J Cancer Res Clin Oncol (2009) 135:13771386 DOI 10.

1007/s00432-009-0579-3

ORIGINAL PAPER

Antitumor activity of the HER2 dimerization inhibitor pertuzumab on human colon cancer cells in vitro and in vivo
Michael Pohl I. Stricker A. Schoeneck K. Schulmann S. Klein-Scory I. Schwarte-WaldhoV M. Hasmann A. Tannapfel W. Schmiegel A. Reinacher-Schick

Received: 13 July 2008 / Accepted: 16 March 2009 / Published online: 2 April 2009 Springer-Verlag 2009

Abstract Purpose The monoclonal antibody pertuzumab represents the Wrst HER2 dimerization inhibitor with unknown activity in colon cancer treatment. We examined the antitumor activity of pertuzumab as a single agent or in combination with erlotinib or irinotecan in human colon cancer cells in vitro and in vivo. Methods Colon cancer cell lines were tested for HER1/ HER2 expression by western blot analysis. The eVect of pertuzumab on cell cycle distribution was analyzed by FACS. Nude mice bearing xenograft tumors were treated with pertuzumab alone, or in combination either with irinotecan or with erlotinib. Tumor volume was measured repeatedly. Tumor histology was analyzed for necrosis. Results Six of nine cell lines showed high expression of HER1/HER2. Pertuzumab inhibited cell cycle progression in various cell lines. Pertuzumab showed minor antitumor activity in xenograft tumors, but signiWcantly inhibited
M. Pohl (&) A. Schoeneck K. Schulmann S. Klein-Scory I. Schwarte-WaldhoV W. Schmiegel A. Reinacher-Schick Department of Medicine, Knappschaftskrankenhaus, Ruhr University, In der Schornau 23-25, 44892 Bochum, Germany e-mail: Michael.Pohl-4@rub.de I. Stricker A. Tannapfel Institute of Pathology, BG University Clinics Bergmannsheil, Ruhr University, Brkle-de-la-Camp-Platz 1, 44789 Bochum, Germany M. Hasmann Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Germany W. Schmiegel Department of Gastroenterology and Hepatology, BG University Clinics Bergmannsheil, Ruhr University, Brkle-de-la-Camp-Platz 1, 44789 Bochum, Germany

tumor growth when combined with erlotinib (P < 0.001). Combination of pertuzumab with irinotecan had no additional eVect on growth of additional tumors. Pertuzumab treated DLD-1 xenograft tumors did not show enhanced necrosis, which, however, was found in HCT116 derived xenografts. Conclusions Pertuzumab has some antitumor activity on human colon cancer cells in vitro and in vivo, in particular when combined with erlotinib. In vivo, pertuzumab combination treatment was not superior to irinotecan monotherapy. These data warrant further investigation of simultaneous HER1/EGFR TKI inhibition and HER1/ HER2 dimerization inhibition for colorectal cancer therapy. Keywords Colorectal cancer Pertuzumab HER dimerization inhibitor Dual ErbB inhibition Irinotecan Abbreviations HER Human epidermal growth factor receptor EGFR Epidermal growth factor receptor MAPK Mitogen-activated protein kinase TKI Tyrosine kinase inhibitor

Introduction Colorectal cancer constitutes the second leading cause of death from cancer in Europe and North America and is responsible for approximately one million new cases and half a million deaths per year worldwide (Ferlay et al. 2001). The human epidermal growth factor receptor (EGFR, HER1, ErbB-1) belongs to the ErbB or HER family of receptor tyrosine kinases and is overexpressed or dysregulated in many gastrointestinal cancer types, including 6080% of colon cancers, which in turn correlates with

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poor prognosis and early disease progression (Hemming et al. 1992; Mayer et al. 1993; Porebska et al. 2000; Salomon et al. 1995). HER2 (ErbB-2) overexpression has been previously reported in up to 85% of colon cancers; however, estimates vary depending on the methods used (Dursun et al. 2001; Ross and McKenna 2001). HER2 overexpression is associated with poor prognosis in some studies (Kapitanovic et al. 1994, 1997), but few studies have examined the therapeutic potential of HER2 inhibition in colon cancers (Mann et al. 2001). In contrast, the HER1/ EGFR has been established to be an important therapeutic target in colon and other human cancers. In addition to HER1/EGFR and HER2, the HER family of receptor tyrosine kinases (TK) includes HER3 (ErbB-3) and HER4 (ErbB-4) (Carpenter and Cohen 1990; Real et al. 1986). They are usually activated by ligand binding to the receptors and subsequent dimerization. However, there are two exceptions: there is no ligand for HER2 and the TK domain of HER3 is inactive. When HER3 is stimulated by its ligands heregulin-1 or -2, it preferentially forms heterodimers with HER2. The HER2/HER3 heterodimer is known to provide a potent oncogenic stimulus by activating both, proliferation and survival signaling cascades (Mendelsohn 2002), such as the Ras/Raf/MEK (mitogenactivated protein kinase or MAPK) and PI3 kinase/Akt/ mTOR signaling pathways (Baselga and Arteaga 2005). Both HER1/EGFR and HER2 are co-expressed in colorectal cancer cells and simultaneous targeting of these receptors enhanced apoptosis in preclinical studies (Fields et al. 2005; Zhou et al. 2006). Pertuzumab (rhu MAb 2C4; Genentech Inc., San Francisco, CA) is the Wrst in the new class of targeted therapeutic agents known as HER2 dimerization inhibitors (Adams et al. 2006). Pertuzumab is a recombinant, humanized monoclonal antibody that speciWcally binds to an epitope on the dimerization domain of HER2 (Franklin et al. 2004). Binding of pertuzumab prevents HER2 homo- and heterodimerization (Agus et al. 2002). Because of this mechanism of action, pertuzumab antitumor activity is not restricted to tumors with HER2 overexpression and therefore diVers from the therapeutic monoclonal antibody trastuzumab (Herceptin, Genentech Inc., San Francisco, CA), which binds to a nonoverlapping juxtamembrane region of HER2s extracellular domain, cannot inhibit HER2 heterodimerization and requires HER2 overexpression for antitumor activity (Friess et al. 2002; Cobleigh et al. 1999). Pertuzumab is also active in low-to-moderate HER2 expressing ovarian and breast cancer cell lines. Preclinical and early phase I/II activities have been identiWed in several cancer types, including breast and ovarian cancer (Agus et al. 2002, 2005; Jackson et al. 2004). The topoisomerase I inhibitor irinotecan (CPT-11) is a water soluble, semisynthetic derivative of camptothecin that has shown activity against a number of diVerent tumor types

in preclinical models and in clinical trials of patients with various human cancers (Saijo 2000). Despite clinical improvements attributed to the addition of CPT-11 therapy for metastatic colorectal cancer, nearly all patients will become refractory to CPT-11. Thus, new treatment options are needed to improve survival in patients with CPT-11 refractory colorectal cancer. Cetuximab (Erbitux, Imclone, New York, NY), a chimeric monoclonal antibody that blocks liganddependent EGFR receptor activation, is highly synergistic with irinotecan in refractory disease of colorectal cancer patients (Saltz et al. 2001, 2004). Cetuximab is FDA approved for second line therapy in combination therapy and will soon aquire Wrst line approval in combination with chemotherapy. Lastly, erlotinib (OSI-774, Tarceva, Genentech Inc., San Francisco, CA) is a selective, orally available low molecular weight inhibitor that binds competitively to the ATP-binding site at the kinase domain of EGFR. Preclinical studies with erlotinib have shown potent antitumor activity in a variety of cultured tumor cell lines as well as human colon cancer xenograft tumors (Akita and Sliwkowski 2003; Grunwald and Hidalgo 2003). Favorable clinical studies have led to the approval of erlotinib for use in the treatment of advanced non-small cell lung cancer (NSCLC) and advanced pancreatic cancer (Johnson et al. 2005; Moore et al. 2007). Erlotinib in colorectal cancer remains investigational. A phase II study presented a 39% SD rate, as the best response, with rash and diarrhea as the main toxicity events (Townsley et al. 2006). This evidence of single agent erlotinib activity in metastatic CRC patients led to the design of several trials in combination with chemotherapy. The drug showed encouraging results when used in combination with capecitabine and oxaliplatin in previously treated disease in a phase II trial. In 32 patients pretreated with an irinotecan-containing regimen, 25% of the patients experienced a partial response and 44% had stable disease for at least 12 weeks (Meyerhardt et al. 2006). In summary, the HER1/EGFR pathway proves to be a valid target in GI cancers, especially in colorectal cancer. Here we examined whether multi-targeting of HER family receptors is a successful approach in colorectal cancer. Given the diVerences in the mechanisms of action of these drugs, we aimed to examine the antitumor activity of single agent therapy with pertuzumab and in combination with either erlotinib or irinotecan in human colon cancer cells in vitro and in a murine xenograft model.

Materials and methods Test agents and vehicles Erlotinib hydrochloride was supplied as solution (Roche Diagnostics GmbH, Penzberg, Germany) and stored at 4C. A stock solution of pertuzumab (25 mg/ml) was obtained

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from Roche Diagnostics GmbH (Penzberg, Germany). The formulation vehicles were Captisol (sulfobutyl ether hcyclodextrin, sodium salt, 6% solution in water) and PBS for erlotinib and pertuzumab. Dosing preparations of both agents were prepared on the day of use. Irinotecan hydrochloride 3H2O (100 mg/5 ml) was obtained from PWzer (New York, NY, USA). Colorectal carcinoma cell lines and culture conditions The cell lines WiDr, SW480, and SW620 were obtained from the American Type Culture Collection (Rockville, MD, USA). All other lines were kindly provided by M Strauss/Berlin. Cells were maintained in Dulbeccos modiWed Eagles Medium (DMEM) supplemented with antibiotics and 10% fetal calf serum (FCS) (Gibco, Germany). The cells were incubated at 37C in a humidiWed atmosphere containing 10% CO2. Cells were passaged using trypsin/ EDTA (Gibco, Germany). On the day of tumor cell injection, cells were detached with trypsin/EDTA and washed in culture medium. Cells were resuspended in DMEM, and cell concentration and cell size were determined. Preparation of proteins and western blot analysis Colon cancer cell lines were tested for HER1/EGFR and HER2 expression by western blots. Cells were lysed in NP40 lysis buVer (25 mM Tris HCl, pH 7.4, 0.5% NP-40, 100 mM NaCl, 1 mM EDTA), containing a protease inhibitor cocktail (Roche, Germany) and 1 mM PMSF. Proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon membranes (Millipore). HER1/EGFR, HER2 were detected using primary anti-HER1/EGFR and antiHER2 monoclonal antibodies from Santa Cruz Biotechnology Inc. (Heidelberg, Germany) and Cell Signaling Technology Inc. (Danvers, MA, USA). Additional goat anti-mouse and anti-rabbit secondary antibodies labeled with Alexa Fluor 680 (700 nm, Molecular Probes, Invitrogen Corp) were detected using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Analysis of cell cycle distribution after pertuzumab and EGF treatment The eVect of pertuzumab on growth and cell cycle distribution was analyzed after stimulation with EGF ligand (R&D Systems, Rsselsheim, Germany). Unstimulated cells and stimulated cells incubated with BSA served as controls. Cells were seeded onto 6-well plates and allowed to adhere for 24 h. After serum starvation, cells were incubated alone or with 10 g/ml pertuzumab for 30 min, then 100 ng/ml EGF ligand was added. Cells were incubated with the

respective compound for 24 h and were harvested by trypsinization. At the end of the treatment period, cells were Wxed in 100% methanol for 30 min at 20C, centrifuged for 10 min at 2000 rpm, resuspended in 0.1% Triton-X 100 in PBS containing propidium iodide (40 g/ l; Sigma Chemicals, Deisenhofen, Germany) and RNase (60 g/ l; Sigma Chemicals) and were incubated at 4C for a minimum of 1 h. Subsequently, DNA content was measured using a Xow cytometer (Beckman Coulter, Krefeld, Germany) and the cell cycle distribution was calculated using the Phoenix Multicycle for Windows cell cycle analysis software. A minimum of 10,000 events were measured for each sample. Animals Female mice, Wve weeks old, were purchased from Harlan Winkelmann (Borchen, Germany). Animals were housed in suitable cages under speciWed pathogen-free conditions, in rooms maintained at 23C and 50% humidity, with a 12-h light/12-h dark cycle. The mice were quarantined during the acclimatization period of at least a week. Standard food (Altromin, Lage, Germany) and water were available ad libitum. Regular health checks were done. Animal experiments were performed in accordance with the Principles of laboratory animal care (NIH publication no. 85-23, revised 1985) and according to local Committee Guidelines (GVSolas, Felasa, TierschG). The experimental study protocol was reviewed and approved by the local government. Growth inhibition studies in vivo For establishment of xenograft tumors, mice were anesthetized with ether (Otto Fischer GmbH & Co KG, Saarbrcken, Germany). Two hundred-microliter suspensions of SW948, DLD-1 or HCT116 tumor cells (Wnal concentration: 106 cells/200 l) were transplanted s.c. into both Xanks of NMRI nu/nu nude mice using a 1.0 ml tuberculin syringe (needle 26 G 1 in., 0.45 25 mm; Becton Dickinson, Drogheda, Ireland). Tumor-bearing mice were randomized (n = 12 per group) when the mean tumor volume was <50 mm3. Treatment began 510 days after cancer cell injection. Tumor volume was determined twice weekly and compared to untreated control tumors. Each xenograft tumor was measured in two dimensions (a = length; b = width) with a digital caliper. Tumor volume (V) was determined by the following equation: V = /6 (a b b), a > b (Prewett et al. 2002). At the end of the studies, all animals were sacriWced humanely by cervical dislocation under anesthesia and tumors were evaluated histo-pathologically by light microscopy. Randomized groups of tumor-bearing mice (n = 12) were treated by oral gavage with vehicle (Captisol: 10 ml/kg

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per day) as control, or with erlotinib (25 mg/kg per day), using 1 ml tuberculin syringes Wtted with feeding needles with round tip and Luer lock hub (FST, Heidelberg, Germany). The dose of erlotinib were based on previous tolerability data. The i.p. dosing equipment was a 26 G 1 in., 0.45 25 mm, needle attached to a 1.0 ml tuberculin syringe. The choice of pertuzumab dose and schedule was based on previously reported data (Friess et al. 2005). Pertuzumab was administered i.p. at a loading dose of 12 mg protein/kg, followed by once weekly doses of 6 mg/ kg. In combination studies, groups of tumor-bearing mice received erlotinib (25 mg/kg per day, p.o.) or irinotecan (50100 mg/kg once a week, i.p.) and pertuzumab (6 mg/kg once a week, i.p, with a twofold loading dose). Control animals were given vehicles, PBS i.p. for pertuzumab, NaCl 0.9% i.p. for irinotecan and captisol solution orally for erlotinib. Local animal welfare regulations require animals to be terminated when tumors reach a certain size or show surface ulceration. All animals were observed daily for clinical signs of toxicity and weighed twice a week. Statistical analysis KruskalWallis test and Dunnett test were applied to compare treatment groups with a level of 5% considered signiWcant using GraphPad Prism version 4 for Windows (GraphPad Software, San Diego, CA, USA) Histo-pathological analysis of explanted tumors Samples of explanted tumors were Wxed in 4% buVered formaldehyde (Kabe Labortechnik, Nmbrecht-Elsenroth, Germany) and embedded in Paraplast (Sherwood Medical, Norfolk, NE). From selected blocks, sections were cut, put on coated slides (Superfrost plus, R. Langenbrinck, Teningen,
Fig. 1 Western blots reveal HER1/EGFR and HER2 expression of colon cancer cell lines. Expression of HER1/ EGFR (a) and HER2 (b) in colon cancer cells lines SW480, HT 29, DLD-1, LS174T, HCT116, SW948, Lovo, SW48 and WIDR was analyzed in lysates of conXuent monolayers of cancer cell lines

Germany), and dried for 12 h at 37C. The sections were deparaYnized in xylene (J.T. Baker) and rehydrated through graded concentrations of ethanol to distilled water. For histopathologic examination, sections were stained with H&E and examined by light microscopy (Leica Microsystems, Wetzlar, Germany), by an experienced histopathologist (IS.). Semiquantitative analysis of the tumor slides using DISKUS software (Fa. Carl H. Hilgers, Knigswinter, Germany) included measurement of cell necrosis and invasive growth.

Results Various colon cancer cell lines express HER1/EGFR and HER2 Nine diVerent colon cancer cell lines were tested for their expression of HER1/EGFR (Fig. 1a) and HER2 (Fig. 1b) by western blotting. The HER1/EGFR is a 170 kDa transmembrane receptor encoded by the human HER1/EGFR gene and HER2 was detected as a protein of approximately 185 kDa. The cell lines SW480, HT29, DLD-1, LS174T, HCT116 and Lovo showed the highest expression of the receptors (Fig. 1a, b). Modest expression was seen in SW48 and SW948 colon cancer cells, and no expression was detected in WIDR cell line. We detected robust HER1/ EGFR and HER2 overexpression in 67% of the nine colon cancer cell lines. EGF-induced cell cycle progression of colon cancer cells is blocked by pertuzumab We next tested the eVect of pertuzumab on cell cycle progression in colorectal cancer cells. First, we identiWed cell

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J Cancer Res Clin Oncol (2009) 135:13771386 Fig. 2 InXuence of pertuzumab on cell cycle distribution. Cell cycle distribution was determined by Xow cytometry 24 h after treatment of colon cancer cell lines SW480 (a), HT115 (b), SW948 (c) and Lovo (d). The graphs illustrate representative results of cell cycle distribution of untreated controls, and cells treated either with EGF, or with both EGF and pertuzumab

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SW480, HT115 and SW948 approximating cell cycle distribution of control cells. Cell cycle progression in EGF refractory HT29 cancer cells was not altered by pertuzumab (Fig. 2c). The expression levels of HER1/EGFR and HER2 obviously did not predict the EGF response and the antiproliferative eVect of pertuzumab in vitro. Pertuzumab inhibits growth of colon cancer xenograft tumors in nude mice EGF-induced cell cycle progression is blocked by pertuzumab most markedly in SW480 colon cancer cells, but no stable growth of xenograft tumors was obtained with this colon cancer cell line. The cell line SW948, on which pertuzumab was eVective in vitro in reducing EGF-induced cell cycle progression, was then successfully used to induce

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xenograft tumors in nude mice. After tumors were established (approximately 510 days post injection into both Xanks of NMRI nu/nu), mice were treated with either pertuzumab i.p. alone once a week, irinotecan i.p. alone once a week, or with the combination of the two drugs, while PBS served as control (n = 12/treatment arm with both Xanks of mice injected). Pertuzumab antitumor activity on human colon cancer xenograft tumors was examined in SW948 (Fig. 3a) and HCT116 (Fig. 3b) in combination with irinotecan, and in DLD-1 xenograft tumors (Fig. 3c) in combination with erlotinib. Tumor volume was measured twice weekly. While irinotecan strongly reduced tumor growth in

nude mice, the addition of pertuzumab to irinotecan in SW948 (Fig. 3a) and HCT116 (Fig. 3b) xenograft tumors did not enhance tumor growth reduction in vivo (Fig. 3a and b). In these cell lines, pertuzumab monotherapy had a small, non-signiWcant eVect on growth of xenograft tumors. Subsequently, pertuzumab was combined with erlotinib p.o. to further reduce HER1/EGFR signaling in colon cancer cells. Erlotinib was administered daily, while the monotherapy of both agents or captisol served as control. Pertuzumab showed minor antitumor activity in xenograft tumors from DLD-1 cells (Fig. 3c), but highly signiWcantly inhibited tumor growth when combined with the TKI erlotinib

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Fig. 3 a Growth inhibition of SW948 colon cancer xenografts in nude mice. SW948 cells were established as xenograft tumors in female NMRI nu/nu mice. Tumors were allowed to establish growth after implantation before initiation of treatment. Vehicle, CPT-11 (100 mg/ kg i.p. once weekly) and pertuzumab (12 mg/kg loading dose, followed by 6 mg/kg i.p. once weekly), or CPT-11 with pertuzumab was administered for the duration of the study. Tumor volume was determined twice weekly. Mean tumor volume (mm3) was plotted against time (days). Symbols mean tumor volume (mm3), bars SE (n = 12 mice per group). b EVect of pertuzumab and/or irinotecan (CPT-11) on growth of HCT116 human colon cancer cell xenograft tumors. HCT116 cells were implanted s.c. (both Xanks) on anesthetized NMRI nu/nu mice. Tumors were allowed to establish growth after implantation before initiation of treatment. Vehicle, CPT-11 (50 mg/kg i.p. once weekly) and pertuzumab (12 mg/kg loading dose, followed by 6 mg/kg i.p. once weekly), or CPT-11 with pertuzumab was administered for the duration of the study. Mean tumor volume (mm3) was plotted against time (days). Symbols mean tumor volume (mm3), bars SE (n = 12 mice per group). c EVect of pertuzumab and erlotinib in mono- and combination therapy on growth of DLD-1 human colon cancer xenograft tumors. DLD-1 xenograft tumors were allowed to establish growth after implantation before initiation of treatment. Treatment of mice consisted of oral gavage of erlotinib or vehicle [erlotinib (25 mg/kg per day, orally), Captisol], pertuzumab i.p. (12 mg/kg loading dose, following 6 mg/kg once weekly) or PBS i.p., or erlotinib with pertuzumab for the duration of the study. Mean tumor volume (mm3) was plotted against time (days). Symbols mean tumor volume (mm3), bars SE (n = 12 mice per group)

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(25.7%) was not increased compared with the control group (37.8%). DiVerent results were obtained with colon cancer cells HCT116 xenograft tumors (Fig. 5b), showing enhanced tumor necrosis up to 57.4% in pertuzumab monotherapy, followed by necrosis up to 42% and 40.6% in the CPT-11 mono and combination treatment arms. Here, the controls showed the smallest amount of necrosis with 29.9%.

Discussion Novel therapy strategies with targeting the ErbB network may be achieved by inhibiting the tyrosine kinases catalytic domain with small molecules (TKIs) or by inhibiting the extracellular receptor domain with monoclonal antibodies. In patients with colon cancer, epidermal growth factor receptor (HER1/EGFR) is overexpressed or up-regulated in 6080% and HER2 overexpression has been previously reported in up to 85% (Dursun et al. 2001; Mayer et al. 1993; Porebska et al. 2000; Ross and McKenna 2001). We detected HER1/EGFR and HER2 overexpression in six of nine colon cancer cell lines (Fig. 1). In a phase II trial in metastatic colon cancer, HER2 was overexpressed in 8% (11/138) of the patients tumor tissues. Partial response (PR) was seen in Wve of seven evaluable patients treated with irinotecan and trastuzumab. The study was closed due to low accrual (Ramanathan et al. 2004). The partial responses seen warranted the investigation of HER2 as a potential therapy target in colon cancer. Pertuzumab represents the Wrst HER2 dimerization inhibitor tested in clinical trials of patients with various solid tumors (Adams et al. 2006). Its antitumor activity is not restricted to tumors with HER2 overexpression and is diVerent from the therapeutic monoclonal antibody trastuzumab (Cobleigh et al. 1999; Franklin et al. 2004). Therefore, our choice of cell lines was not limited by the HER2 expression status. Cell cycle analysis showed an EGF induced cell cycle progression that was almost completely abolished by pertuzumab in EGF-dependent colon cancer cell lines SW480 (Fig. 2a), HT115 (Fig. 2b) and SW948

Fig. 4 Histo-pathological examination of colon cancer xenograft tumors. H.E. staining showing intratumoral necrosis of xenograft tumors after 34 weeks treatment. The amount of necrosis was determined using light microscopy

(mean tumor volume control: 1,065 mm3, erlotinib mono: 687 mm3, pertuzumab mono: 592 mm3, combination therapy: 432 mm3, P < 0.001). Histo-pathological examination of colon cancer xenograft tumors At the end of the 34 week treatment period, the xenograft tumors were harvested and examined histologically after H.E. staining showing intratumoral necrosis (Fig. 4). The amount of necrosis was estimated upon light microscopy using the DISKUS software. Necrosis was measured in DLD-1 xenograft tumors after treatment with pertuzumab alone and in combination with erlotinib. Untreated tumors served as negative controls (Fig. 4). Histological examination after H&E staining of DLD-1 xenograft tumors showed a slightly enhanced tumoral necrosis in pertuzumab monotherapy, but not in combination therapy (Fig. 5a). The amount of tumor necrosis in the treatment groups of pertuzumab (34.8%), erlotinib (33.8%) and in combination
Fig. 5 Amount of necrosis in colon cancer xenograft tumors after therapy. The amount of tumor necrosis per total tumor volume (%) in the diVerent treatment combinations was measured upon light microscopy in DLD-1 (a) and HCT116 (b) xenograft tumors. The number of examined tumors/group varied between 19 and 24

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(Fig. 2c). As expected, the antiproliferative eVect of pertuzumab in colon cancer cell lines in vitro was independent from their HER1/EGFR and HER2 status. Kuwada et al. demonstrated for the combination of cetuximab and trastuzumab, an additive inhibition of cell proliferation in EGF-responsive colon cancer cell lines Caco-2 and HCA-7, compared to either antibody alone, but showed no eVect on the cell proliferation of EGF refractory cancer cells DLD-1 and HT29 (Kuwada et al. 2004). Our study of colon cancer cells in vitro revealed that treatment with pertuzumab decreased the proliferation in colon cancer cells. The examination of cell cycle analysis by Xow cytometry was restricted to EGF responsive cancer cells. We further examined the antitumor activity of pertuzumab in vivo in colon cancer xenograft models based on various colon cancer cell lines. The combination therapy with pertuzumab and irinotecan was not superior to the standard chemotherapy agent irinotecan. In xenograft tumors from SW948 and HCT116 cell lines, pertuzumab monotherapy had a modest eVect on tumor growth which was statistically not signiWcant, probably because the ranges of tumor sizes using these two cell lines were large and the numbers of mice per treatment arm were too small for pertuzumab monotherapy to reach signiWcance. While irinotecan alone was very eVective in reducing tumor growth, pertuzumab could not enhance the irinotecan eVect on xenograft size. Targeted therapies in clinical trials for colon cancer patients failing standard therapy have gained at best a 10% response rate, despite the fact that 80% of colon cancers potentially use the HER network (Black et al. 2003; Normanno et al. 2003). HER1/HER2 or HER2/HER3 heterodimers may be responsible for the limited eYcacy and targeting HER2 alone by monoclonal antibody might not be suYcient for colon cancer treatment. A previous study has shown that targeting HER2 with pertuzumab failed to block GEO colon cancer cell proliferation when EGF is present in the medium (Jackson et al. 2004). More recently, a compensatory mechanism involving HER1/EGFR and HER2 for dimerization with HER3 was observed in GEO cells, when HER2 activation was downregulated by a speciWc antibody or the selective HER2 TKI AG879 (Hu et al. 2005). Because mabs and TKIs target the HER network at diVerent sites, we decided to explore whether the combined administration of such compounds is superior to monotherapy. The oral quinazoline erlotinib reversibly inhibits EGF receptor tyrosine kinase and reduces intratumoral HER1/EGFR autophosphorylation with no eVect on HER1/EGFR expression or surface receptor density (Pollack et al. 1999). In GEO, FET and HCT116 human colorectal cancer cell lines, erlotinib diminished HER1/EGFR activation but did

not aVect total expression compared with controls. In contrast, HER2 activation was increased in all cell lines. The inhibition of HER1/EGFR led to increased activation of HER2. This result suggests a possible mechanism which may rescue the cells after loss of anti-apoptotic signals resulting from HER1/EGFR blockade. The inhibition of multiple HER family members may yield stronger responses than single receptor blockade (Learn et al. 2006). The convenient oral application of erlotinib and its proven activity in phase II trial in colon cancer disease suggested to test the combination approach of pertuzumab with erlotinib (Meyerhardt et al. 2006). Clinical studies with erlotinib have shown that response to HER1/EGFR-targeted therapy is not correlated with HER1/EGFR expression (Prez-Soler et al. 2004). We examined the eVect of pertuzumab and erlotinib in mono- and combination therapy on growth of a well-established DLD-1 xenograft tumor model. Pertuzumab alone reduced tumor growth after 4 weeks of treatment in DLD-1 cells. Furthermore, when combined with erlotinib pertuzumab reduced tumor growth signiWcantly suggesting a synergistic eVect of the two agents. The data presented here suggest that the combination of diVerent classes of HER inhibitors can augment the antitumor response over that realized with a single HER inhibitor. The combination of erlotinib and pertuzumab was shown to be active against human non-small cell lung cancer (NSCLC) (Calu-3, QG56) and breast cancer cell (KPL-4) xenograft tumors, independently of their HER1/EGFR or HER2 expression status (Friess et al. 2005). Arpino et al. showed that combined treatment with geWtinib, trastuzumab, and pertuzumab blocked signals from all HER homo- and heterodimers and inhibited growth of HER2 overexpressing breast cancer xenografts of MCF7/ HER2-18 (HER2-transfected) or BT474 (HER2-ampliWed) cells, signiWcantly better than single agents and dual combinations. In the presence of the two HER2 antibodies trastuzumab and pertuzumab, geWtinib was needed for a complete HER signaling blockade indicating that HER1/ EGFR signaling may be important in activating HER pathways, even at low levels of HER1/EGFR expression. After blocking one HER pathway, tumors appeared to use an alternative HER pathway (Arpino et al. 2007). In the present study, the histo-pathological examination of the HCT116 xenograft tumors revealed enhanced necrosis in pertuzumab monotherapy, which constituted approximately 55% of the area of the tumor sections, compared with smaller amounts of necrosis in the other therapy and control groups (Fig. 5a). These results are in line with previous preclinical Wndings. e.g., histo-pathological analysis of ovarian carcinoma cell OVCA433 tumors from untreated mice revealed approximately 20% necrotic areas in the tumor sections. In contrast, 5060% of each of the tumor

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J Cancer Res Clin Oncol (2009) 135:13771386

1385 Black JD, Brattain MG, Krishnamurthi SA, Dawson DM, Willson JK (2003) ErbB family targeting. Curr Opin Investig Drugs 4:1451 1454 Carpenter G, Cohen S (1990) Epidermal growth factor. J Biol Chem 265:77097712 Cobleigh MA, Vogel CL, Tripathy D et al (1999) Multinational study of the eYcacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 17:26392648 Dursun A, Poyraz A, Suer O, Sezer C, Akyol G (2001) Expression of Bcl-2 and c-ErbB-2 in colorectal neoplasia. Pathol Oncol Res 7:2427 Ferlay J, Bray F, Pisani P et al (2001) GLOBOCAN 2000: cancer incidence, mortality and prevalence worldwide, version 1.0. IARC cancerbase no. 5. IARC, Lyon, France Fields LA, Rinaldi D, Henderson CA et al (2005) An open-label multicenter phase II study of oral Lapatinib (GW572016) as single agent, second-line therapy in patients with metastatic colorectal cancer. J Clin Oncol 23:3583 Franklin MC, Carey KD, Vajdos FF, Leahy DJ, deVos AM, Sliwkowski MX (2004) Insights into ErbB signaling from the structure of the ErbB-2-pertuzumab complex. Cancer Cell 5:317 328. doi:10.1016/S1535-6108(04)00083-2 Friess T, Bauer S, Burger AM (2002) In vivo activity of recombinant humanized monoclonal antibody 2C4 in xenografts is independent of tumor type and degree of HER-2 overexpression. Eur J Cancer 38:149156 Friess T, Scheuer W, Hasmann M (2005) Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy. Clin Cancer Res 11:53005309. doi:10.1158/1078-0432.CCR-04-2642 Grunwald V, Hidalgo M (2003) Development of the epidermal growth factor receptor inhibitor Tarceva (OSI-774). Adv Exp Med Biol 532:235246 Hemming AW, Davis NL, Kluftinger A et al (1992) Prognostic markers of colorectal cancer: An evaluation of DNA content, epidermal growth factor receptor, and Ki-67. J Surg Oncol 51:147152. doi:10.1002/jso.2930510304 Hu YP, Venkateswarlu S, Sergina N et al (2005) Reorganization of ErbB family and cell survival signalling following knockdown of ErbB-2 in colon cancer cells. J Biol Chem 280:2738327392. doi:10.1074/jbc.M414238200 Jackson JG, St. Clair P, Sliwkowski MX, Brattain MG (2004) Blockage of epidermal growth factor- or heregulin-dependent ErbB-2 activation with the anti-ErbB-2 monoclonal antibody 2C4 has divergent downstream signalling and growth eVects. Cancer Res 64:26012609. doi:10.1158/0008-5472.CAN-03-3106 Johnson JR, Cohen M, Sridhara R et al (2005) Approval summary for erlotinib for treatment of patients with locally advanced or metastatic non-small cell lung cancer after failure of at least one prior chemotherapy regimen. Clin Cancer Res 11:64146421. doi:10.1158/1078-0432.CCR-05-0790 Kapitanovic S, Spaventi R, Poljak L et al (1994) High c-erbB-2 protein level in colorectal adenocarcinomas correlates with clinical parameters. Cancer Detect Prev 18:97101 Kapitanovic S, Radosevic S, Kapitanovic M et al (1997) The expression of p185 (HER-2/neu) correlates with the stage of disease and survival in colorectal cancer. Gastroenterology 112:11031113. doi:10.1016/S0016-5085(97)70120-3 Kuwada SK, Scaife CL, Kuang J et al (2004) EVects of trastuzumab on epidermal growth factor receptor-dependent and -independent human colon cancer cells. Int J Cancer 109:291301. doi:10. 1002/ijc.11686 Learn PA, Krishnegowda N, Talamantez J, Kahlenberg MS (2006) Compensatory increases in Her-2/neu activation in response to

sections of mice treated with pertuzumab revealed necrosis, histologic changes of apoptosis and Wbrosis involved approximately 30% of the tumor area (Takai et al. 2005). The histo-pathological examination of the DLD-1 xenograft tumors in the present study did not indicate enhanced tumor necrosis by combination therapy with pertuzumab and erlotinib, in comparison to the monotherapy regimens. Previous preclinical data using the EGF refractory cell line DLD-1 and HT29 have shown no increased cancer cell necrosis after 2 weeks of therapy with HER signaling blocking antibodies trastuzumab and cetuximab (Kuwada et al. 2004). However, Prewett el al. showed enhanced tumor necrosis, tumor cell apoptosis and a reduction in tumor vasculature in DLD-1 and HT29 xenograft tumors treated with a combination of cetuximab and CPT-11 (Prewett et al. 2002). Large areas of necrosis in xenografts may be indicative of ischemia due to the compromised tumor vascularization following inhibition of HER1/EGFR and HER2. We showed in this preclinical study that pertuzumab reduces the growth of colon cancer cells in vitro and in vivo, in particular when combined with erlotinib. Both drugs are known to have less severe side eVects than standard chemotherapeutic regimens. Pertuzumab treatment in combination with irinotecan standard chemotherapy in the murine xenograft model was not superior to monotherapy. These preclinical data warrant further investigation of simultaneous HER1/EGFR and HER2 dimerization inhibition for colorectal cancer treatment.
Acknowledgments The compounds erlotinib and pertuzumab were supplied by Roche Diagnostics GmbH, Penzberg, Germany. This work was supported by grants from the Forschungsfrderung der RuhrUniversitt (FoRUM), Germany.

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