inhibit the effects of these ZD1839), given alone or in combination with a VEGFR growth factors is predominantly mediated by blocking endo- inhibitor (PTK787/ZK222584; ref. 24). We show that combi- thelial cell VEGFR activity. The lack of complete inhibition of nation therapy exerts a cooperative antitumor and antiangio- the effects of bFGF by PTK787/ZK222584 is probably due to genic effect in several experimental tumor models. The DU145 the documented ability of bFGF to stimulate endothelial cell prostate cancer cell line was further used to investigate the migration and sprout formation, in a VEGFR-independent mechanisms underlying this cooperativity. The importance of fashion (40). PTK787/ZK222584 inhibited EGF-stimulated ErbB1 signaling in progression of prostate cancer has been VEGF production, suggesting that ErbB1 and VEGFR crosstalk extensively described. Prostate carcinomas and their derived may occur (Fig. 4D). tumor cell lines display autocrine activation of the receptor, due The effects of ZD1839 on sprouting (Fig. 6) and VEGF release to expression of the ligands EGF and transforming growth (Supplementary Fig. S3) were also examined. In the presence of factor-a (37, 43, 44) and addition of EGF further stimulates ZD1839, EGF-stimulated (h) but not VEGF- (g), bFGF- (i), or proliferation (45) and invasiveness (46). Conversely, blockade PDGF-stimulated (j) sprout formation was reduced (Fig. 6B-C; of ErbB1 inhibits growth of cell lines and (47) prostate tumor total length of sprouts, P = 0.014 versus control within EGF xenografts (48). We show here that PKI166 treatment caused group). With respect to VEGF release from ZD1839-treated extensive apoptosis in tumor xenografts and a dose-dependent HPASMCs, only EGF-treated cultures showed a significant inhibition of EGF-induced intracellular signaling and cell decrease (Supplementary Fig. S3). These results suggest that proliferation in vitro. ErbB1 inhibition abrogated VEGF the effects of ZD1839 on the coculture assay are, at least secretion, both in the in vivo context and in cultured cells partially indirect, resulting from blockade of ErbB1 activity in in vitro, suggesting that both inhibition of the tumor angiogenic HPASMCs. Moreover, these experiments highlight ZD1839’s response as well as a direct effect on tumor cell proliferation selectivity because it has no effect upon sprouting induced by and survival, underlie the in vivo efficacy of ZD1839 and the other ligands. PKI166 (7, 8, 13). Recently, active ErbB1 has been detected on ZD1839, and to a greater degree PTK787/ZK222584 (P < tumor-associated endothelial cells, an effect ascribed to tumor 0.05), reduced basal sprouting (Fig. 6A, f and k), without cell expression of EGF or transforming growth factor-a (9). In lowering VEGF release from HPASMCs (Supplementary Fig. the studies presented here, we show that the EGF- and S3). The PTK787/ZK222584-mediated reduction is likely due transforming growth factor-a–expressing DU145 cancer cells to inhibition of the action of HPASMC-released VEGF on (37) give rise to tumors associated with ErbB1 expressing endothelial cell VEGFR. The ZD1839-mediated block must endothelial cells. As we show here, in normal culture occur by alternative mechanisms because HUVECs do not conditions, both HUVECs and human dermal microvascular produce VEGF, either basally or in response to EGF, nor do endothelial cells express ErbB1. However, we have observed they express VEGF mRNA (data not shown). However, we that in vitro treatment of these endothelial cells with EGF- have detected expression of the ErbB1 ligand heparin binding- related ligands failed to induce a further increase in ErbB1 EGF in a gene expression profile analysis (data not shown) mRNA (data not shown), suggesting that alternative mecha- raising the possibility that ErbB1 might be activated by an nisms underlie the increased receptor levels observed in the

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endothelium proximal to the ligand expressing tumors (9). We show here that treatment with either an ErbB1 or Several explanations could be envisaged, an important one VEGFR inhibitor resulted in apoptosis of endothelial cells being that a localized concentration of a particular ligand could within the prostate tumor. In vitro studies suggested that EGF favor the proliferation or migration of endothelial cell clones protects endothelial cells from apoptosis both by directly that express higher levels of its cognate receptor. acting on the endothelial cell compartment as well as via

Fig. 5. Effect of PKI166 and PTK787/ ZK222584 on tumor cell and endothelial cell survival. A, representative immunofluorescent images for terminal deoxynucleotide transferase ^ mediated nick-end labeling (apoptosis) of DU145 xenografts sections from mice treated with vehicle (Control)orPKI166.B,tumor sections were double stained for CD31/ PECAM-1 (red) and activated caspase-3 (green) and images captured for endothelial cells (CD31, red) were overlapped with images captured for activated caspase-3(apoptosis); colocalization of the two markers (yellow). Treatment with PKI166 or PTK787/ ZK222584 induced apoptosis of tumor (green) and endothelial cells (yellow). C, tumor sections from mice treated with vehicle (Control), PKI166, or PTK787/ ZK222584 were stained for CD31 and the vessel density was determined as described in Materials and Methods; bars, SE. *, P < 0.05 versus control (ANOVA and Tukey test). D, quiescent HUVECs were treated with the indicated concentration of ZD1839 in the presence of EGF for 48 hours at 37jC. Apoptotic cell death was analyzed using the Cell Death Detection ELISA kit, as described in Materials and Methods. Columns, averages of quadruplicate determination; bars, SE. *, P < 0.05 versus control; j, P < 0.05 versus EGF 100 ng/mL (ANOVA and Tukey test).

www.aacrjournals.org 4529 Clin Cancer Res 2005;11 (12) June 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical paracrine induction of survival factors (e.g., VEGF). ErbB1 production of VEGF by the smooth muscle cell monolayer activation also led to increased endothelial cell proliferation, seemed to underlie the growth factor mediated increase in albeit to a lesser extent than stimulation with VEGF. Notably, sprout formation. Notably, inhibition of ErbB1 activity the maximal concentration of VEGF used in these in vitro blocked both basal as well as EGF-stimulated sprouting. studies was far in excess of that estimated to occur within the Sprouting is likely due to proliferation and migration of tumor environment. When suboptimal, but more physiolog- endothelial cells. Extensive studies failed to show a role for ically relevant, quantities of VEGF were used, an additive EGF in endothelial cell migration (P. Sini, data not shown) increase in proliferation was observed upon coadministration suggesting that the effects of EGF are largely due to promotion of EGF. An in vitro angiogenesis assay system was used to of cell division. The effect of ZD1839 on basal sprouting was show that EGF plays additional roles in endothelial cell very similar to the decrease observed upon treatment with biology. Endothelial cells cultured on a smooth muscle cell PTK787/ZK222584, indicating that both growth factors are monolayer undergo morphologic changes resulting in the required for morphologic changes of endothelial cells under formation of sprouts. Although sprout formation was ob- these conditions. In a similar manner, tumors from ErbB1 served under basal conditions, increased sprouting occurred inhibitor treated mice seemed to contain fewer vessels (data upon stimulation with EGF, bFGF, and PDGF. Paracrine not shown). Although this may be partly a consequence of

Fig. 6. Effect of ZD1839 and PTK787/ ZK222584 on sprouting in a coculture system of HUVECs and HPASMCs. A, HUVECs were cocultured with HPASMCs for 9 days in the presence of the indicated growth factors and inhibitors. At days 1, 3, 5, and 7, the culture medium was replaced with fresh medium and at day 9 cells were fixed and immunostained with anti-CD31/PECAM-1antibody specific for endothelial cells. Bar,1mm. CD31-stained HUVECs were analyzed using the Cellemger software.

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Fig. 6 continued. B, total area of sprouts (Am2/mm2). C, total length of sprouts (Am/mm2). Bars, SE. *, P < 0.05 versus controls within growth factor group (ANOVA and Tukey test).

blocking VEGF production by the tumor, these data suggest underlies the cooperative antitumor effect observed upon that ErbB1 may also have a direct role in tumor angiogenesis. coadministration of ErbB1 and VEGR inhibitors. Our studies In conclusion, we have provided evidence that EGF acts on suggest that combination treatment with ErbB1 and VEGFR multiple components of the tumor environment, directly on inhibitors may have enhanced beneficial effects in cancer tumor and endothelial cell proliferation and survival as well as patients compared with treatment with either agent alone. indirectly, via release of proangiogenic factors from tumor and smooth muscle cells. Furthermore, we propose that, in conditions mirroring those found within the tumor, EGF Acknowledgments

cooperates with VEGF to induce angiogenesis (Supplementary We thank A. Theuer, B. Probst, M. Walker, and H.P. Muller for technical assis- Fig. S4). It is likely that inhibition of the complex crosstalk that tance; M.A. Pearson for critically reading the article; and P.Herrling for his invaluable occurs between the tumor and endothelial cell compartment support to this work.

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