Transcriptional Gene Expression Profiling of Small Cell Lung Cancer Cells1

[CANCER RESEARCH 63, 1943–1953, April 15, 2003] Transcriptional Gene Expression Profiling of Small Cell Lung Cancer Cells1

Nina Pedersen,2 Shila Mortensen,2 Susanne B. Sørensen, Mikkel W. Pedersen, Klaus Rieneck, Lone F. Bovin, and Hans Skovgaard Poulsen3 Department of Radiation Biology, The Finsen Centre [N. P., S. M., M. W. P., S. B. S., H. S. P.] and Institute for Inflammation Research, IIR 7521 [K. R., L. F. B.], National University Hospital, Copenhagen DK-2100, Denmark

ABSTRACT expression profiles, which can distinguish between these subclasses, have been revealed by microarray analyses (1–3). A global gene expression analysis using oligonucleotide microarrays Because of the aggressive behavior of SCLC and the very poor was performed on many human small cell lung cancer (SCLC) cell lines in outcome of present treatments, new therapeutic methods for systemic cell culture and/or as xenografts. The expression was compared with the expression profiles of 18 normal tissues. treatment of SCLC are in high demand. Using global gene expression In a hierarchical cluster analysis the cell lines clustered distinctly from analysis we have searched for genes that are highly and/or specifically normal tissues and grouped into four clusters. One cluster consisted of two expressed in all or most of the tumor cells with the aim to identify related cell lines and was markedly different from the other SCLC cell novel potential targets for the development of new therapeutic agents. lines, whereas the rest of the clusters grouped together. Two subclusters These could be surface molecules for direct targeting in radio-, toxin, contained the classical SCLC types and one subcluster the variant SCLC or gene therapy, or molecules to which development of cancer vac- type, thus identifying many genes with differential expression between the cines could be used. Other potential targets are molecules involved in two variants of SCLC. All of the xenografts clustered closest to the cell maintenance of the malignant phenotype, such as oncogenes and lines from which they originated and had the same expression levels as the antiapoptotic molecules, to which inhibitors can be applied or lost cells grown in culture for the majority of genes. activity restored. The analysis confirmed the high expression of many genes identified previously as highly expressed in SCLC cells including neuroendocrine A characteristic of all of tumors and their metastases, both between markers, oncogenes, and genes involved in cell proliferation and division. patients and within a tumor, is their heterogenicity, making the de- The analysis furthermore identified a number of molecules not identified velopment of therapeutic strategies difficult, as some cells invariably previously as expressed in SCLC. Several of these are expressed in low or can escape the treatment. Sufficient material of SCLC tumors from undetectable amounts in the majority of normal tissues and, therefore, are patients is extremely difficult to obtain both in number of specimens potential targets for new therapeutic approaches. By including the pub- and sufficient amounts for microarray analysis. Therefore, we used an lished array profiles of six ressected SCLC tumors from Bhattacharjee et alternative approach and used the expression profiles of 21 SCLC al. (A. Bhattacharjee et al., Proc. Natl. Acad. Sci. USA, 98: 13790–13795, cells lines obtained from five different laboratories and 8 xenografted 2001.), the analysis revealed that most of the novel potential targets tumors from these cell lines to compare to the expression profiles of expressed by SCLC cell lines and xenografts were also expressed in the 17 normal adult tissues. By this analysis, we identified several genes tumors. This analysis demonstrates the value of using cell lines and xenografts highly and specifically expressed by all or most of the SCLC cell for expression profiling, when a limited quantity of tumor material is lines, xenografts, and 6 ressected tumors with no or little expression available. in normal tissues, which could be candidates for therapeutic targeting. In addition, the analysis clearly divided the SCLC cell lines into two INTRODUCTION distinct subclasses with different expression profiles. SCLC4 is an aggressive disease, which is generally disseminated at MATERIALS AND METHODS the time of diagnosis. Initially the cancer is responsive to chemotherapy, but almost always recurs in a resistant form resulting in a 5-year Cell Culture. The following human SCLC cell lines were used: CPH 54A, survival rate of Ͻ5%. SCLC is generally correctly identified by CPH 54B (4), GLC-2, GLC-3, GLC-14, GLC-16, GLC-19, GLC-26, GLC-28 pathological means, wherefore identification of new markers for clas- (5–7), DMS 53, DMS 79, DMS 92, DMS 114, DMS 153, DMS 273, DMS 406, sification of this tumor type is not pertinent. This is in contrast to the DMS 456 (8), NCI H69, NCI N417 (9), MAR 24H and MAR 86MI (10, 11). situation for discrimination of subclasses of other lung tumor forms, CPH 54 A and B were propagated in MEM (Eagle), all of the GLC, NCI, MAR such as adenocarcinomas, for which therapeutic response and survival cell lines and DMS 79 were propagated in RPMI 1640, and all DMS (except DMS 79) were propagated in Waymouth medium, all supplemented with 10% rates can differ markedly despite similar pathology. Differences in FCS. All of the serum and media were obtained from Invitrogen (Tåstrup, Denmark). Received 8/28/02; accepted 2/14/03. Xenografts. Cells (0.5–1.2 ϫ 107) from the cell lines CPH 54A, GLC-3, The costs of publication of this article were defrayed in part by the payment of page GLC-14, DMS 273, NCI H69, NCI N417, and MAR 24H were inoculated charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. bilaterally in the flanks of 12–13-week-old BALB/c nude mice. The mice were 1 Supported by Odin Medical A/S, the Danish Cancer Society, the Danish Medical sacrificed, and the xenografted tumors were harvested when one of the tumors Research Council, The Danish Rheumatism Association, and the A. P. Møller Foundation had reached a maximal diameter of 1 cm. The cell line CPH 136A was only for the Advancement of Medical Science. propagated in nude mice by inoculation of a 2-mm tumor block. Necrotic tissue 2 These two authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Radiation was removed, and the tumors were either processed immediately or stored in Biology, Finsen Centre, Section 6321, National University Hospital, Blegdamsvej 9, RNAlater (Ambion, Cambridgeshire, United Kingdom) for RNA extraction. DK-Copenhagen 2100, Denmark. Phone: 45-35-45-63-03; Fax: 45-35-45-63-01; E-mail: Isolation of RNA. Total RNA from normal, human tissues were obtained skovgaard @rh.dk. from either Clontech (Brøndby, Denmark; fetal brain, brain, lung, kidney, 4 The abbreviations used are: SCLC, small cell lung cancer; RT-PCR, reverse transcription-PCR; MAGE, melanoma-associated antigen; DDC, L-dopa decarboxylase; GRP, gastrin- heart, trachea, adrenal gland, prostate, salivary gland, and thyroid) or from releasing peptide; SGNE1, secretory granule, neuroendocrine protein 1; ASCL1, achaete scute Ambion (lung, liver, brain, pancreas, spleen, small intestine, skeletal muscle, homologous protein; NCAM1, neural cell adhesion molecule 1; GRIA2, ionotropic glutamate colon, stomach, and testis). Only one sample was analyzed in duplicate (lung receptor 2; GRM8, metabotropic glutamate receptor 8; NPTXR, neuronal pentraxin receptor; RNA from Clontech and Ambion) and one in triplicate (two different batches ITGAE, integrin subunit ␣ E; PTTG1, pituitary tumor transforming gene (securin); TIMP, tissue inhibitor of metalloproteinase; MMP, metalloproteinase; pRB, retinoblastoma protein; brain RNA from Clontech and one from Ambion). The duplicates and tripli- INSM1, insulinoma-associated antigen 1; ENO2, neuron-specific enolase 2 (NSE). cates showed similar expression profiles, and only the results using the RNA 1943

from Ambion are shown here. RNA from cell lines in exponential growth was cell lines were distributed in 4 subclasses (A, B, C, and D). The cluster harvested (after trypsinization for adherent cells), and total RNA from ϳ107 D only contains the CPH 54 cell lines and has an expression pattern cells was isolated using the RNeasy kit (Qiagen) according to the manufac- distinctly different from the remainder of the SCLC lines. The 6 turer’s instructions. RNA from xenografted tumors were homogenized in ressected tumors clustered closely together, and closest to the normal TRIzol (Invitrogen) and purified according to the manufacturer’s instruction. tissues and the CPH 54 lines. It has been shown previously that these The TRIzol isolated RNA was additionally purified using the RNAeasy kit SCLC tumors cluster distinctly apart from other forms of lung cancers (Qiagen, Albertslund, Denmark). Affymetrix Oligonucleotide Array. The preparation of biotin cRNA was and normal lung (1). prepared essentially as described in the Affymetrix Expression Analysis Tech- The cell lines GLC-14, -16, and -19 were established from the same nical Manual. Briefly,10 ␮g of RNA was used as template to generate double- patient during longitudinal follow-up. These cell lines have been

stranded cDNA using a T7-(dT)24 primer (Genset, Paris, France) using Super- compared with the biopsies from which they were derived, and Script RnaseHϪ Reverse Transcriptase and subsequent second-strand synthesis showed a good match among morphological, biochemical, and im- (Invitrogen). The cDNA was transcribed into biotin-labeled cRNA using the munohistological findings (6). GLC-14, established from a tumor BioArray, High Yield RNA transcript labeling kit (Enzo Diagnostics, Farm- before treatment, was in the same subcluster as GLC-16 and -19, ingdale, NY). Fragmentation, hybridization, and scanning were according to which were established after relapse after chemotherapy and on re- the Affymetrix protocol using the Human U95Av2 array (Affymetrix, Santa occurrence after radiotherapy, respectively. GLC-26 was established Clara, CA) and the antibody amplification protocol. The data were analyzed from 1 patient from the primary tumor and GLC-28 from a metastasis using Affymetrix Microarray Suite version 5. Data from each chip was scaled to a global scaling of 100. In figures displaying the microarray signals (array (13), and were found in different subclusters. The line CPH 136A, signal) as bar diagrams, only genes with expression scored as present (P) are which has only been passaged in vivo as xenografts, clustered with the included. cell lines in cluster A. Gene Selection and Hierarchical Clustering. As the basis for the clus- A fetal brain sample was included in the analysis for comparison, tering analysis, genes were selected that had a sum of signals across all of the as this sample would be expected to have the expression pattern of a samples Ͼ5000, and a SD threshold of 200 expression units to select the 1620 proliferative, neuronal phenotype with many similarities to the endo- most variable transcripts. We used the CLUSTER and TREEVIEW programs crine and oncofetal characteristics typical for SCLC. In fact all of the (12) for clustering and visualization of the dataset. Median centering and SCLC cell lines and tumors clustered closest to fetal brain, adult brain, normalization of the data were performed before clustering. and testis, thus confirming the neuroendocrine characteristics of RT-PCR. Semiquantitative RT-PCR was performed using cDNA from all SCLC. The clustering close to testis presumably reflects the prolifer- of the normal tissues, cell lines, and xenografts prepared as described above. The PCR was performed using cDNAs from 150 ng of total RNA using ative status of testis, and that a large variety of cancer cells express oligonucleotide primers for INSM1, CDKN2A, PTTG1, and ASCL1 (Official cancer/testis antigens, such as NY-ESO-1 (CTAG1), MAGEs, and Gene Symbol annotations) with 25 cycles of PCR amplification. GAGEs G antigens (14). All of the xenografts clustered closest to the Western Blots. Whole cell lysates were prepared from cell lines and corresponding cell line, showing that the conditions of cell culture xenografts by homogenization in ice-cold 20 mM Tris-Cl (pH 7.5), 2% Triton does not significantly change the expression pattern compared with X-100 containing Protease Inhibitor mixture set II and III (Calbiochem, Al- the more physiological growth condition as xenografts. bertslund, Denmark). Western blots were performed on 5–15 ␮g of lysate. For Many distinct clusters were observed. Several of these are tissue- probing with anti-NCAM1 antibodies, the lysates were pretreated for 5 min at specific and contain genes specifically expressed by, e.g., brain, liver, ␮ 37°C with 40 ng/ l recombinant EndoN-HIS (gift from E. Bock, Protein adrenal gland, skeletal muscle, and heart. Several sets of clusters are Laboratory, University of Copenhagen, Denmark) to remove polysialylation highly expressed in normal tissues and the SCLC tumors, but not the residues. The antibodies used were mouse monoclonal anti-NCAM1 clone 123C3 (Santa Cruz, Århus, Denmark), polyclonal anti-mGluR8 (GRM8; Up- cell lines or xenografts, as indicated (Normal tissues) in Fig. 1. Some state Biotechnology, Frederikssund, Denmark), goat polyclonal anti-integrin of these gene clusters may reflect the similar expression profiles ␣E (N-19; Santa Cruz), mouse monoclonal anti-GluR2 clone 3A11 (PharM- between SCLC and bronchial epithelial cells as found by Anbazhagan ingen, Brøndby, Denmark), and goat polyclonal anti-NPTXR (C-17; Santa et al. (15), and some reflect the influence of the tumor microenviron- Cruz). ment on gene expression. In addition, there are several gene clusters containing genes of the immune system and extracellular matrix RESULTS AND DISCUSSION proteins, which probably reflect some infiltration by immune and stromal cells. One tumor (no. 6) has distinctly less expression of these Cluster Analyses. The expression profiles of 21 SCLC cell lines, genes but does not differ significantly in the SCLC specifically 8 xenografted tumors, and 18 normal tissues (17 adult tissues and 1 expressed genes. fetal brain) were analyzed. The xenografted tumors were included in There is a large group of genes exclusively expressed by the cell the analysis to be able to identify genes expressed preferentially lines and xenografts, but not by the tumors. Many of these gene because of the conditions of cell culture rather than the more physi- products are involved in replication, cell cycle regulation, and prolif- ological condition of propagation in nude mice. eration. Therefore, these may not be cancer relevant, but rather arti- Of the 12,000 genes represented on the U95Av2 array, a selection facts of established cell lines and are, therefore, not potential targets of differentially expressed genes was performed on 1,620 genes for or markers. There is a distinct cluster of genes expressed by the brain cluster analysis. A two-dimensional hierarchical cluster analysis of all but not in other tissues of which there is expression in most of the of the median levels of expression of SCLC cell lines, xenografts, and tumors and cell lines. A part of this cluster is shown in detail in Fig. normal tissues was performed. In the analysis, it was possible to 2 (Box 3). In addition, there is a cluster of neuronal or neuroendocrine include the expression data of 6 ressected SCLC tumors from Bhat- genes expressed by many of the tumors and cell lines. A section of this tacharjee et al. (1), as their analyses were performed on the same cluster is shown in Fig. 2 (Box 2). Finally there is a cluster of genes Affymetrix Microarray Chips. Comparison of the genes highly ex- expressed by almost all of the tumors and some cells lines, and only pressed in normal lung in Bhattacharjee et al. (1) with the expression in few normal tissues and, therefore, to some extent SCLC-specific. A levels measured for lung in this analysis showed good agreement, thus part of this cluster is shown in detail in Fig. 2 (Box 1). The total justifying a direct comparison of the data. The total cluster analysis is cluster analysis clearly demonstrates the neuronal or neuroendocrine shown in Fig. 1. All of the normal tissues clustered together, and all phenotype of SCLC. The analysis reveals that there is some hetero- of the SCLC lines, except CPH 54A and B, clustered together. The geneity in expression of the cell lines. The cell lines have a set of 1944

Fig. 1. Two-dimensional hierarchical clustering of 21 SCLC cell lines, 8 xenografted tumors, 17 normal tissues, 1 fetal tissue, and 6 SCLC tumors based on the array analysis. The data from the 6 SCLC tumors is from Bhattacharjee et al. (1). A, row shows the expression pattern of a specific gene for all samples of tissues, tumors, cell lines, and xenografts. A, column shows the expression of all of the genes in each sample. Normal tissues are shown in black, tumors in green, cell lines in culture in red, and xenografts in blue. The expression levels of each gene is shown in color coding, where the colors indicate higher, equal or lower expression than the median level of expression of all samples as displayed at the bottom of the figure. The boxed A, B, C, and D indicate the four subclasses of SCLC cell lines and xenografts. The boxed 1, 2, and 3 at the left of the figure show the positions of the clusters displayed in Fig. 2. 1945

Fig. 2. Clusters of genes expressed relevant to specific SCLC and/or neuronal or neuroendocrine expression extracted from the total cluster as indicated in Fig. 1. Cluster 1 shows cancer-specific genes and clusters 2 and 3 display genes highly expressed in both the SCLC and brain and/or neuronal tissues. The names in parentheses are the official gene symbol annotations. expressed genes, which are not expressed in the tumors, and the SCLC Cell Line Subclasses. The grouping into four distinct clus- tumors have expression of contaminating normal tissues and cells, but ters of SCLC cell lines and xenografts (cluster A, B, C, and D in Fig. by comparing both sets of data, SCLC-specific gene expression can be 1) reveals that there may be distinct subclasses of these cell lines. identified. Cluster D is discussed below. SCLC cell lines have been divided 1946

therefore, be expected to cluster together, although they differ in DNA index and sensitivity to radiation therapy (13). Although the cell lines express several neuronal genes, the expression pattern was distinct from the other SCLC lines and xenografts. A cluster of genes highly expressed by the CPH 54 lines and a xenograft thereof, but not any other SCLC line or tumor is indicated in Figs. 1 and 2. It is noticeable that in the cluster of genes highly expressed by many SCLC cells and tumors, the expression in the CPH 54 lines is low or absent (Fig. 2). In the cluster (Fig. 1) specific for the CPH 54 cell lines there is high expression of GAGE 1, 2, 3,4, 5, 6, and 7, which are known tumor- associated testis-specific antigens (18). Only one other cell line (DMS 153) expresses GAGEs at high levels, thus demonstrating that the CPH 54 lines differ markedly from the majority of the other SCLC lines. In the CPH 54 cluster there is also high expression of extracellular matrix proteins, such as a large variety of collagens, fibronectin, Fig. 3. Expression of GRP and DDC. Expression levels determined by the array analysis of all SCLC cell lines, xenografts, and tumors are displayed as bars. and laminin, which are not expressed to the same level by the rest of the SCLC cell lines. This indicates that the cell lines may be of fibroblastoid origin rather than SCLC, although the xenografted tu- previously into two classes: the variant type and the classic type. The mors from the CPH 54A cell line preserved the pathologically deter- variant type is associated with higher growth rates and a more ag- mined features of SCLC (4). In addition, these are the only cell lines gressive phenotype. Indeed, the growth rates of cell lines in cluster B that do not have mutated p535 or loss or mutated pRB, two of the (average doubling time 27 h) is higher than for clusters A and C (72 characteristics of SCLC (19–22). and 67 h, respectively), whereas there is no obvious distinction SCLC, Brain, and Neuroendocrine Clusters. There are three between the morphology of different cell lines in the clusters in vitro. clusters of genes either predominantly expressed by the SCLC cell The classic type express DDC and bombesin-like immunoreactivity, lines and tumors or also expressed in the brain. A section of these are such as GRP, whereas the variants do not (9, 16). Almost all of the displayed in Fig. 2 with the gene annotations included. In Fig. 1 is cells in cluster C, which contains the classic type NCI H69, express indicated the localization of the three clusters. In Table 1 the genes are mRNA for GRP and DDC, whereas cells in cluster B, to which the listed according to functions. Cluster 1 contains genes, which are variant form NCI N417 belongs, do not (Fig. 3). All of the cells in expressed primarily by SCLC tumors and cell lines. This cluster cluster A also express DDC and GRP. The expression in the tumors contains many genes known to be highly expressed by a variety of indicate that these may include both types. Another suggested marker tumor cells. Many of these are genes involved in cell proliferation, for distinction between classic and variant types is expression of the signal transduction, cell division, or high cell motility. The cluster also SGNE1, which has been found expressed in all of the classic, but few contains genes reflecting high metabolic activity. Noteworthy is the variant cell lines. SGNE1 is expressed in most cell lines of clusters A presence of ASCL1 in this cluster, because this is regarded as a and C, but few in cluster B (data not shown). Therefore, it is probable specific neuroendocrine tumor marker negatively regulated in other that clusters A and C contain the classical type and cluster B the tissues (23). The function of ASCL1 is not clarified, but its expression variant type of SCLC, and this analysis identifies many genes differ- is highly correlated to the snail family of transcription factors in- entially expressed in these subclasses of tumors. volved in cell migration and, therefore, may contribute to the invasive In the selected clusters shown in Fig. 2 it is clear that cluster B has phenotype (24). High expression of ASCL1 was found in the 6 a distinctly lower expression of many tumor, brain, neuronal, and neuroendocrine-specific genes. Some of the typical neuroendocrine ressected tumors in Bhattacharjee et al. (1) and 5 ressected tumors in markers such as chromogranin C (SCG2), ENO2, and NCAM1 are Garber et al. (2). The expression levels of ASCL1 in the cell lines and reduced in cluster B, whereas others such as synaptophysin (SST) and normal tissues were verified by semiquantitative RT-PCR (Fig. 4D) PGP 9.5 (USHL1) are not. A large group of clusters of genes rela- confirming the expression levels on the array and showing low ex- tively specific for the cell lines containing many genes involved in pression only in fetal and adult brain in normal tissues. There is only replication, cell cycle regulation, and proliferation do not differ be- very low expression in one line of cluster B (variant type) and very tween the cell line clusters. An exception is a small cluster, which high expression in all but one cell line in clusters A and C (classic appears more prominent in the cluster B and contains many ribosomal type). Therefore, ASCL1 is probably one of the best novel markers for proteins and the molecular chaperone HSP90, and may, therefore, distinction between the variant and classic type of SCLC. ASCL1 is reflect the higher proliferation rate of cell lines in cluster B. not expressed by the CPH 54 cell lines. It should be mentioned that the grouping in classical and variant Cluster 2 contains many neuroendocrine genes, including typical types of SCLCs has been performed mainly on cell lines, and it is not neuroendocrine markers, neuronal developmental markers, and neu- quite clear how these relate to the mixed small cell-large cell carci- rotransmitters. This cluster also contains other genes often associated noma or variant type of SCLC-combined described in the new WHO with tumor cells coding for proteins involved in cell proliferation, classification of lung tumors (17). As this present analysis now reveals signal transduction, cell adhesion, and motility. Cluster 2 contains many new, intracellular markers to which antibodies are available, it NCAM1, a molecule used as a marker for endocrine tumors and cell would be possible to make a retrospective analysis on historical lines (25, 26), and is primarily expressed in brain in the adult. The paraffin sections to determine whether the grouping has clinical analysis was verified by Western blotting revealing that all but one of significance. the SCLC cell lines express the two predominantly fetal isoforms (Mr

Characterization of the CPH 54 Cell Lines. Two cell lines, CPH 140,000 and Mr 180,000 isoforms) of NCAM1 (Fig. 5), thus confirm- 54A and B clustered distinctly apart from the other SCLC cell lines ing earlier studies performed on several of the cell lines (27). Cell and closest to normal tissues (Figs. 1 and 2, cluster D). These two cell lines are subclones from the same original tumor 54A (4) and would, 5 H. S. Poulsen, unpublished observations. 1947

Table 1 Functional listing of the genes shown in the clusters displayed in Fig. 2 Column 1 indicates in which cluster the gene appears, column 2 the name of the gene with the Official Gene Symbol annotations in parenthesis, and column 3 the (putative) functions of the gene products. All genes in italic are highly expressed in the adult and/or fetal brain, and/or known to be neuroendocrine specific or involved in neuronal differentiation or development. Cluster Gene Function(s) Tumor suppressors and regulators of apoptosis 1 Programmed cell death 6 (PDCD6) Inducer of apoptosis 1 Exostoses (multiple)-like 3 (EXTL3) Putative tumor suppressor Cell cycle and replication 1 DEAD/H (DDX11) RNA helicase involved in translation and splicing 1 Extra spindle poles like 1 (S. cerevisiae) (ESPL1) Cell cycle regulation? 1 Cell division cycle 25 B (CDC25B) Positive cell cycle control 2 H1 histone family, member X (H1FX) Chromatin component 2 H2A histone family, member X (H2AFX) Chromatin component 2 Nucleosome assembly protein 1-like 4 (NAP1L4) Assembly of chromatin Cell adhesion, cytoskeleton, and motility 1 Immunoglobulin superfamily 4 (IGSF4) Putative cell adhesion molecule 1 Cofilin 1 (CFL1) Controls actin de- and polymerisation 2 Thymosin, ␤, neuroblastoma cells (TMSNB) Inhibitor of actin polymerisation, neuronal 2 Neural cell adhesion molecule (NCAM1) Cell adhesion, neurite outgrowth and migration 2 Rho GDP dissociation inhibitor (GDI)␣ (ARHGDIA) Inhibitor of cell adhesion 2 Rh-related antigen (CD47) Cell adhesion, migration and integrin signalling 2 Dynein, cytoplasmic, intermediate polypeptide 1 (DNCI1) Tubulin-cytoskeleton associated motor protein 2 Rho guanine nucleotide exchange factor 7 (ARHGEF7) Cell adhesion and motility via Cdc42- and Rac1 2 Secretogranin II (chromogranin C) (SCG2) Chemotaxic neuroendocrine secretory protein 3 Doublecortin (DCX) Tubulin associated in brain development 3 Tubulin ␤ (TUBB) Component of the tubulin-cytoskeleton 3 Internexin neuronal intermediate filament protein, ␣(INA) Neuron-specific intermediate filament protein 3 Kinesin family member 5C (KIF5C) Neuron-specific microtubule motor 3 Doublecortin and CaM kinase-like 1 (DCAMKL1) Brain kinase involved in neuronal migration 3 L1 cell adhesion molecule (L1CAM) Cell adhesion, neurite outgrowth and migration Signal transduction and proliferation 1 Hepatoma-derived growth factor (HDGF) Growth factor and mitogen for fibroblasts 1 Gastrin-releasing peptide (GRP) Growth factor 1 Neurite growth-promoting factor 2 (MDK) Inducer of cell proliferation 1 Tyrosine phosphatase type IVA, member 3 (PTP4A3) Signalling molecule, induces motility and growth 1 VEGF related factor isoform VRF186 (VEGFB) Stimulator of angiogenesis and cell growth 2 Guanine nucleotide binding protein (GNB1) Signal transducer from G protein-coupled receptors 2 Guanine nucleotide binding protein 4 (GNG4) Signal transducer from G protein-coupled receptors 2 Macrophage myristoylated alanine-rich C kinase substrate Signal transducer (MACMARCKS) 2 Growth hormone releasing hormone (GHRH) Growth hormone releasing factor 2 NEL-like 1 (chicken) (NELL1) Putative growth factor 2 Secretagogin (SCGN) Ca-flux and cell proliferation 3 Nel-related protein 2 (NELL2) Possibly involved in cell growth regulation 3 Glutamate receptor, ionotropic, AMPA 2 (GRIA2) Neurotransmitter receptor Development and differentiation 1 Thyroid transcription factor 1 (TITF-1) Activates thyroid specific genes. 1 Achaete scute homologous protein (ASCL1) Transcription factor in neuronal differentiation 1 Inhibitor of DNA binding 2 (ID2) Transcription factor in embryonic development 1 Sex determining region Y-box 2 (SOX2) Transcription factor in embryonic development 2 Neuronal protein (NP25) Central nervous system development 2 SRY (sex determining region Y)-box 4 (SOX4) Transcription factor in embryonic development 2 Distal-less homeo box 5 (DLX5) Transcription factor in neuronal development 2 ISL1 transcription factor, (islet-1) (ISL1) Transcription factor, endocrine 2 Homeo box B2 (HOXB2) Transcription factor in embryonic development 2 Distal-less homeo box 2 (DLX2) Transcription factor in neuronal development 3 Dihydropyrimidinase-like 3 (DPYSL3) Probably involved in axonal outgrowth 3 Ectodermal-neural cortex (with BTB-like domain) (ENC1) Nuclear protein involved in neurogenesis 3 Stathmin-like 2 (STMN2) Neurite outgrowth and differentiation 3 Zinc finger protein 238 (ZNF238) Transcriptional repressor in neuronal development 3 Collapsin response mediator protein 1 (CRMP1) Growth cone guidance in neural development 3 Brain abundant, membrane attached signal protein 1 (BASP1) Growth cone guidance and nerve sprouting Metabolism 1 Neutral amino acid transporter B (SLC1A5) Solute carrier 1 Thiopurine S-methyltransferase (TPMT) Causes methylation of thiopurine drugs 1 Sodium channel, nonvoltage-gated 1 alpha (SCNN1A) Sodium channel 1 Uncoupling protein homologue (UCP2) Mitochondrial membrane transporter 1 Lysophosphatidic acid acyltransferase-␣ (AGPAT1) De novo phospholipid biosynthesis 2 Neuron specific (␥) enolase (ENO2) Neuronal glycolysis 3 Fatty acid binding protein 7, brain (FABP7) Fatty acid uptake and metabolism in the brain Unknown or other functions 1 Hypothetical protein FLJ12443 Possibly belonging to the chaporonin family 1 Nuclear receptor co-repressor N-Cor (NCOR1) Inhibitor of transcription by hormone receptors 1 Calmodulin 1 (CALM1) Phosphorylase kinase 1 Tyr 3-/Trp 5-monooxygenase activation protein ␨ (YWHAZ) Signal transduction 1 Nuclear receptor subfamily 2, group F, member 1 (NR2F1) Hormone regulated transcription factor 1 Inositol polyphosphate-5 phosphatase-like 1 (INPPL1) Phosphatase 1 Tripartite motif-containing 28 (TRIM28) Transcription co-repressor 1 Transport-secretion protein 2.2 (TTS-2.2) Unknown 1 Protective protein for ␤-galactosidase (PPGB) Lysosomal protective protein

1948

Table 1 Continued. Cluster Gene Function(s)

Unknown or other functions—Continued 2 GDP dissociation inhibitor 1 (GDI1) Intracellular vesicle transport in neurons 2 Ubiquitin specific protease 11 (USP11) Deubiquitinating enzyme 2 General transcription factor II (GTF2I) Transcriptional activator 2 Coxsackie virus and adenovirus receptor (CXADR) Receptor for coxsackievirus and adenovirus 2 Dopa decarboxylase (DDC) Synthesis of neurotransmitters 2 FLJ30781 fis clone FEBRA2000874 Unknown, brain specific 3 Core-binding factor, runt domain, ␣ subunit 2 (CBFA2T1) Putative transcription factor 3 Likely homolog of rat kinase D-interacting substance (KIDINS220) Unknown 3 P311 protein (P311) (neuronal) Unknown 3 Y09836: mRNA for 3UTR of unknown protein Unknown 3 AL036744:DKFZp564I1663_r1 Unknown 3 Cytoplasmic linker associated protein 2 (CLASP2) Unknown 3 Reticulon 1 (RTN1) Possibly involved in neuroendocrine secretion 3 AF038185:Homo sapiens clone 23700 Unknown lines in clusters A and C (the classical type) have high expression, Other Potential Target Molecules Identified by the Microarray whereas cell lines in cluster B (variant type) have low expression. Analysis. Specific expression of other surface molecules not associ- Therefore, the level of expression of NCAM1 may also be a new ated previously with SCLC were identified by the analysis, and some marker for determination of which subclass a cell line or tumor of these are potential candidates as surface targets for therapy. The belongs to. However, as most SCLCs express NCAM1 (although at NPTXR was found expressed by the array analysis in more than half different levels), it has been a frequent candidate for targeted therapy of the SCLC cell lines, and only in brain and prostate in normal against SCLC. Indeed, 5 of the 6 of the tumors from Bhattacharjee et tissues. The expression of the protein by the SCLC cell lines was al. (1) also show high expression of NCAM1. Attempts have been confirmed by Western blotting (Fig. 5), which demonstrated expres- made to use NCAM1 for radioimmunotherapy (28–31) or for immu- sion in Ͼ90% of the cell lines and xenografts. This receptor is notoxin therapy (32–35), but none of these have reached clinical use. normally only expressed in synapses, where it mediates uptake of A number of small peptides, which bind NCAM1, have been identi- extracellular material via one of its two ligands, neuronal pentraxin 1 fied recently (36), and these could alternatively be tried for use in and 2 (40). Another receptor, the apolipoprotein E receptor 2 (LRP8), NCAM1-targeted therapy against SCLC. was also expressed in many cell lines with no detectable expression in Cluster 3 demonstrates the neuronal phenotype of SCLC, as this normal tissues except for brain and testis (data not shown). This cluster contains molecules almost exclusively expressed by SCLC, receptor internalizes lipoproteins associated with ApoE in addition to and fetal and/or adult brain. Many of the molecules are involved in a specific ligand, reelin (41, 42). These two receptors are internalizing neurite outgrowth or neuronal development. A neuronal molecule not and, therefore, good candidates for a number of therapeutic ap- associated previously with SCLC is the GRIA2, an excitatory neuro- proaches involving natural ligands, synthetic ligands, or antibodies. transmitter receptor, is included in Cluster 3. The array analysis Another candidate surface molecule which has not been identified identified expression of GRIA2 by Ͼ50%, and RT-PCR verified 86% previously as expressed by SCLC is the ITGAE. ITGAE interacts with (data not shown) of the SCLC lines or xenografts and among normal the ␤7 subunit forming an E-cadherin receptor and is normally only tissues only expressed in brain. Validation by Western blotting expressed on intraepithelial lymphocytes (43, 44). The expression of showed the presence of protein in Ն35% of the cell lines or xenografts the protein by the cell lines was verified by Western blotting, which (Fig. 5). Another glutamate receptor, the GRM8, was also identified demonstrated expression by at least 76% of the cell lines (Fig. 5). as expressed by 30% of the SCLC cell lines by the array analysis, Other receptors such as the nicotinic acetylcholine receptor ␣ 5 and whereas Western blotting revealed expression in all of the cell lines GRP49, a glycoprotein hormone receptor, were also found highly (Fig. 5). The presence of glutamate receptors on SCLC provides expressed in most SCLC lines and xenografts. potential new targets for radiotherapy, because there are many spe- Cancer-related Gene Expression. A newly identified oncogene, cific, small molecular weight ligands (agonist or antagonists) for these PTTG1 (securin), expressed by various cancers was highly expressed receptors (37, 38) and receptor-specific antibodies. As many of these in all of the SCLC lines, tumors, and testis, and weakly expressed in ligands do not penetrate the blood brain barrier, therapy could be spleen, thyroid, and trachea. The analysis was verified on the cell lines limited to areas outside the brain, thus avoiding adverse effects on and normal tissues by RT-PCR (Fig. 4A), which revealed low expres- glutamate receptors expressed in the brain. sion in more normal tissues. The gene product has transforming Other neuroendocrine markers not included in the clusters dis- activity in vitro and tumorigenic activity in vivo (45, 46). Several played in Fig. 2 were also found highly expressed in all or most SCLC different functions have been attributed to this molecule relating to its lines and tumors. Of these can be mentioned ubiquitin carboxyl- oncogenic nature. One function is as an inhibitor of chromatid sepa- terminal esterase L1, which is a neurospecific peptide that protects ration causing chromosomal instability (47) and a different function as against targeted protein degradation; secretonin I (chromogranin B), a a transcription factor. The latter is believed to induce expression of neuroendocrine secretory protein, and creatine kinase BB, a suppos- c-myc, which subsequently induces proliferation and expression of edly brain-specific enzyme, although the array analysis showed high basic fibroblast growth factor (FGF2), which, in turn, stimulates expression also in many normal tissues. A newly identified marker angiogenesis (45). Indeed, increased serum levels of basic fibroblast highly expressed exclusively in neuroendocrine cells and tumors is growth factor have been found in patients with SCLC (48). The effect INSM1, a transcription factor involved in pancreatic and neuronal of the expression of PTTG1 on cell proliferation, vascularization, and development (39). INSM1 was highly expressed by most cell lines, chromatin stability in SCLC should therefore be performed to assess and by the 6 SCLC tumors from Bhattacharjee et al. (1) and the 5 whether drugs targeted to this molecule may offer new approaches for tumors from Garber et al. (2). The array analysis for expression of therapy. INSM1 in the cell lines and normal tissues was verified by RT-PCR Protooncogenes of the myc family are highly expressed by many (Fig. 4C). INSM1 was not expressed by the CPH 54 lines. cancer types, in particular lung cancers. Elevated expression of at least 1949

many trials using immunotherapy, as peptides from these molecules are displayed by cytotoxic T lymphocytes (53). Only few studies of MAGE expression in lung cancer have been performed (54). As the array contains probe sets for all 11 of the known MAGEs, a more complete profiling of MAGE expression in SCLC could be determined. The analysis revealed very high expression of many members of the MAGE family in most SCLC cell lines with high expression of MAGE-2, -3, and -6, and intermediate expression of MAGE- 1, -5a, and -12. Expression of MAGE-3 is shown in Fig. 6. However, in all of the cases the tumors had a markedly lower expression, supporting the theory that cell culture may lead to activation of MAGE genes (53). Therefore, MAGE-based immunotherapy may not appear to be the first choice for alternative treatment of SCLC. A group of cancer invasion-associated molecules are the MMPs and their inhibitors (TIMPs), as these can supply the extracellular prote- olysis necessary for vascularization, tumor invasion, and tumor dis- semination. Expression of several MMPs and TIMPs has been found in several cancer types including SCLC (55, 56). However, it is well established that components of extracellular protease systems can be expressed by either stromal and tumor cells or both, and involve cellular interactions in the microenvironment of the tumor (57). In- deed, the array analysis showed that neither the SCLC cell lines nor xenografts have elevated MMP or TIMP expression compared with normal tissues, whereas the tumors had elevated expression of many MMPs and TIMPs (in particular MMP-9, MMP-11, MMP-12, MMP- 14, and TIMP-1), thus demonstrating one of the limitations of using cell lines and xenografts for this type of analysis. The array analysis shows that of the multidrug resistance and resistance-associated protein genes, only ABC C5 (MRP5) is highly expressed by all of the SCLC cell lines and tumors. High expression of an unspecified MRP type in SCLC has been reported previously (58). The knowledge of the most prominently expressed multidrug resistance gene could affect the choice of anticancer drugs for the disease. Proliferation and Replication-related Molecules. The analysis revealed high and relatively specific expression in most or all of the cell lines and xenografts of a number of molecules involved in cell proliferation, cell cycle control, or chromosome separation; all molecules that are potential targets for therapy. A number of these are listed in Table 2. All of the shown genes are also expressed in at least 50% of the tumors. Some of the genes have not been associated previously with SCLC but have found to be up-regulated in other tumors. One puzzling novel observation is the high expression in Fig. 4. Validation of microarray analysis by RT-PCR. Displayed is the RT-PCR of SCLC of mitotic arrest deficient, a component of the mitotic spindle expression of 4 selected genes using total RNA from normal tissues, SCLC cell lines, and assembly checkpoint, which is generally regarded as a tumor suppres- xenografts compared with the expression levels detected by the array analysis. The bar sor. Reduced expression has been reported in some cancers (59), diagrams show the signals on the microarray. Below the diagram is shown the electro- phoresis bands after RT-PCR performed on the equal amounts of total RNA from each sample. Expression analysis is shown for PTTG1; A, p16INK4/p14ARF (CDKN2A; B), INSM1; C, and ASCL1; D.InE is displayed the sequence of the normal tissues and SCLC samples. one of the three mycs (c-myc, n-myc, or l-myc) has been found in many SCLC cell lines (13, 49, 50). The high levels of expression and the form of myc expressed as determined by the array analysis correlated completely with the data published previously for the cell lines. The high expression has in many cases been correlated to amplification of the genes, but is generally more prevalent after establishment as xenografts (51). This is in concordance with the observation that the ressected tumors showed expression of c-myc but not at the same high level as the cell lines and xenografts. A family of cancer-related molecules are the MAGEs, which are Fig. 5. Validation of protein expression by Western blotting. Equal amounts of whole expressed exclusively in testis in normal tissue, and often highly cell lysates from SCLC cell lines and xenografts were analyzed by SDS-PAGE and immunoblotting. Expression analysis is shown for NCAM1, NPTXR, GRM8, ITGAE, and expressed in cancer cell lines and tumors (14, 52). They are targets for GRIA2. 1950

Fig. 6. Microarray expression levels of the tumor/testis antigen MAGE-3.

although elevated expression has been reported for gastric cancer. The similar to the array analysis (Fig. 4B) demonstrating the presence of microarray analysis did not indicate reduced expression of its down- this mRNA. p14ARF transcription is stimulated by E2F transcriptions stream component, cdc20. factors (67), and the analysis shows high expression of E2F1 and However, this is the case for another tumor suppressor, CDKN2A E2F3 in all of the SCLC cell lines, as has been found previously for (p16INK4), which is a negative regulator of cell proliferation by most SCLC tumors by immunohistochemistry (68). However, an stabilizing the tumor suppressor pRB. Deletions, reduced expression, immunohistochemical analysis has shown that despite high levels of or mutations of p16INK4 are observed frequently in a variety of mRNA expression, the p14ARF protein was not present in the ma- tumors. However, the microarray analyses showed high expression by jority of SCLC tumors, indicating a regulation at the translational most SCLC lines and tumors, which is consistent with previous level, whereas p16INK4A protein was present in most tumors (60). observations of high expression in SCLC tumors (60, 61) and cell This observation demonstrates the importance of validating results lines (62). p16INK4 leads to cell cycle arrest in the presence of obtained by array analysis on a protein level. functional pRB. pRB has been found to be absent or mutated in the In the search for new therapeutic targets for therapy and prognosis majority of SCLC tumors and cell lines (20, 22, 63, 64). In fact, of SCLC, an expression screening of many SCLC cell lines and absence of p16INK4 has been found to be restricted to lung cancer xenografts was performed using oligonucleotide microarrays. The cell lines that retain wild-type pRB (65). There are two major forms analysis revealed several novel potential targets and confirmed the of CDKN2A, p16INK4 and p14ARF, derived by alternative splicing. expression of the majority of known targets. These genes include p14ARF is likewise regarded a tumor suppressor by stabilizing p53, surface receptors, oncogenes, antiapoptotic genes, and other cancer- but also has p53-independent cell cycle regulatory functions (66). The related genes, which all may be targets for a wide variety of thera- probe sequences on the microarray will recognize both forms. RT- peutic approaches. Comparison with the published expression profiles PCR using a p14ARF specific primer set showed expression levels of 6 ressected SCLC tumors (1) demonstrated that the expression

Table 2 Highly expressed genes in SCLC involved in cell replication and proliferation The names in parenthesis are the Official Gene Symbols. Signal is the average of the hybridisation signals from the array of the all cell lines/xenografts or all tumors from (1). Expression in normal tissues Cell lines Tumors Gene Function(s) Signal Signal High Medium Low Not previously described in SCLC fms-related tyrosine kinase 3 Stimulates proliferation/differentiation of 479 416 Colon, spleen, (STK-12) stem cells testis Possibly involved in chromosome segregation Mitosin (CENPF) Involved in chromosome segregation 182 134 Testis during mitosis Cyclin-selective ubiquitin carrier Involved in completion of mitosis 983 543 Spleen, testis protein (UBE2C) Kinesin-like spindle protein Motor protein, functions in chromosome 198 100 Colon, testis Small intestine (KNSL1) separation Ribonuclease H1 large subunit Degradation of RNA:DNA hybrids 263 142 Spleen, testis (RNASEH1) during replication Mitotic arrest deficient (MAD2) Mitotic spindle assembly checkpoint 192 49 Brain, colon, small intestine, spleen, stomach, testis, thyroid, trachea Lamin B1 (LMNB1) Nuclear structure and interaction with 490 270 Colon, spleen PKC Previously described in SCLC Topoisomerase II ␣ (TOP2A) Involved in replication and transcription 520 574 Testis Colon, heart, lung, liver, spleen, thyroid, trachea, small intestine Cyclin B1 (CCNB1) Involved in cell cycle progression 363 105 Testis Colon, spleen, trachea Cyclin B2 (CNB2) Involved in cell cycle progression 273 102 Testis Spleen, trachea Proliferating cell nuclear antigen Replication factor 1090 513 All tissues (PCNA) p16INK4 (CDKN2A) Involved in cell cycle progression 263 76 1951

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Nina Pedersen, Shila Mortensen, Susanne B. Sørensen, et al.

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