Pharmacoproteomics Study of Cetuximab in Nasopharyngeal

Carcinoma. Fion L. Sung,† Ronald T. K. Pang,§ Brigette B. Y. Ma,‡ May M. L. Lee,§ Shuk Man Chow,‡. Terence C. W. Poon,§ and Anthony T. C. Cha...
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Pharmacoproteomics Study of Cetuximab in Nasopharyngeal Carcinoma Fion L. Sung,† Ronald T. K. Pang,§ Brigette B. Y. Ma,‡ May M. L. Lee,§ Shuk Man Chow,‡ Terence C. W. Poon,§ and Anthony T. C. Chan*,†,‡ Hong Kong Cancer Institute, Department of Clinical Oncology, and Department of Medicine and Therapeutics, The Sir Y. K. Pao Center for Cancer, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China Received December 12, 2005

Epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC). Our recent in vitro study has demonstrated that cetuximab (an antibody drug against EGFR) inhibits the growth of NPC cell lines, HK1 and HONE-1. The present study investigates the effect of cetuximab on protein expressions of NPC cell lines. NPC cells were cultured in the absence or presence of cetuximab at the IC50 concentrations (3 nM for HK1 and 0.3 nM for HONE-1) for 48 h, and total cell lysates were extracted. The cell lysates were then subjected to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and the 2D gel images were compared to discover the protein changes caused by cetuximab treatment. The common differentially expressed proteins in NPC cell lines were identified by peptide mass fingerprinting. We found that heat shock protein gp96 was down-regulated, while R-enolase, tumor suppressor protein maspin, and p97 valosin containing protein were up-regulated after cetuximab treatment. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that the changes in protein levels of gp96, maspin, and p97 coincided with mRNA levels, indicating that these proteins were regulated at transcriptional levels. Up-regulation of gp96 has been observed in various cancers and reported to have tumor protective effects. P97 is a multifunctional AAA (ATPase associated with a variety of activities) protein and is involved in numerous cellular activities including membrane transport, protein folding, protein degradation, and cell division. Maspin has been shown to increase apoptosis, and block the growth, invasion, and metastatic properties of many tumors. The comparative tumor suppression effects of cetuximab and maspin suggest that cetuximab might exert its antitumor effects partly by up-regulation of maspin expression. The study also indicates that proteomic analysis is a promising approach to elucidate the functional mechanisms of anticancer drugs. Pharmacoproteomic study may also help to identify clinical responders for drug treatment and provide insight for new drug development. Keywords: epidermal growth factor receptor • C225 • maspin • p97/VCP • heat shock protein gp96

Introduction During the past decade, a large number of studies indicated that the epidermal growth factor receptors (EGFR) and their ligands are important factors in regulating cell cycle, angiogenesis, metastasis, and apoptosis in tumor cells.1,2 EGFR is frequently overexpressed in human tumors; examples include cancers of the lung, breast, head and neck, bladder, colorectal, ovary, and prostate.3 Increased EGFR expression correlates with a poorer clinical outcome in malignancies including bladder,4 breast,5 lung,6 and head and neck.7 In addition, the increased expression of the receptor is often associated with the increased * Corresponding author. Tel.: (852) 2632 2119. Fax: (852) 2649 7426. E-mail: [email protected]. † Hong Kong Cancer Institute. ‡ Department of Clinical Oncology. § Department of Medicine and Therapeutics.

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amount of ligands production within the same tumor cell that forms an autocrine stimulatory pathway.4 These observations have led to the development of anti-EGFR strategies that target different components of the EGFR signaling network or cells that express EGFRs. One of the strategies that has promising results is the use of a human-mouse chimeric monoclonal antiEGFR antibody (cetuximab, C225). This antibody shows high specificity against the EGFR.9 It is a potent inhibitor of the growth of cultured cancer cells that have an active autocrine EGFR mechanism and is able to induce the regression of wellestablished human tumor xenografts that overexpress EGFR.10 It has been applied into phase III trial in combination with cisplatin for patients with metastatic or recurrent head and neck cancer11 and in combination with gemcitabine for patients with locally advanced unresectable or metastatic adenocarcinoma of the pancreas.12 It has also been approved by the U.S. 10.1021/pr050452g CCC: $33.50

 2006 American Chemical Society

Proteomic Changes of Cetuximab-Treated NPC Cells

Food and Drug Administration (FDA) to be used for the treatment of EGFR-expressing metastatic colorectal cancer under specified clinical situation.13 The antitumor effects of cetuximab against many other EGFR-expressing tumors, in a single modality or in combination with radiation or chemotherapy, are still under active investigation in laboratory and clinic. Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China, especially in the Cantonese region around Guangzhou, where the incidence rate is as high as 15-50 per 100 000 people per year.14 Overexpression of EGFR is common in NPC.15-19 Our previous immunohistochemical study indicated that there is moderate to strong expression of EGFR in tumor among 85% of the Chinese patients with undifferentiated NPC.20 Moreover, strong expression of EGFR was associated with poor overall survival in patients with advanced tumor. These findings urge us to investigate the effect of cetuximab in NPC cell lines. Our recent in vitro study has demonstrated that cetuximab inhibits the growth of NPC cell lines, HK1 and HONE-1.21 We have also carried out a phase II study of cetuximab in combination with carboplatin in patients with recurrent or metastatic nasopharyngeal carcinoma. Cetuximab in combination with carboplatin demonstrates clinical activity and an acceptable safety profile in those patients.22 In this study, we aimed to investigate the effect of cetuximab on protein expression of the NPC cell lines by comparative proteomic analysis. The identified proteins may play a significant role in the signaling machinery of cetuximabinduced tumor suppression effects.

Methods and Materials Cell Culture and Cetuximab Treatment. Human NPC cell lines including HONE-1 and HK1 were studied. The HONE-1 cell line was established from poorly differentiated nasopharyngeal carcinomas,23 and the HK1 cell line was established from well-differentiated squamous nasopharyngeal carcinomas.24 HONE-1 cells and HK1 cells were cultured in RPMI-1640 medium with antibiotics (100 units/mL penicillin and 10 µg/ mL streptomycin) and supplemented with 5% and 10% fetal bovine serum (FBS), respectively. Cell cultures were maintained at 37 °C in a humid atmosphere of 5% carbon dioxide in air. All culture medium and reagents were purchased from GIBCO BRL (GIBCO BRL, Grand Island, NY). For experiments, NPC cells were cultured in the absence and presence of cetuximab with a concentration close to IC50 (3 nM for HK1 and 0.3 nM for HONE-1) for 48 h. Cell Lysate Preparation. The NPC cells were washed twice with Hanks buffer supplemented with 20 mM HEPES (pH 7.0) and once with 10 mM Tris-Cl (pH 7.0) containing 250 mM sucrose. Next, the cells were lysed in cell lysis buffer (8 M urea, 4% w/v CHAPS, 40 mM Tris, and 65 mM DTT) by the sample grinding kit (Amersham Biosciences, Piscataway, NJ) with the assistance of a hand-held motor. The protein concentrations of each sample were quantified with the RC DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). All of the samples were stored at -20 °C until use. Two-Dimensional Polyacrylamide Gel Electrophoresis. The first dimensional isoelectric focusing (IEF) was performed on 11 cm immobilized pH 3-10 nonlinear gradient strips (BioRad Laboratories, Inc., Hercules, CA). Eighty micrograms of total cellular protein was mixed with IEF rehydration buffer (Bio-Rad Laboratories, Inc., Hercules, CA), to a total volume of 180 µL. The mixture was passively rehydrated into the IPG

research articles strips for 16 h. IEF was carried in 20 °C under the following conditions: 100 V, 50 V h; 200 V, 200 V h; 500 V, 500 V h; 1000 V, 1000 V h; 8000 V, 24 000 V h. After IEF, the gels were washed briefly with water and equilibrated for 15 min in 6 M urea, 0.51 mM EDTA, 141 mM Tris-base, 106 mM Tris-Cl, 2% SDS, 20% glycerol, and 1% DTT (Sigma, St. Louis, MO). Next, the strips were equilibrated in 6 M urea, 0.51 mM EDTA, 141 mM Trisbase, 106 mM Tris-Cl, 20% glycerol, and 4% iodoacetamide (Sigma, St. Louis, MO). Finally, the strips were equilibrated in 6 M urea, 0.51 mM EDTA, 141 mM Tris-base, 106 mM Tris-Cl, 2% SDS, and 20% glycerol for an additional 10 min. The second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with the Criterion gel system (Bio-Rad Laboratories, Inc., Hercules, CA) in 4-12% gradient gel. The gels were silver stained with the Plus-one silver staining kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. All of the samples were run at least in duplicate. Image Acquisition and Data Analysis. The gels were scanned with a densitometer GS-700 (Bio-Rad Laboratories, Inc., Hercules, CA) as raw 2-DE images. After scanning, all of the gels were stored in 4 °C before further processing. The gel images were processed and compared using the PDQuest 2-D Analysis Software (version 7.11, Bio-Rad Laboratories, Inc., Hercules, CA). The Spot Detection Wizard was used to select the parameters for detecting spots in the gel images. By using the Spot Detection Wizard, the faintest spot, smallest spot, and largest spot were selected by naked eye. Next, a box was drawn around a large representative region of the most representative gel image to allow the PDQuest to select the correct noise filter. For background subtraction, streak removal, and speckle removal, the default settings were used. Before spot detection, the original gel images were filtered and smoothed to clarify the spots. Next, the three-dimensional Gaussian spots were created from the clarified spots. The normalized volume of a Gaussian spot was directly proportional to the concentration of the corresponding protein in the sample preparation. The gel image containing the most number of spots was used to generate a Master template for spot indexing and matching. The unique spots in the other gel images were added to the Master template while matching the spots of individual gel images against the Master template. The auto-matching function was used for spot indexing and matching automatically, followed by menu-editing to remove the wrong matchings and add the right matchings. The quantities of all of the spots were normalized as total valid spots in each of the images. Protein quantities of 2-fold up- or down-regulation between the two groups were considered as differentially expressed spots. Protein Identification by Peptide Mass Fingerprinting. After gel images analysis, proteins of interests were excised from the gels and placed into Eppendorf tubes. The gel pieces were first washed with milli-Q water, and then destained with a freshly prepared mixture of 100 mM sodium thiosulfate and 30 mM potassium ferricyanide(III) in a 1:1 ratio. Next, the gels pieces were washed with milli-Q water, 50% methanol, 10% acetic acid, and 25 mM NH4HCO3, respectively. Afterward, the samples were reduced and alkylated with 10 mM DTT and 50 mM IAA in 25 mM NH4HCO3, respectively. Next, the gel pieces were washed again with 25 mM NH4HCO3, dehydrated with 80% ACN, 25 mM NH4HCO3. After drying, the gel pieces were rehydrated with digestion buffer (0.01% n-octylglucoside in 25 mM NH4HCO3, pH 8.0) containing 50 ng of sequencing grade Journal of Proteome Research • Vol. 5, No. 12, 2006 3261

research articles trypsin (Sigma, St. Louis, MO). Digestion was carried out at 37 °C overnight. Digested peptides were extracted twice with 70% ACN in 0.1% TFA, and the pooled samples were vacuum-dried. The recovered peptides were spotted on a MALDI-TOF MS sample plate and overlaid with R-cyano-4-hydroxycinnamic acid (CHCA) as matrix. All of the samples were detected by the Voyager DE-Pro MALDI-TOF MS system (Applied Biosystem, Foster city, CA). The acquired masses were calibrated with trypsin autolyzed peptides as internal references masses. The acquired masses were submitted to Internet programs ProFound (http://prowl.rockefeller.edu/profound_bin/WebProFound.exe) for searching the NCBInr database. Proteins were considered to be successfully identified when the ProFound probability was 1.0 and the estimated Z score was higher than 1.65.25 RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from NPC cells with TRIzol reagent (Invitrogen, CA). Two micrograms of DNase-treated total RNA was used for RT with Superscript III (Invitrogen, CA). Four microliters of each one-tenth-fold cDNA was amplified by PCR using FastStart Taq DNA polymerase (Roche Diagnostic GmbH, Mannheim, Germany). The forward primer 5′-CTT TCT ACA GAG TTC ATC AGC-3′ and reversed primer 5′-CAA TCT TCT CCA AGC CTG TG-3′ were used for maspin amplification. The forward primer 5′-ATC TGC TCC ACG TGG TCT G-3′ and reversed primer 5′-ACC ATT CAA GGG GAG ATC AT-3′ were used for gp96 amplification. The forward primer 5′-CCC AAG TCA TTG TTT TTC TCG-3′ and reversed primer 5′-CTC ATG GGT CAC TGA GGC T-3′ were used for R-enolase amplification. The forward primer 5′-GAG AGG ATG AGG AAG AGT CC-3′ and reversed primer 5′-CTC CAA GTC CAC ATC CTT GG-3′ were used for p97 amplification. The forward primer 5′-GTG GGG CGC CCC AGG CAC CA-3′ and reversed primer 5′-CTC CTT AAT GTC ACG CAC GAT TT-3′ were used for β-actin amplification as the loading control. The PCR of maspin, gp96, and enolase is composed of denaturation at 95 °C for 30 s, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min, followed by a final incubation at 72 °C for 7 min. The PCR of p97 and β actin have similar conditions except for the annealing at 50 and 56 °C for 1 min, respectively. The amplified product was analyzed by electrophoresis on agarose gel and stained with the SYBR Safe DNA gel stain (Invitrogen, OR). Next, the individual gene expression was normalized by β-actin expression, and the relative ratio of gene expression of cetuximab-treated to nontreated cell line was calculated for each NPC cell line sample.

Results NPC cells were incubated in the absence or presence of cetuximab, and cell lysates were then subjected to 2D gel electrophoresis followed by silver staining. Figure 1 shows a representative 2D gel image of the NPC cell lysates after silver staining. Around 900 protein spots were detected on the 2D gels and localized in the ranges of pI 3-10 and Mr 10-250 kDa. The gel images were then subjected to computer analysis by PDQuest 2-D Analysis Software. Individual gels were normalized relative to the total density in each images, and the quantitative analysis is based on a 2-fold cutoff. The results revealed that two proteins were down-regulated and eight proteins were up-regulated in NPC cells after cetuximab treatment. The positions and the spot identification numbers of some of the differentially expressed proteins are shown in Figure 1. 3262

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Figure 1. Representative 2D gel images of the NPC cell lines. Control and cetuximab-treated NPC cells were lysed, and cellular proteins were separated by 2D PAGE. Proteins in the resulting gels were detected by silver-staining. The upper panel shows a representative 2D gel image of the NPC cell lysates. Differentially expressed protein spots were labeled by their standard spot number determined by computer software PDQuest. The lower panel shows the zoom view images of the differentially expressing spots.

These differential expressed protein spots were excised from the gels and subjected to trypsin peptide mass fingerprinting. Figure 2 shows the representative MALDI-TOF trypsin peptide mass spectra of the differentially expressed proteins. The identities of the proteins were finally determined by the Internet identification tool “ProFound”. The protein identities of the differentially expressed proteins and their relative abundances are summarized in Table 1. All of the proteins identified have a probability of matching equal to 1 and Z score higher or equal to 1.65 (less than 5% of random matching can generate score higher than the peptides). The down-regulated protein identified was heat shock protein gp96, and the three up-regulated proteins identified were valosin containing protein (p97 or VCP), MASP human tumor suppressor protein maspin, and R-enolase. RT-PCR analyses were performed for the four identified differentially expressed proteins. The results showed that the mRNA levels of gp96 were decreased and those of p97 and maspin were increased in NPC cells cetuximab treatment. The changes in mRNA levels of these three proteins coincided with the protein expression in the two NPC cell lines tested. However, R-enolase mRNA levels were only increased in HK1 cells but decreased in HONE-1 cells (Figure 3). The results from RT-PCR analysis suggested that the changes in the protein levels of gp96, p97, and maspin were mediated at least partly by a transcriptional-dependent pathway derived from the change of mRNA levels.

Discussion Cetuximab, which is also known as C225, is a chimeric human-mouse monoclonal antibody against EGFR. It has been shown to be a potent anticancer drug that specifically inhibits the ligand binding of the EGFR. It has already been approved

Proteomic Changes of Cetuximab-Treated NPC Cells

research articles mechanisms for its antitumor effects are not fully understood. Some of the molecular pathways identified for its antitumor effects include down-regulation of the angiogenic factors VEGF, bFGF, IL-8, and MMP-9, which reduce the tumor-induced angiogenesis and metastatic potential,29-33 up-regulation of the cyclin-dependent kinase inhibitor p27Kip1, leading to cell cycle arrest at the G1-S checkpoint,34,35 and blockage of EGF-induced autophosphorylation of EGFR33 in which the downstream signaling pathway is blocked. Our previous studies have shown that EGFR was expressed in more than 85% of NPC20 and cetuximab exhibits antitumor effect in NPC cell lines HK1 and HONE-1.21 In this study, the molecular effects of cetuximab in NPC cell lines were elucidated through proteomic approaches. We have identified one down-regulated protein, heat shock protein gp96, and three up-regulated proteins, p97 valosin containing protein (p97 or VCP), MASP human tumor suppressor protein maspin, and R-enolase in cetuximab-treated NPC cells. RT-PCR analyses showed that the mRNA levels of gp96, p97, and maspin matched the changes of protein levels in NPC cells after cetuximab treatment. Heat shock protein gp96, abbreviated as gp96, belonged to the HSP90 family. Heat shock proteins are molecular chaperones and are involved in proper folding of nascent polypeptides, protecting polypeptides from denaturing during cell stress, antigen presentation.36 In general, the expression level of heat shock protein increases during cellular stress. However, over-production of heat shock proteins protects malignant cells from apoptotic cell death and fosters resistance to chemotherapeutic drugs and irradiation.37 Gp96 up-regulation has been reported in several cancers including colorectal cancer,38 esophageal cancer,39 malignant mesothelioma,41 and hepatocellular carcinoma (HCC).41 The tumorigenicity of colon carcinoma cells was increased by gp96.42 In addition, the degree of gp96 elevation was significantly correlated with the progression of hepatitis B virus induced disease, being the highest in HCC patients, the lowest in chronic HBV infection, and cirrhosis in the middle.43 In the present study, we showed that the expression of gp96 was down-regulated in cetuximabtreated NPC cells. It is possible that cetuximab-mediated gp96 reduction could help to reduce the tumorigenicity and the viability of NPC cells.

Figure 2. MALDI-TOF peptide mass spectrum of tryptic digest of the differentially expressed proteins in cetuximab-treated NPC cells. (A) Heat shock protein gp96, (B) valosin containing protein, (C) human tumor suppressor protein maspin, and (D) R-enolase.

by the U.S. FDA to be used in combination with irinotecan for the treatment of EGFR-expressing, metastatic colorectal cancer in patients who had failed to improve with irinotecan-based chemotherapy and used as a single agent in the treatment of patients with EGFR-expressing, metastatic colorectal cancer who are intolerant to irinotecan-based chemotherapy.13 Cetuximab also has efficacy in cancers of lung and head and neck26,27 and does not exacerbate the side effects of co-administrated cytotoxic chemotherapy.28 The uses of cetuximab to treat patients with other EGFR-expressing cancers are actively investigated in preclinical studies as well as clinical trials. The

Valosin-containing protein (VCP), which is also called p97, is a member of the AAA protein (ATPase associated with a variety of cellular activities) superfamily. VCP couples ATP hydrolysis to the unwinding, disassembly, unfolding, or extraction of substrates. The N-terminal domain of VCP can bind polyubiquitinated proteins. It is a multifunctional protein that participates in many cellular processes including membrane transport, membrane fusion, protein folding, ubiquitin-proteasome degradation, cell division, and apoptosis.44,45 VCP is an abundant protein and expressed ubiquitously in all tissues.46 Alternation of VCP expression might interfere with numerous cellular activities. VCP may participate in the DNA damagerepair function, which is regulated by phosphorylation47 and interaction with BRCA1 protein.48 Some functions of VCP might be cell type specific and dependent on physiological conditions. RNA interference (RNAi) of VCP induced apoptosis in HeLa cells.45 Expression of the mutant VCP led cells to undergo apoptosis in murine BAF/B03 pro-B cells49 and increased cell death in rat neuronal PC12 cells.46 In this study, VCP was found to be up-regulated in both NPC cell lines after treatment with cetuximab. It is probably that up-regulation of VCP in NPC cells would help to remove mutated protein and increase apoptosis, Journal of Proteome Research • Vol. 5, No. 12, 2006 3263

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Table 1. Summary of the Profound Matching Scores and the Relative Abundance Ratio of the Differentially Expressed Proteins in Cetuximab-Treated NPC Cells

SWISS-PROT accession no.

SWISS-PROT entry name

spot number

protein description

Proteins Down-regulated in C225-Treated Cell Lines P14625 ENPL_HUMAN 1901 heat shock protein gp96 Proteins Up-regulated in C225-Treated Cell Lines P55072 TERA_HUMAN 2807 P97 valosin containing protein P36952 MASP_HUMAN 4402 maspin P06733 ENOA_HUMAN 6606 R-enolase 7601 R-enolase

profound profound estimated probability Z score

HONE-1

%

Mr/pI

Mr/pI

1.83

0.3

0.4

14

92.4/4.8

96.0/4.2

1.0

2.40

2.9

2.7

17

89.3/5.1

77.5/5.1

1.0 1.0 1.0

2.35 1.82 2.28

2.3 3.2 2.1

3.2 2.0 2.6

37 53 33

42.1/5.7 47.2/7.0 47.2/7.0

38.6/6.0 49.2/7.4 48.7/7.6

Figure 3. Expression of mRNA of differentially expressed protein in NPC cells after treatment with cetuximab. NPC cells were incubated in the absence or presence of cetuximab for 48 h. At the end of incubation, cells were collected and total RNA was extracted. RT-PCR was then performed. The PCR amplified cDNA were separated by agarose gel electrophoresis and staining with the SYBR Safe DNA gel stain. The β-actin was used as the loading control. These are representative gel pictures taken from two separate experiments. Journal of Proteome Research • Vol. 5, No. 12, 2006

HK1

1.0

leading to decreased viability of the tumor cells. The exact roles of VCP in NPC cells need further investigation. Maspin is a 42 kDa cytoplasmic protein and belongs to the serine protease inhibitor (serpin) family. Maspin was first identified as a tumor suppressor protein that may play a role in breast cancer. Its expression was detected in epithelia of many human organs but not in most mammary carcinoma cell lines.50,51 Low levels of maspin have also been found in other cancers such as prostatic cancer,52 pulmonary adenocarcinoma,53 bladder carcinoma,54 and non-small-cell lung cancers.55 Higher tumoral maspin expression is associated with improved survival of patients with oral squamous cell carcinoma56 and pulmonary adenocarcinoma.53 Maspin can inhibit the invasion, motility, metastasis, and angiogenesis when overexpressed. For example, loss of maspin gene expression in breast cancer increases its invasiveness and metastasis in vitro and in vivo.51 Maspin-transfected carcinoma cells demonstrated reduced tumorigenicity, an increased rate of spontaneous apoptosis, and reduced cell invasion in vitro and metastatic spread in vivo.57 Maspin also dramatically reduced the density of tumorassociated microvessels in a prostate cancer xenograft mouse model.58 In addition, maspin is involved in tumor cell apoptosis.59-61 In the present study, we found that maspin was up-regulated in NPC cells treated with cetuximab. In vitro

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abundance ratio: sequence cetuximab to control coverage theoretical experimental

studies and xenograft models have also demonstrated that cetuximab has an antitumor effect against various cancers by inhibiting tumor growth, inducing apoptosis, and reducing angiogenesis and metastatic potential.10,62,63 The tumor suppression effects of maspin share similarities with the antitumor effects of cetuximab; therefore, maspin may play a key role in the functional mechanisms of antitumor effects of cetuximab. Binding of cetuximab to EGFR has been shown to block the activation of PI3K/PTEN/AKT and RAS/Raf/MEK/ERK signaling pathways,64,65 and this binding may also modulate other signaling pathways. The modulation of the EGFR signaling pathways may lead to the regulation of transcription factors including p53 activation.66 Zou et al. have demonstrated that activated p53 can bind to the promoter region of maspin gene,67 which may lead to an increase in the transcription of the maspin gene and protein level. Maspin may help to exert the antitumor effects of cetuximab by regulating the expression of other proteins or interacting with other pre-existing proteins. Recent studies have shown that maspin can reduce the level of anti-apoptotic protein Bcl-2 and increase the level of proapoptotic protein Bax.68,69 A similar observation has been also seen when cells of head and neck squamous cell carcinomas are exposed to cetuximab.70 In addition, maspin can interact with glutathione S-transferase (GST),71 interferon regulatory factor-6 (IRF-6),72 and urokinase-type plasminogen activator/ urokinase-type plasminogen activator receptor (uPA/uPAR)73 to exert antitumor effects at cellular levels. In addition, Hendrix et al. showed that maspin was phosphorylated on tyrosine moieties in normal epithelial cells.74 They also demonstrated that recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the EGFR in vitro. Therefore, maspin may also act on other signaling pathways in its tyrosinephosphorylated form. The involvement of maspin in other signaling pathways and the functional activities between different forms of maspin remain to be further elucidated. Taking together, maspin may play an important role in the functional mechanisms of antitumor effects of cetuximab in NPC. Preclinical and clinical studies indicate that cetuximab demonstrates antitumor effects in a variety of cancers either as a single agent or in combination with chemotherapeutic agents or radiation.21,26-28,75-79 Study also indicates that the expression of EGFR in tumors is not the only criteria for predication of response for EGFR inhibitions. In studies of nonsmall-cell lung cancers, the EGFR expression levels showed no correlation with sensitivity to ZD1839 and cetuximab.26,80 Our in vitro study of cetuximab in NPC cell lines demonstrated different activity in two NPC cell lines (HONE-1 and CNE-2) with similar EGFR expression.21 A phase II clinical study of

Proteomic Changes of Cetuximab-Treated NPC Cells

single agent cetuximab in colon cancer also suggested that the clinical activity of cetuximab did not seem to correlate with the level of EGFR expression.81 In our clinical phase II study of cetuximab plus carboplatin in patients with recurrent or metastatic NPC resistant to platinum treatment, all patients enrolled demonstrated EGFR expression in target tumor. The study has showed that cetuximab in combination with carboplatin demonstrates clinical activity with a 60% disease control rate. However, there were a significant number of nonresponders, and no obvious association between the level of EGFR expression and best response or survival time was found.22 It seems that the level of EGFR is not a sufficient predictive marker of cetuximab response. Other factors may also affect cetuximab responses. By studying the functional mechanism of cetuximab, it helps to identify potential responder for cetuximab treatment. In the present study, maspin, a tumor suppressor protein, may play a key role in the functional mechanisms of antitumor effects of cetuximab. Maspin may be a potential indicator for biological efficacy after cetuximab treatment. It is also interesting to know whether a low level of maspin is a prerequisite for cetuximab responders. In addition, pharmacoprotemic study may also provide insight in new drug development. By studying the proteome changes after an antitumor drug treatment, it provides information for the signaling mechanism and effectors involved in its tumor suppression effects. Potential drug or improved analogues with higher efficacy and lower adverse effects may be developed along this signaling pathway. Furthermore, for regimen with combination of different drugs or treatments, they should target different mechanisms of action to further enhance the tumor suppression effect when compared to single drug or treatment is used. Pharmacoproteomics study of drugs can also help to combine the drugs most effectively. By investigating the proteomic changes of NPC cells affected by cetuximab treatment, we identified maspin as a potential molecular pathway for the observed phenotypic changes in cetuximab-treated tumor cells. The study also indicates that proteomic analysis is a promising approach to elucidate the functional mechanisms of anticancer drugs.

Acknowledgment. This work was supported by a Direct Grant for Research and a Strategic Research Area Grant from the Chinese University of Hong Kong, and a Central Allocation Grant from the University Grants Committee of Hong Kong. We thank Merck KgaA for supplying the Cetuximab. References (1) Baselga, J. The EGFR as a target for anticancer therapy - focus on centuximab. Eur. J. Cancer 2001, 37, S16-S22. (2) Baselga, J. Why the epidermal growth factor receptor? The rationale for cancer therapy. Oncologist 2002, 7, Suppl 4, 2-8. (3) Salomon, D. S.; Brandt, R.; Ciardiello, F.; Normanno, N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit. Rev. Oncol. Hematol. 1995, 19, 183-232. (4) Neal, D. E.; Sharples, L.; Smith, K.; Fennelly, J.; Hall, R. R.; Harris, A. L. The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer 1990, 65, 1619-1625. (5) Sainsbury, J. R.; Farndon, J. R.; Sherbet, G. V.; Harris, A. L. Epidermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985, 1, 364-366. (6) Pavelic, K.; Banjac, Z.; Pavelic, J.; Spaventi, S. Evidence for a role of EGF receptor in the progression of human lung carcinoma. Anticancer Res. 1993, 13, 1133-1137. (7) Santini, J.; Formento, J. L.; Francoual, M.; Milano, G.; Schneider, M.; Dassonville, O.; Demard, F. Characterization, quantification, and potential clinical value of the epidermal growth factor receptor in head and neck squamous cell carcinomas. Head Neck 1991, 13, 132-139.

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