Global Gene Expression Profile of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Complementary DNA Microarrays

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4944 Vol. 10, 4944–4958, August 1, 2004 Clinical Cancer Research

Featured Article Global Gene Expression Profile of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Complementary DNA Microarrays

Virote Sriuranpong,1,3 Apiwat Mutirangura,4 expressed in tumors included genes involved in apoptosis John W. Gillespie,2 Vyomesh Patel,1 (B-cell CLL/lymphoma 6, secretory leukocyte protease in- Panomwat Amornphimoltham,1 hibitor, and calpastatin), cell structure (keratin 7 and car- 1 cinoembryonic antigen-related cell adhesion molecule 6), Alfredo A. Molinolo, and putative tumor suppressor genes (H-Ras-like suppres- 5 Veerachai Kerekhanjanarong, sor 3, retinoic acid receptor responder 1, and growth ar- Siripornchai Supanakorn,5 rested specific 8) among others. Gene expression patterns Pakpoom Supiyaphun,5 Samreung Rangdaeng,6 also suggested alterations in the Wnt/␤-catenin and trans- ␤ Narin Voravud,3 J. Silvio Gutkind1 forming growth factor pathways in nasopharyngeal carcinoma. Thus, expression profiles indicate that aberrant 1Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, and 2Laboratory of Pathology, National Cancer expression of growth, survival, and invasion-promoting Institute, NIH, Bethesda, Maryland; 3Medical Oncology Unit, genes may contribute to the molecular pathogenesis of Department of Medicine, 4Genetics Unit, Department of Anatomy, nasopharyngeal carcinoma. Ultimately, this approach may and 5Department of Otolaryngology, Faculty of Medicine, 6 facilitate the identification of clinical useful markers of dis- Chulalongkorn University, Bangkok, Thailand; and Department ease progression and novel potential therapeutic targets for of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand nasopharyngeal carcinoma.

ABSTRACT INTRODUCTION A number of genetic and epigenetic changes underlying Nasopharyngeal carcinoma poses a major public health the development of nasopharyngeal carcinomas have re- challenge in Southeast Asia, and at least 2000 new cases of cently been identified. However, there is still limited infor- nasopharyngeal carcinoma are diagnosed each year in the mation on the nature of the genes and gene products whose United States (1, 2). Although nasopharyngeal carcinoma is aberrant expression and activity promote the malignant classified as a subtype of head and neck squamous cell carci- conversion of nasopharyngeal epithelium. Here, we have noma, its unique epidemiology, clinical characteristics, etiology, performed a genome-wide transcriptome analysis by prob- and histopathology warrant separate efforts for the study of its ing cDNA microarrays with fluorescent-labeled amplified underlying molecular mechanisms of carcinogenesis (1). For RNA derived from laser capture microdissected cells pro- example, nasopharyngeal carcinoma patients tend to present at a cured from normal nasopharyngeal epithelium and areas of more advanced stage of disease because the primary anatomical metaplasia-dysplasia and carcinoma from EBV-associated site of tumor growth is located in a silent area, and they exhibit nasopharyngeal carcinomas. This approach enabled the higher metastatic potential when compared with other head and identification of genes differentially expressed in each cell neck squamous cell carcinoma (3–5). In addition, a strong population, as well as numerous genes whose expression can association of EBV and nasopharyngeal carcinoma has been help explain the aggressive clinical nature of this tumor type. widely accepted (1, 6). Indeed, nasopharyngeal carcinoma can For example, genes indicating cell cycle aberrations (cyclin be EBV or non-EBV associated, being the latter with a higher D2, cyclin B1, activator of S-phase kinase, and the cell cycle prevalence in the endemic regions, as reflected by the detection checkpoint kinase, CHK1) and invasive-metastatic potential of EBV gene products in the vast majority of the nasopharyn- ␤ geal carcinoma patients (7). The persistence of the viral genome (matrix metalloproteinase 11, v-Ral, and integrin 4) were highly expressed in tumor cells. In contrast, genes under- in tumor cells is usually associated with nonkeratinized squamous cell carcinoma or undifferentiated carcinoma (8, 9). Ex- tensive investigations on the contribution of EBV to carcinogenesis have identified at least one viral gene product, the latent membrane protein 1, which acts as a transforming protein in Received 12/17/03; revised 4/23/04; accepted 5/4/04. The costs of publication of this article were defrayed in part by the many cellular contexts, and thus represents a candidate molecule payment of page charges. This article must therefore be hereby marked for the initiation and/or maintenance of nasopharyngeal carci- advertisement in accordance with 18 U.S.C. Section 1734 solely to noma (6, 10, 11). indicate this fact. A number of studies have recently identified multiple ge- Requests for reprints: J. Silvio Gutkind, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, 30 netic and epigenetic alterations that occur in nasopharyngeal Convent Drive, Building 30, Room 211, Bethesda, MD 20892-4330. carcinoma. For example, extensive and high-resolution allelo- Phone: (301) 496-6259; Fax: (301)402-0823; E-mail: [email protected]. typing studies have revealed potential tumor suppressor gene

loci in nasopharyngeal carcinoma on chromosomes 3p, 9p, 9q, Table 1 Clinical information and isolated cell populations from each 11q, 12q, 13q, 14q, and 16q (12, 13). Candidate approach patient specimen studies have also identified aberrant hypermethylation of genes WHO residing in these loci such as RASSF1A, RAR␤2, and p16INK4A Sample histological Tumor-node- Microdissected cell (14–16). However, for most of these loss of heterozygosity ID diagnosis† metastasis Stage populations‡ (LOH) loci, the presence of candidate tumor suppressor genes 563 II T1N2M0 III N, T 566 III T N M IVB N, T has not been thoroughly investigated. These genetic approaches 4 3b 0 567* NA NA NA N can eventually lead to the identification of critical genes that are 568 III T2aN2M1 IVC R, T important for nasopharyngeal carcinoma development and pro- 575 II T2N1M0 IIB R, T gression. Complementary efforts to characterize the gene ex- 582* II T2aN1M0 IIB N 589 II T N M IIB R, T pression pattern during the malignant conversion of nasopha- 2 1 0 594 III T N M IVA R, T ryngeal carcinoma and, in particular of those genes involved in 4 1 0 key intra- and extracellular molecular pathways, could ulti- Abbreviations: ID, identification number; NA, Nonavailable. * Sample contained inadequate number of tumor cells for micro- mately lead to the identification of functional interactions dissection. among genes and their protein products, which in turn may † II, non-keratinizing squamous cell carcinoma; III, undifferenti- explain the unique biological features and aggressive nature of ated carcinoma. this particular cancer type. The comprehensive analysis of the ‡ N, normal; R, metaplasia-dysplasia; T, tumor. gene expression profile of nasopharyngeal carcinoma may help identify aberrantly expressed or mutated genes, which may thus represent novel molecular targets for therapeutic intervention in this disease. tion. We selected eight clinical samples based on adequacy of Relatively limited information is currently available on the tissue, the presence of nonneoplastic epithelium, and the gene expression in nasopharyngeal carcinoma, which has been appropriate status of tissue preservation. Clinical data, including generated using bulk nasopharyngeal carcinoma tissues or na- diagnosis, staging, and cell populations isolated from each sam- sopharyngeal carcinoma-derived cells in culture and using low- ple, are included in Table 1. As patient specimens were too density cDNA arrays (17, 18). Recent technological progress small to allow further sectioning, for immunohistochemical using laser capture microdissection (LCM) has now made it staining a set of eight archival paraffin-embedded nasopharyn- possible to enrich populations of cells from heterogeneous tis- geal carcinoma samples unrelated to the frozen samples were sues (19–21), which is particularly relevant to nasopharyngeal selected. carcinoma as primary tumors often include numerous infiltrat- Confirmation the Presence of EBV. All specimens ing inflammatory cells, nonneoplastic nasopharyngeal epithe- were confirmed for EBV positivity by either in situ hybridiza- lium, and stroma. In this regard, the LCM platform enables the tion of EBV-encoded small nonpolyadenylated RNA (EBER) or isolation of homogeneous cell populations for further down- by PCR of the EBV BamHI-W region from genomic DNA. stream applications. In this study, we have focused on EBV- EBER in situ hybridization was performed by using fluorescein- associated nasopharyngeal carcinoma, given its higher preva- conjugated EBER PNA probe (Dako, Glostrup, Denmark) ac- lence and thus clinical relevance, and used LCM to procure cells cording to the manufacturer’s instruction. Primers for PCR of from tumors or adjacent nonneoplastic tissues from nasopha- the BamHI-W region were described elsewhere (22). ryngeal carcinomas for transcriptional profiling by high-density LCM. Isolation of tumor and nasopharyngeal epithelial cDNA microarray. This comparative analysis between normal cells by LCM was performed as described previously (21). nasopharyngeal epithelium, metaplastic-dysplastic epithelium, Briefly, 6–8-␮m cryostat sections stained with H&E were pre- and tumor tissue revealed numerous differentially expressed pared for LCM. The Arcturus Pixell II apparatus was used to genes. Validity of the microarray results was confirmed by harvest cells of interest. Target cell number for each sample was quantitative reverse transcription-PCR and immunohistochemi- ϳ2500–5000 cells. cal evaluation of gene products whose levels were assessed to be Nucleic Acid Isolation and mRNA Amplification. For differentially expressed in normal and tumor cells. These efforts PCR of genomic DNA, DNA was extracted from samples by may ultimately provide a better understanding of the molecular incubating overnight at 55°C with lysis buffer containing 10 mM mechanisms underlying nasopharyngeal carcinoma carcinogen- Tris-HCl, 1 mM EDTA, 1% Tween 20, and 0.1 mg/ml proteinase esis, as well as help identify suitable targets for the development K (pH 8.0). Total RNA was extracted using a modified version of novel treatment strategies for this aggressive cancer type. of the Stratagene RNA microisolation kit (Stratagene, La Jolla, CA), as described previously (21). Two rounds of mRNA amplification were performed for each sample by using the Mes- MATERIALS AND METHODS sageAmp kit for T7-based amplification (Ambion, Austin, TX), Tissue Samples. Informed consent was obtained from according to the manufacturer’s protocol (23). Universal Human each patient before collecting pretreatment specimens from na- Reference RNA (Stratagene) was used as a source of common sopharyngeal carcinoma patients at the Chulalongkorn Univer- reference RNA and was also subjected to mRNA amplification sity Hospital. Each biopsy sample was divided into two sections, in parallel to each nasopharyngeal carcinoma sample. one that was submitted for routine histological diagnosis for cDNA Microarrays and Analysis. Human GEM2 nasopharyngeal carcinoma, and the remaining section was flash cDNA clones were obtained from Incyte Genomics, Inc. (Palo frozen and stored in liquid nitrogen until ready for microdissec- Alto, CA), and these were arrayed onto precoated glass slides at

the Advanced Technology Center (National Cancer Institute, melting at 95°C for 15 s; and annealing/extension at 60°C for 1 Gaithersburg, MD). Each array consisted of 9128 cDNA clones, min. A negative control without template was run in parallel to which included 7102 known genes, 1179 expressed sequence assess the overall specificity of the reaction. The PCR products tag cluster, and 122 Incyte-expressed sequenced tags clones. were analyzed on a 1.2% agarose gel to confirm the size of the Preparation of fluorescent-conjugated cDNA targets were per- amplified product. The comparative threshold cycle (CT) formed by an indirect labeling approach using amplified RNA (⌬⌬CT) method, which compares differences in CT values of as template. Briefly, 1 ␮g of amplified RNA was used for each common reference RNA and nasopharyngeal carcinomas or reverse transcription with random hexamer primers and a mix- normal tissues, were used to achieve the relative fold changes in ture of nucleotides at a final concentration of 0.5 mM dATP, gene expression between normal and tumor. The experiments dCTP, and dGTP, 0.1 mM dTTP, and 0.15 mM 5-(3-amino- were repeated in triplicate and the mean fold changes and SE allyl)-2Ј deoxyuridine-5Ј-triphosphate (Sigma, St. Louis, MO). between normal and nasopharyngeal carcinoma are reported. cDNA targets were then coupled with fluorescent dye, Cy3, or Oligonucleotide Primers. Gene-specific primers were Cy5 (Amersham, Piscataway, NJ) under 0.1 M NaHCO3 for1h generated using the Primer 3.0 program (provided by White- in the dark. The probes were appropriately mixed, purified, and head/Massachusetts Institute of Technology Center for Genome concentrated before hybridization to arrays at 42°C overnight. Research, Cambridge, MA). The primer sequences were as After washing, the arrays were scanned with GenePix scanner follows: CD83, forward 5Ј-AGAAGGGGCAAAATGGTTCT-3Ј, (Axon Instruments, Union City, CA). Most samples were re- reverse 5Ј-CACCTGTATGTCCCCGAGTT-3Ј; MMP11, forward peated at least twice using independent cDNA hybridization in 5Ј-TAGGTGCCTGCATCTGTCTG-3Ј, reverse 5Ј-AGAATAC- which tumor and noncancerous tissues were labeled with green CCCTCCCCATTTG-3Ј; CCND2, forward 5Ј-AGGCAGCTGAC- (Cy3) and reference RNA-labeled with red (Cy5). TATATCA-3Ј, reverse 5Ј-CTGCTGGCCAACTCTTAC-3Ј; SLPI, Statistical analysis of the microarray data were performed forward 5Ј-GGGAAGTGCCCAGTGACTTA-3Ј, reverse 5Ј-GG- using the BRB ArrayTools software developed by Dr. Richard CAGGAATCAAGCTTTCAC-3Ј; PIGR, forward 5Ј-AGCCTC- Simon and Amy Peng from Biometric Research Branch of the TTCGATCACTCAGG-3Ј, reverse 5Ј-TGGACTGGAGCAGG- 7 National Cancer Institute. We used both supervised and unsu- AAGTCT-3Ј; and KRT7, forward 5Ј-CAGGATGTGGTGGAG- pervised hierarchical clustering analyses with median center GACTT-3Ј, reverse 5Ј-TTGCTCATGTAGGCAGCATC-3Ј.In correlation and average linkage. We also used a supervised parallel, a human 18 s rRNA forward 5Ј-CGCCGCTAGAGGT- Class Comparison Tool based on univariate F tests to identify GAAATTC-3Ј, reverse 5Ј-TTGGCAAATGCTTTCGCTC-3Ј was differentially expressed genes between predefine cell popula- used as an endogenous control for normalization. tions according to their histology. A stringent criteria was used Immunohistochemistry. Paraffin-embedded tissue sec- to define the statistical significance of each observed change in tions were deparaffinized in xylene and hydrated through graded gene expression, using F statistics (P Ͻ 0.001), the significance alcohols and distilled water. Sections were subjected to antigen of which was confirmed by 2000 random permutations (24). unmasking with AUF solution (Vector Laboratories, Burl- Quantitative Reverse Transcription-PCR. RNA from ingame, CA) for cyclin D2, antigen retrieval with 0.25% trypsin the second round of amplification was used to generate cDNA. (Invitrogen, Inc.) for carcinoembryonic antigen-related cell ad- Briefly, 1 ␮g of aRNA was used as starting material, to which hesion molecule 6 (CEACAM6), or their combination for ker- we added 1 ␮l(3␮g/␮l) of random primer and diethyl pyro- atin 7 (KRT7). Slides were incubated in 3% hydrogen peroxide carbonate-treated water to total volume 25 ␮l, then heated the in ethanol to quench the endogenous peroxidase. The sections mixture at 65°C for 5 min and chilled on ice. The other com- were incubated in blocking solution, 2% BSA. We used bio- ponents were added as follows: 10 ␮lof5ϫ first strand buffer; tinylated secondary antibody (Vector Laboratories) and Vec- 5 ␮lof0.1M DTT; 1 ␮lof25mM deoxynucleoside triphos- tastain kit (Vector Laboratories). The slides were developed in phates; 1 ␮l of RNase inhibitor; and 6 ␮l of diethyl pyrocar- 3,3-diaminobenzidine (Sigma). The primary antibodies used in bonate-treated water. The samples were incubated at 42°C for 2 this study were KRT7 Ab-1, CEA/CD66e Ab-2, and MMP11 min. Then 1 ␮l of Superscript II (40 units/␮l; Invitrogen, Inc., (Neomarker, Fremont, CA); and cyclin D2 (M-20) (Santa Cruz Carlsbad, CA) was added, and the samples were incubated at Biotechnology, Santa Cruz, CA). Methyl green [2% in acetate 42°C for 50 min. The reaction was inactivated at 70°C for 5 min, buffer (pH 4.8)] was used as nuclear counterstain. and 1 ␮l (2 units/␮l) of RNAase H was added to degrade the RNA strand by incubating at 37°C for 20 min. Real-time PCR using the ABI prism 7700 sequencer detector system and Qia- RESULTS gen’s Quantitect SYBR Green PCR kit (Qiagen, Valencia, CA) We first confirmed the homogeneity of tumor origin of the was performed following the manufacturer’s protocol. In brief, sample sets by confirming the presence of EBV genome. All the reaction mixture (50 ␮l of total volume) contained 500 ng of tumor specimens were positive for EBV as judged by either cDNA, gene-specific forward and reverse primers at 1 ␮M final EBER in situ hybridization (Fig. 1) or genomic DNA PCR for concentration, and 25 ␮lof2ϫ Quantitect SYBR Green PCR EBV BamHI-W region (data not shown). EBER was found in Master mix. The real-time cycler conditions were as follows: the nuclei of tumor cells but absent in the normal nasopharyn- PCR initial activation step at 95°C for 10 min; 40 cycles each of geal epithelial cells (Fig. 1, A and B). In the adjacent metaplastic and dysplastic nasopharyngeal epithelium, EBER was mainly localized to the basal and suprabasal layers (Fig. 1C), which is consistent with previous studies supporting an involvement of 7 Internet address: http://linus.nci.nih.gov/BRB-ArrayTools.html. EBV in nasopharyngeal carcinoma carcinogenesis (25).

Fig. 1 Confirmation of EBV- associated nasopharyngeal carcinoma. In situ hybridization detection of virally encoded RNA EBER or PCR of genomic DNA for the EBV BamHI-W region was used to confirm the presence of EBV in the nasopharyngeal carcinoma tissue samples. Repre- sentative samples from nasopharyngeal carcinoma with adjacent normal nasopharyngeal epithelium (A and B) and nasopharyngeal carcinoma with adjacent metaplastic epithelium (B and D) are shown. Typical nuclear expression of EBER was demonstrated in nasopharyngeal carcinoma cells in both samples (B and D). EBER expression was also found in metaplastic lesions (C) but was not detectable in normal epithelium (A). Not all tumor cells showed detectable EBER. Similarly, in metaplastic epithelium, nuclear EBERs were limited to basal and suprabasal layer representing the proliferative zone of epithelium.

According to their cytologic features, we isolated three ited correlation with their histological characteristics. Thus, we populations of cells from each nasopharyngeal biopsy, normal performed additional analyses by a supervised class comparison pseudostratified columnar ciliated respiratory epithelium (N), based on the predefined histological characteristics (24) to de- squamous metaplastic or dysplastic epithelium (R), and carci- tect genes differentially expressed according to each of the noma (T). Within these clinical specimens, we characterized observed histological groups. By this approach, we identified normal epithelium (4), metasplasia or dysplasia (4), and carci- 650 genes that were differentially expressed among the three noma (Ref. 6; Table 1). Each of these cell populations were groups (P Ͻ 0.001). Reanalysis of the data by a supervised isolated by LCM and subsequently subjected to total RNA hierarchical clustering of samples based on these 650 genes isolation. To overcome the problem of the limited amount of revealed three clusters that matched well with each cell popu- ϳ mRNA isolated from LCM procured samples ( 50–100 ng of lation (Fig. 2B). Among these genes, clustering analyses re- total RNA), we chose to increase target mRNA by linear am- vealed four major clusters: underrepresented in normal; highly plification with a T7 polymerase-based in vitro transcription represented in normal; underrepresented in tumor; and highly system. This method has been shown to exhibit high fidelity and represented in tumor. reproducibility and to yield RNA that is suitable for microarray analysis (26–28). A universal human reference RNA was se- We next performed pairwise comparisons among the three lected as the common reference for the microarray study be- cell populations using a supervised class comparison approach. cause this frequently used RNA reference may afford the pos- In normal versus tumor comparison, 477 genes were differen- sibility of future comparisons with other data sets. To avoid bias tially expressed, of which, 402 and 83 genes were highly ex- in hybridization to microarrays, the reference RNA was ampli- pressed in normal and tumor, respectively (Fig. 3). When meta- fied in parallel to those generated from nasopharyngeal carci- plastic-dysplastic tissues were compared with tumor cells, 169 noma samples and used as a reference for each nasopharyngeal genes were differentially expressed, of which, 121 and 48 genes carcinoma-derived sample for their hybridization to cDNA mi- were highly expressed in metaplastic-dysplastic cells and tumor, croarray chips. All arrays included for additional data analyses respectively (Fig. 3). By comparing RNA extracted from normal had a mean target signal intensity of at least 4-fold over the and metaplastic-dysplastic cells, 658 genes were found to be background intensity, and each array was normalized by sub- differentially expressed, of which, 457 and 201 genes were tracting the median log-ratio from log-ratio of each spot. The highly expressed in normal and metaplastic-dysplastic tissues, initial analysis of the fluorescence intensity data by global respectively (Fig. 3). Thus, the transcriptional profiles of meta- hierarchical clustering of all of the tissue samples showed lim- plasia-dysplasia and nasopharyngeal carcinoma were both quite

Fig. 2 Molecular classification of nasopharyngeal carcinoma tissues. Nasopha- ryngeal carcinoma samples were microdissected from normal nasopharyngeal epithelium, metaplastic or dysplastic epithelium, and carcinoma. Individual cell populations were subjected to mRNA isolation, amplification, and hybridization to cDNAs microarrays. Analysis of a subset of 670 genes that were found to be differentially expressed by supervised class comparison analysis showed a good correlation with each histological group (A). The same set of genes was further classified by clustering analysis of genes according to the relative expression levels in each histological type. In B, the image array on the left shows color plot of gene expression clusters. Dendrogram of the cluster correlation is shown on the right. Pseudocolors indicate differential expression (green, transcript levels greater than the median; black, transcript levels equal to median; red, transcript levels below the median; gray, missing data). N, normal nasopharyngeal epithelium; R, metaplastic or dysplastic epithelium; T, carcinoma.

distinct from those of adjacent normal nasopharyngeal epithe- of nasopharyngeal carcinoma (Tables 2 and 3). Highly repre- lium. sented genes in the carcinoma group included numerous genes Although the complete list of genes differentially ex- involved in cell cycle regulation and DNA replication, including pressed is provided (see supplemental information), we focused Check 1 Kinase checkpoint homologue (CHK1), cyclin D2 our attention on those genes of known function from normal and (CCND2), cyclin B1 (CCNB1), likely ortholog of mouse gene tumor cells whose aberrant (increased or decreased) expression rich cluster, C8 gene (GRCC8), and centromere protein F may play critical roles in determining the malignant phenotypes (CENPF). Other set of differentially expressed genes function in

Fig. 3 Pairwise analyses between individual histological groups revealed unique patterns of gene expression. Differentially expressed genes identified from a supervised class comparison analysis were subjected to hierarchical clustering analysis. Each comparison consisted of two major groups, under- and overrepresented genes, as demonstrated by green and red, respectively. A–C show comparison between normal (N) and tumor (T), metaplasia or dysplasia (R) and tumor (T), and normal (N) and metaplasia or dysplasia (R), respectively. The full list of genes and analyses are provided in the supplemental tables.

cell adhesion and thus may potentially confer the invasive- (RARRES1), H-Ras-like suppressor 3 (HRASLS3), mutated ␤ metastatic property of tumor cells such as integrin 4 (ITGB4), in colorectal cancers (MCCs), and LOH 11 chromosomal matrix metalloproteinase 11 (MMP11), and syndecan 2 (SDC2). region 2 gene A (LOH11CR2A). Down-regulated apoptosis- Interestingly, genes involved in Ras/Ral signaling pathways, related genes included in this group were B-cell CLL/lym- RalA, as well as in the Wnt/␤catenin pathways, frizzle 7 (FZD7) phoma 6, calpastatin, calpain 3, caspase10, and serine leuko- and claudin1 (CLDN1), were also up-regulated in the tumor cyte protease inhibitor (SLPI). Genes related to the Notch compartment. pathway numb homologue (NUMB) and transducin-like en- Among the genes underrepresented in the tumor cell hancer of split 2 (TLE2) were also underrepresented in tu- population, there were a number of potential tumor suppres- mors. Structural genes in this group included KRT7, sor genes such as epidermal growth factor receptor pathway CEACAM6, mucin 1, and mucin 5b. Within the down-regu- substrate 8-related protein 1, retinoic receptor responder 1 lated gene group, this analysis identified polymeric immuno-

‘Table 2 Partial list of genes with known function that are highly represented in tumor cells when compared with normal nasopharyngeal epithelium by a supervised class comparison analysis Fold difference of Parametric geometric means UniGene Gene Functional group P* (T/N)† Description cluster symbol Map Cell adhesion 0.00003 2.931 Claudin 1 Hs.7327 CLDN1 3q28-q29 0.00016 1.531 Integrin, ␤ 4 Hs.85266 ITGB4 17q11-qter 0.00057 1.525 Integrin, ␣ 7 Hs.74369 ITGA7 12q13 Cell cycle 0.00002 2.274 Discs, large homologue 7 (Drosophila) Hs.77695 DLG7 14q22.1 0.00005 3.104 Cyclin D2 Hs.75586 CCND2 12p13 0.00008 2.149 Centromere protein F, 350/400ka (mitosin) Hs.77204 CENPF 1q32-q41 0.00017 2.147 Cyclin B1 Hs.23960 CCNB1 5q12 0.00029 1.860 Activator of S-phase kinase Hs.152759 ASK 7q21.3 0.00035 2.049 CHK1 checkpoint homologue (S. pombe) Hs.20295 CHEK1 11q24-q24 0.00054 3.753 MAD2 mitotic arrest deficient-like 1 (yeast) Hs.79078 MAD2L1 4q27 0.00064 1.281 Cyclin 1 Hs.79933 CCN1 4q21.21 Chaperone 0.00083 1.954 Prefoldin 4 Hs.91161 PFDN4 20q13 DNA replication 0.00001 2.740 Exonuclease 1 Hs.47504 EXO1 1q42-q43 0.00031 2.754 Topoisomerase (DNA) II alpha 170 kDa Hs.156346 TOP2A 17q21-q22 Immunology 0.00006 1.734 T-Cell receptor delta locus Hs.2014 TRD@ 14q11.2 0.00085 3.147 CD83 antigen (activated B lymphocytes, immunoglobulin Hs.79197 CD83 6p23 superfamily) Ion homeostasis 0.00030 1.376 Solute carrier family 6 (neurotransmitter transporter, Hs.553 SLC6A4 17q11.1-q12 serotonin), member 4 Metabolism 0.00004 1.814 Exostoses (multiple) 1 Hs.184161 EXT1 8q24.11- q24.13 0.00064 2.968 Steroidogenic acute regulatory protein Hs.3132 STAR 8p11.2 Nuclear protein 0.00010 1.848 Thymopoietin Hs.406660 TMPO 12q22 Nucleotide 0.00025 2.089 5-aminoimidazole-4-carboxamide ribonucleotide Hs.90280 ATIC 2q35 synthesis formyltransferase/IMP cyclohydrolase 0.00080 2.741 Phosphoribosylaminoimidazole carboxylase, Hs.117950 PAICS 4pter-q21 phosphoribosylaminoimidazole succinocarboxamide synthetase Oncogene 0.00025 1.783 Stathmin 1/oncoprotein 18 Hs.406269 STMN1 1p36.1-p35 Protease/Protease 0.00046 2.317 Matrix metalloproteinase 11 (stromelysin-3) Hs.155324 MMP11 22q11.23 inhibitor 0.00073 1.427 Serine (or cysteine) proteinase inhibitor, clade F (␣-2 Hs.159509 SERPINF2 17p13 antiplasmin, pigment epithelium-derived factor), member 2 RNA splicing 0.00056 1.353 Splicing factor, arginine/serine-rich 3 Hs.388623 SFRS3 6p21 0.00056 1.866 Splicing factor, arginine/serine-rich 10 (transformer 2 Hs.30035 SFRS10 3q26.2-q27 homologue, Drosophila) Signal 0.00003 1.965 V-ral simian leukemia viral oncogene homologue A Hs.6906 RALA 7p22-p15 transduction (ras related) 0.00006 1.600 RAB21, member RAS oncogene family Hs.184627 RAB21 12q15 0.00015 2.499 Frizzled homologue 7 (Drosophila) Hs.173859 FZD7 2q33 0.00027 1.811 Lymphocyte antigen 64 homologue, radioprotective 105 Hs.87205 LY64 5q12 kDa (mouse) 0.00033 2.521 Diacylglycerol O-acyltransferase homologue 2 (mouse) Hs.334305 DGAT2 11q13.3 0.00076 1.523 Tumor necrosis factor receptor superfamily, member 9 Hs.73895 TNFRSF9 1p36 0.00088 1.797 Dual-specificity tyrosine-(Y)-phosphorylation-regulated Hs.38018 DYRK3 1q32 kinase 3 Structural Ͻ0.00001 2.681 Kinesin family member 14 Hs.3104 KIF14 1pter-q31.3 protein 0.00025 1.900 Troponin T2, cardiac Hs.296865 TNNT2 1q32 0.00029 2.156 Syndecan 2 (heparan sulfate proteoglycan 1, cell surface- Hs.1501 SDC2 8q22-q23 associated, fibroglycan) 0.00060 2.242 H2A histone family, member Z Hs.119192 H2AFZ 4q24 Transcription 0.00033 1.835 E2F transcription factor 3 Hs.1189 E2F3 6p22 factor 0.00034 1.491 Activator of basal transcription 1 Hs.109428 ABT1 6p21.33 0.00048 2.276 Zinc finger protein 281 Hs.59757 ZNF281 1q32.1 0.00065 1.713 Interleukin enhancer binding factor 2, 45 kDa Hs.75117 ILF2 1q21.3 0.00072 1.718 Zinc finger protein 230 Hs.193583 ZNF230 19q13.31 0.00079 1.469 General transcription factor IIIC, polypeptide 4, 90 kDa Hs.22302 GTF3C4 9q34.3 0.00081 2.951 Spi-B transcription factor (Spi-1/PU.1 related) Hs.192861 SPIB 19q13.3- q13.4 Abbreviations: N, normal nasopharyngeal epithelium; T, tumor. * Parametric P indicates statistical significance based on univariate F test by supervised class comparison with 2000 random permutations. † Fold difference of geometric mean is derived from transcript level of each histologically defined cell population in relation to the common reference.

Table 3 Partial list of genes with known function that are underrepresented in tumor cells when compared with normal nasopharyngeal epithelium by a supervised class comparison analysis Fold difference of Parametric geometric means UniGene Gene Functional group P* (T/N)† Description cluster symbol Map Apoptosis related Ͻ0.00001 0.360 B-Cell CLL/lymphoma 6 (zinc finger protein 51) Hs.155024 BCL6 3q27 0.00009 0.418 Calpastatin Hs.359682 CAST 5q15-q21 0.00022 0.394 Calpain 3, (p94) Hs.293267 CAPN3 15q15.1- q21.1 Cell adhesion 0.00022 0.380 Caspase 10, apoptosis-related cysteine protease Hs.5353 CASP10 2q33-q34 Ͻ0.00001 0.081 Carcinoembryonic antigen-related cell adhesion Hs.73848 CEACAM6 19q13.2 molecule 6 (nonspecific cross-reacting antigen) 0.00001 0.138 Claudin 10 Hs.26126 CLDN10 13q31-q34 0.00074 0.293 Claudin 7 Hs.278562 CLDN7 17p13 Cell cycle Ͻ0.00001 0.235 Serum-inducible kinase Hs.3838 SNK 5q12.1- q13.2 0.00005 0.609 CDC16 cell division cycle 16 homologue Hs.1592 CDC16 13q34 (S. cerevisiae) 0.00043 0.404 Cyclin A1 Hs.79378 CCNA1 13q12.3- q13 0.00059 0.491 Cyclin-dependent kinase (CDC2-like) 10 Hs.77313 CDK10 16q24 Immunology Ͻ0.00001 0.043 Polymeric immunoglobulin receptor Hs.205126 PIGR 1q31-q41 Ͻ0.00001 0.077 B-factor, properdin Hs.69771 BF 6p21.3 Ͻ0.00001 0.426 Complement component 5 receptor 1 Hs.2161 C5R1 19q13.3- (C5a ligand) q13.4 Ͻ0.00001 0.072 Serum amyloid A4, constitutive Hs.1955 SAA4 11p15.1- p14 0.00004 0.168 Complement component 4A Hs.170250 C4A 6p21.3 0.00008 0.149 Complement component 3 Hs.284394 C3 19p13.3- p13.2 0.00009 0.198 EBNA1 binding protein 2 Hs.346868 EBNA1BP2 1p35-p33 0.00017 0.189 Leukocyte immunoglobulin-like receptor, Hs.77062 LILRB5 19q13.4 subfamily B (with TM and ITIM domains), member 5 Ion homeostasis Ͻ0.00001 0.061 Selenium binding protein 1 Hs.334841 SELENBP1 1q21-q22 Ͻ0.00001 0.097 S100 calcium binding protein P Hs.2962 S100P 4p16 Ͻ0.00001 0.083 Solute carrier family 22 (organic cation Hs.77239 SLC22A4 5q31.1 transporter), member 4 Ͻ0.00001 0.139 Solute carrier family 27 (fatty acid transporter), Hs.11729 SLC27A2 15q21.2 member 2 Ͻ0.00001 0.136 Hermansky-Pudlak syndrome 3 Hs.282804 HPS3 3q24 0.00001 0.179 Ceruloplasmin (ferroxidase) Hs.296634 CP 3q23-q25 0.00001 0.274 Calcium channel, voltage-dependent, beta 3 Hs.250712 CACNB3 12q13 subunit 0.00002 0.232 Calcium channel, voltage-dependent, gamma Hs.7235 CACNG3 16p12- subunit 3 p13.1 0.00017 0.160 Solute carrier family 26, member 4 Hs.159275 SLC26A4 7q31 0.00017 0.285 Calcium and integrin binding 1 (calmyrin) Hs.10803 CIB1 15q25.3- q26 0.00025 0.129 ATPase, Class V, type 10B Hs.109358 ATP10B 5q34 0.00043 0.128 Lactotransferrin Hs.105938 LTF 3q21-q23 0.00065 0.393 Solute carrier family 13 (sodium-dependent Hs.102307 SLC13A2 17p11.1- dicarboxylate transporter), member 2 q11.1 Membrane 0.00005 0.096 Connective tissue growth factor Hs.75511 CTGF 6q23.1 protein/receptor Ͻ0.00001 0.038 Prominin-like 1 (mouse) Hs.112360 PROML1 4p15.33 0.00009 0.289 Transmembrane 4 superfamily member 6 Hs.121068 TM4SF6 Xq22 0.00010 0.511 Receptor (calcitonin) activity-modifying protein 2 Hs.155106 RAMP2 17q12- q21.1 0.00047 0.593 Membrane-bound transcription factor protease, Hs.75890 MBTPS1 16q24 site Metabolism Ͻ0.00001 0.121 Monoglyceride lipase Hs.6721 MGLL 3q21.3 Ͻ0.00001 0.083 Carboxylesterase 1 (monocyte/macrophage serine Hs.76688 CES1 16q13- esterase 1) q22.1 Ͻ0.00001 0.188 Cytochrome P450, family 2, subfamily J, Hs.152096 CYP2J2 1p31.3- polypeptide 2 p31.2 Ͻ0.00001 0.292 Iduronate 2-sulfatase (Hunter syndrome) Hs.172458 IDS Xq28 Ͻ0.00001 0.290 Galactosamine (N-acetyl)-6-sulfate sulfatase Hs.159479 GALNS 16q24.3 (Morquio syndrome, mucopolysaccharidosis type IVA)

Table 3 Continued

Fold difference of Parametric geometric means UniGene Gene Functional group P* (T/N)† Description cluster symbol Map 0.00001 0.083 Aldehyde dehydrogenase 1 family, member A1 Hs.76392 ALDH1A1 9q21.13 0.00004 0.378 Carboxylesterase 2 (intestine, liver) Hs.282975 CES2 16q21 0.00005 0.226 Spermidine/spermine N1-acetyltransferase Hs.28491 SAT Xp22.1 0.00045 0.147 Glutathione S-transferase A3 Hs.102484 GSTA3 6p12.1 Oncogene 0.00001 0.397 V-ral simian leukemia viral oncogene homologue Hs.348024 RALB 2cen-q13 B (ras related; GTP binding protein) 0.00022 0.323 Mdm2, transformed 3T3 cell double minute 2, Hs.170027 MDM2 12q14.3- p53 binding protein (mouse) q15 Protease/Protease Ͻ0.00001 0.023 Secretory leukocyte protease inhibitor Hs.251754 SLP1 20q12 Inhibitor (antileukoproteinase) Ͻ0.00001 0.067 Sodium channel, nonvoltage-gated 1 ␣ Hs.446415 SCNN1A 12p13 Ͻ0.00001 0.225 Serine (or cysteine) proteinase inhibitor, clade B Hs.41072 SERPINB6 6p25 (ovalbumin), member 6 Ͻ0.00001 0.152 Amylase, ␣ 2B; pancreatic Hs.335493 AMY2B 1p21 0.00036 0.061 Serine (or cysteine) proteinase inhibitor, clade B Hs.227948 SERPINB3 18q21.3 (ovalbumin), member 3 0.00074 0.096 Matrix metalloproteinase 10 (stromelysin 2) Hs.2258 MMP10 11q22.3 Secretory protein Ͻ0.00001 0.019 Trefoil factor 3 (intestinal) Hs.82961 TFF3 21q22.3 0.00085 0.449 Trefoil factor 1 (breast cancer, estrogen-inducible Hs.82961 TFF3 21q22.3 sequence expressed in) Signal Ͻ0.00001 0.388 Rho-related BTB domain containing 1 Hs.15099 RHOBTB1 10q21.2 transduction Ͻ0.00001 0.212 Inhibin, ␤ B (activin AB ␤ polypeptide) Hs.1735 INHBB 2cen-q13 Ͻ0.00001 0.198 Arg/Abl-interacting protein ArgBP2 Hs.379795 ARGBP2 4q35.1 Ͻ0.00001 0.432 Transducin-like enhancer of split 2 (E(sp1) Hs.332173 TLE2 19p13.3 homologue, Drosophila) Ͻ0.00001 0.226 Protein tyrosine phosphatase, receptor type, U Hs.19718 PTPRU 1p35.3- p35.1 Ͻ0.00001 0.072 Ras-related associated with diabetes Hs.1027 RRAD 16q22 0.00001 0.445 FK506 binding protein 1B, 12.6 kDa Hs.77643 FKBP1B 2p24.1 0.00001 0.301 Phosphodiesterase 8B Hs.78106 PDE8B 5q13.2 0.00001 0.212 Transforming growth factor, ␤ receptor III Hs.342874 TGFBR3 1p33-p32 (betaglycan, 300 kDa) 0.00002 0.321 Rho GTPase-activating protein Hs.111138 RICS 11q24-q25 0.00003 0.419 RAB, member of RAS oncogene family-like 4 Hs.50267 RABL4 22q13.1 0.00003 0.459 Numb homologue (Drosophila) Hs.78890 NUMB 14q24.3 0.00004 0.249 Axin 2 (conductin, axil) Hs.127337 AXIN2 17q23-q24 0.00006 0.314 EphA2 Hs.171596 EPHA2 1p36 0.00006 0.527 Protein kinase C, zeta Hs.78793 PRKCZ 1p36.33- p36.2 0.00007 0.307 V-erb-b2 erythroblastic leukemia viral oncogene Hs.199067 ERBB3 12q13 homologue 3 (avian) 0.00008 0.253 Retinoic acid induced 3 Hs.194691 RAI3 12p13- p12.3 0.00010 0.242 Serine/threonine kinase with Dbl- and pleckstrin Hs.162189 TRAD 3q21.2 homology domains 0.00016 0.243 Protein tyrosine phosphatase, receptor-type, Hs.78867 PTPRZ1 7q31.3 Z polypeptide 1 0.00046 0.355 Transforming growth factor ␤-stimulated protein Hs.114360 TSC22 13q14 TSC-22 0.00053 0.285 Myeloid differentiation primary response gene Hs.82116 MYD88 3p22 (88) 0.00072 0.183 Tumor-associated calcium signal transducer 2 Hs.23582 TACSTD2 1p32-p31 0.00080 0.350 Interleukin 1 receptor, type I Hs.82112 IL1R1 2q12 Structural protein Ͻ0.00001 0.082 Dynein, axonemal, light intermediate Hs.406050 DNAL11 1p35.1 polypeptide 1 Ͻ0.00001 0.256 Keratin 7 Hs.23881 KRT7 12q12-q13 Ͻ0.00001 0.237 Calmin (calponin-like, transmembrane) Hs.406099 CLMN 14q32.2 Ͻ0.00001 0.259 Kinesin family member 13B Hs.15711 KIF13B 8p12 Ͻ0.00001 0.072 Mucin 1, transmembrane Hs.89603 MUC1 1q21 Ͻ0.00001 0.241 Mucin 5, subtype B, tracheobronchial Hs.102482 MUC5B 11p15 0.00002 0.369 Collagen, type VIII, ␣ 1 Hs.114599 COL8A1 3q12-q13.1 0.00003 0.215 Myosin ID Hs.39871 MYO1D 17q11-q12 0.00022 0.145 Profilin 2 Hs.91747 PFN2 3q25.1- q25.2

Table 3 Continued

Fold difference of Parametric geometric means UniGene Gene Functional group P* (T/N)† Description cluster symbol Map 0.00084 0.264 Tubulin, ␤ polypeptide Hs.336780 TUBB 6p21.3 Transcription Ͻ0.00001 0.204 Meis1, myeloid ecotropic viral integration site 1 Hs.380923 MEIS3 17p11.2 factor homologue 3 (mouse) Ͻ0.00001 0.187 Zinc finger protein 204 Hs.8198 ZNF204 6p21.3 Ͻ0.00001 0.116 Regulatory factor X, 2 (influences HLA class II Hs.100007 RFX2 19p13.3- expression) p13.2 Ͻ0.00001 0.218 Meis1, myeloid ecotropic viral integration site 1 Hs.170177 MEIS1 2p14-p13 homologue (mouse) Ͻ0.00001 0.569 Myeloid/lymphoid or mixed-lineage leukemia Hs.114765 MLLT2 4q21 (trithorax homologue, Drosophila); translocated to, 2 Ͻ0.00001 0.241 E74-like factor 3 (ets domain transcription factor, Hs.166096 ELF3 1q32.2 epithelial-specific) 0.00001 0.273 Microphthalmia-associated transcription factor Hs.166017 MITF 3p14.1- p12.3 0.00002 0.039 Forkhead box J1 Hs.93974 FOXJ1 17q22- 17q25 0.00007 0.124 B-cell CLL/lymphoma 3 Hs.31210 BCL3 19q13.1- q13.2 0.00043 0.367 Kruppel-like factor 4 (gut) Hs.356370 KLF4 9q31 0.00051 0.151 Zinc finger protein 76 (expressed in testis) Hs.29222 ZNF76 6p21.3- p21.2 0.00089 0.349 Interferon-stimulated transcription factor 3, Hs.1706 ISGF3G 14q11.2 ␥ 48 kDa 0.00091 0.375 Bone morphogenetic protein 1 Hs.1274 BMP1 8p21 Tumor suppressor Ͻ0.00001 0.124 Retinoic acid receptor responder (tazarotene Hs.82547 RARRES1 3q25.32 induced) 1 Ͻ0.00001 0.339 Epidermal growth factor receptor pathway Hs.28907 EPS8R1 19q13.42 substrate 8-related protein 1 Ͻ0.00001 0.261 HRAS-like suppressor 3 Hs.37189 HRASLS3 11q12.3 Ͻ0.00001 0.204 Mutated in colorectal cancers Hs.1345 MCC 5q21-q22 0.00001 0.076 Nonmetastatic cells 5, protein expressed in Hs.72050 NME5 5q31 (nucleoside-diphosphate kinase) 0.00011 0.476 Growth arrest-specific 8 Hs.54877 GAS8 16q24.3 0.00014 0.448 Loss of heterozygosity, 11, chromosomal Hs.152944 LOH11CR2A 11q23 region 2, gene A Abbreviations: N, normal nasopharyngeal epithelium; T, tumor. * Parametric P indicates statistical significance based on univariate F test by supervised class comparison with 2000 random permutations. † Fold difference of geometric mean is derived from transcript level of each histologically defined cell population in relation to the common reference.

globulin receptor (pIgR) and EBNA1 binding protein 2, antisera to KRT7, CEACAM6, MMP11, and cyclin D2. All eight which may play role in the EBV pathogenesis in nasopha- samples showed strong cytoplasmic staining of KRT7 and ryngeal carcinoma. CEACAM6 in histologically normal or metaplastic nasopharyngeal To validate the results of microarray studies, we first per- epithelium, which was in striking contrast to its undetectable ex- formed quantitative reverse transcription-PCR analysis of six se- pression in cancer cells (Fig. 4, compare left three panels). All lected genes. Consistent with the microarray data, the expression nasopharyngeal carcinoma samples were strongly immunoreactive levels of CD83, MMP11, and cyclin D2 were higher in tumor cells, for anti-cyclin D2 and anti-MMP11 (Fig. 4, B and C, last two whereas the expression of pIgR, SLPI, and KRT7 was higher in panels), in contrast to nonneoplastic tissues. For cyclin D2, the normal tissues and thus underexpressed in tumor cells (Fig. 4A). To majority of the cancer cells showed strongly positive nuclear stain- study further the relationship between RNA and protein levels, we ing, whereas a somehow weaker staining limited to a few cells selected a representative set of proteins whose mRNAs were shown within the basal and suprabasal epithelial layers was observed in to be over- or underexpressed in the normal versus tumor compar- normal and metaplastic epithelium. All clinical samples showed ison in microarray experiments based on the availability of their stronger and more extensive cyclin D2 immunoreactivity in tumor corresponding antibodies. A total of eight paraffin blocks from cells. For MMP11, a stronger expression in tumor cells was found nasopharyngeal carcinoma patients was selected based on the pres- in five of eight tissues, whereas the rest exhibited only barely ence of neoplastic and nonneoplastic epithelium within the same detectable expression in both nasopharyngeal carcinoma and non- section. Tissue sections were subjected to immunostaining with cancerous nasopharyngeal epithelium. Although these observations

Fig. 4 Genes either under- or overexpressed in tumor cells were selected for quantitative reverse transcription-PCR analysis and immunohistochemistry. A, six differential expressed genes from microarray experiment were selected to perform quantitative reverse transcription-PCR in amplified RNA materials (five normal and four tumor samples). The 18S rRNA gene was used as internal control for normalization. Means of signal intensity and SE for each mRNA were calculated as described in “Materials and Methods” and represented in a log scale. Ratio of means between tumor and normal tissues are also shown. B and C, immunohistochemical analysis of two representative cases of eight is depicted. Staining is indicated on top of each column. B, the top panel shows pseudostratified columnar normal nasopharyngeal epithelium and the bottom panel an area of carcinoma within the same section. C, an area of squamous metaplasia of the nasopharyngeal epithelium is shown in the top panel and the nasopharyngeal carcinoma area from the same sample in the bottom panel. Keratin-7 and CEACAMM6 gene products are present in a higher number of cells in nonneoplastic or metaplastic epithelium, as compared with carcinomas; the opposite is true for MMP11 and cyclin D1.

need to be extended to a large sample collection to establish their DISCUSSION clinical relevance, their strict correlation with the gene expression In this study, we provide a genome-wide analysis of the profiles, as revealed by microarray analysis, support the validity of global pattern of gene expression in nasopharyngeal carcinoma the combined use of LCM and cDNA microarrays as an experi- by using a combination of laser capture isolation of defined cell mental approach for the molecular profiling of nasopharyngeal populations, linear mRNA amplification, and high-density carcinoma. cDNA microarrays. In addition to normal and tumor cells, we

were able to procure cells from adjacent metaplastic and dys- gene dlg1 (32), were highly represented in tumor mRNAs when plastic lesions and thus analyze gene expression patterns cov- compared with normal cells. In addition, favoring deregulated ering the spectrum of nasopharyngeal carcinoma carcinogenic mitotic entry, nasopharyngeal carcinoma cells displayed up- progression. Genes that are differentially expressed in each cell regulation of GRCC8, which is also known as trigger of mitotic phenotype were identified using bioinformatic tools. Overall, entry 1 or Tome-1 (33) cytosolic protein that promotes the the number of genes differentially expressed between normal activation of cyclin-dependent kinase 1/cyclin B and conse- nasopharyngeal epithelium and tumor cells or metaplasia- quently mitotic entry. These tumor cells also displayed in- dysplasia was 477 and 658 genes, respectively. In contrast, only creased expression of cyclin B1 itself, in conjunction with the 169 genes were identified as being differentially expressed down-regulation of CDC16, a protein involved in the anaphase between metaplasia-dysplasia and the tumor cell compartment. promoting complex (34). Taken together, these findings demonstrated a pattern of tran- On the other hand, we have observed an up-regulation of scriptional alterations in nasopharyngeal carcinoma carcinogen- stromelysin-3/MMP11 (35) in tumors. This protease has been esis, as well as revealed the existence of numerous genes whose described to be increased in a variety of cancer types, including expression levels is up- or down-regulated in this cancer type. breast (36), lung (37), and colon (38), particularly in the non- Previous studies addressing the molecular profile of naso- neoplastic fibroblasts surrounding the malignant epithelial cells. pharyngeal carcinoma have been performed using low-density In contrast, expression of MMP11 in nasopharyngeal carcinoma cDNA arrays that included a limited number of genes. For seems to be restricted to the malignant epithelium, as also example, in one study that used pooled nasopharyngeal carci- described in oral squamous carcinomas (39). This suggests that noma and normal tissues the authors described up-regulation of MMP11 represents a specific marker for carcinomas derived cell cycle regulatory genes such as cyclin B1, cyclin D2, and from the head and neck mucosa, a possibility that may warrant cyclin A, which is consistent with our present results (17). Other further exploration. Nasopharyngeal carcinoma cells also over- ␤ genes classified as differentially expressed, however, were not express RalA (40) and integrin 4 (41, 42), combined with significantly different in our experimental analyses. As whole down-regulation of the proapoptotic caspase 10 (43, 44), the tissues were used in this prior study (17), we cannot exclude the anti-inflammatory secretory leukocyte protease inhibitor (45), possibility that some of the detected changes in gene expression and B-cell CLL/lymphoma 6, which can become oncogenic were associated with stromal or infiltrating inflammatory cells either by its persistent expression or upon accidental down- rather than with the tumor compartment. Another study using regulation, as in certain types of human B-non-Hodgkin’s lym- similar low-density cDNA arrays compared nasopharyngeal car- phomas (46). These genes have been shown to contribute to the cinoma cell lines with nasopharyngeal epithelial explants, which oncogenic and metastatic potential of many cancers, which may resulted in the identification of a set of differentially expressed fit well with the aggressive clinical behavior of nasopharyngeal genes poorly related to those described in our current study (18). carcinoma. These discrepancies may arise from the different source of RNA Histologically, EBV-associated nasopharyngeal carcino- used because cultured cells may have adapted to in vitro growth mas often present as poorly differentiated or undifferentiated conditions and thus express genes that may be quite different carcinomas. In agreement, our results revealed down-regulation from those normally transcribed in the tumor microenvironment. of genes for structural proteins that are associated with cellular Instead, to isolate these potentially confounding variables, in the differentiation such as KRT7 (47, 48), mucin1 (49), and mucin current study we chose to use recently developed techniques that 5b (50). These molecules are usually expressed variable levels enable the isolation of pure cell populations, which are opti- in normal epithelium and adenocarcinomas, but their expression mized for the subsequent RNA extraction, linear mRNA ampli- in squamous cell carcinoma arising from various organs, includ- fication, and hybridization to high-density cDNA microarrays, ing lung and head and neck cancer, is often restricted only to thus providing a suitable platform to perform detailed gene more differentiated areas within keratinized foci (50). Down- expression analysis as part of an effort aimed to understand regulation of a cell adhesion molecule gene, CEACAM6 (51), nasopharyngeal carcinoma pathogenesis. which has been shown to be deregulated and to affect the Clinically, nasopharyngeal carcinoma has been recognized differentiation of colon carcinomas, was also detected in naso- as an aggressive head and neck tumor by the fact that the pharyngeal carcinoma. Although these changes may reflect the majority of patients surrender to metastatic disease (1, 3). In- undifferentiated state of nasopharyngeal carcinomas, they may deed, the molecular profiling of nasopharyngeal carcinoma now also be linked to a common molecular alteration underlying revealed the presence of molecules consistent with a highly these cancer lesions. As such, they may result from the observed proliferative and invasive behavior. For example, CENPF (mi- reduction in the expression of the transcription factor Kruppel- tosin), whose expression is usually low in the G0 phase of the like factor 4 (KLF4) (52), which plays a critical role in skin cell cycle, was found to be highly expressed in tumors, which is barrier development in late-stage keratinocyte differentiation, or consistent with the active cycling status of tumor cells (29). by the decreased expression of genes involved in cell fate Moreover, aberrant cell cycle regulation throughout key check- determination related to Notch pathway such as numb (53) and points was strikingly portrayed. For example, cyclin D2, which TLE2 (54), which were under expressed in tumor cells. Overall, activates the G1-phase cyclin-dependent kinases 4 and 6 (30), our results indicate a remarkable perturbation of normal differ- the cell cycle G1-S and G2-M checkpoint kinase Chk1 (31), and entiation programs during nasopharyngeal carcinoma carcino- discs-large homologue 7, which has been proposed to play a role genesis. Whether these alterations in differentiation programs in checkpoint control and/or DNA repair based on its pattern of are caused by EBV and its encoded genes, as well as whether expression and homology with the Drosophila tumor suppressor aberrant cell differentiation plays a causative role in nasopha-

ryngeal carcinoma or are a direct consequence of the unregu- duction, differentiation, and survival, as well as those involved lated cell growth that characterizes this aggressive tumor, is at in tissue invasion and metastatic spread. The emerging molec- the present unknown and warrants additional investigation. ular anatomy of nasopharyngeal carcinoma may now facilitate Of interest, our study revealed the existence of clear dif- the identification of proteins whose expression or activity may ferences in the expression of genes involved in signal transduc- promote uncontrolled growth in this malignancy, as well as tion in nasopharyngeal carcinoma. For example, although the those molecules that correlate with clinical staging, prognosis, Ras-liked GTPase, RalA, was assessed to be up-regulated, RalB or response to treatment, thus representing novel potential di- in contrast was down-regulated in nasopharyngeal carcinoma. agnostic tools and therapeutic targets for nasopharyngeal carci- Although both RalA and RalB are downstream effectors of ras noma. signaling pathway (55), a distinct function for RalA and RalB has been described. For instance, RalA is involved in anchor- age-independent cell proliferation, whereas RalB functions in ACKNOWLEDGMENTS the survival of tumor cells (56). Another gene related to ras We thank the staff of Department of Otolaryngology and the pathway, Rab21 (57), was found up-regulated in nasopharyn- Radiation Oncology Unit, Department of Radiology, Chulalongkorn geal carcinoma, whereas Ras-related associated with diabetes University Hospital, for recruiting nasopharyngeal carcinoma patients. (Rad) (58) was down-regulated in nasopharyngeal carcinoma. We also thank Ju-Seog Lee, National Cancer Institute, NIH, for tech- Frizzle 7 (59), which encodes a seven-transmembrane receptor nical advice and Wichai Pornthanakasem and Sairoong Sakdikul for technical assistance. in the Wnt pathway, was found up-regulated in nasopharyngeal carcinoma. Moreover, claudin-1 (60), a tight junction gene whose expression is positively regulated by the presence of REFERENCES ␤ -catenin, was up-regulated in the tumor cell population. In 1. Vokes EE, Liebowitz DN, Weichselbaum RR. Nasopharyngeal car- agreement with these findings, axin 2 (61), an inhibitory mol- cinoma. Lancet 1997;350:1087–91. ecule of the Wnt pathway, was underrepresented in nasopha- 2. Forastiere A, Koch W, Trotti A, Sidransky D. Head and neck cancer. ryngeal carcinoma. These results are strongly suggestive of a N Engl J Med 2001;345:1890–900. dysregulated hyperactivity of the Wnt pathway in nasopharyn- 3. 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