- Email: [email protected]
Characterization of Gene Expression Profiles of 3 Different Human Oral Squamous Cell Carcinoma Cell Lines With Different Invasion and Metastatic Capacities
J Oral Maxillofac Surg 66:918-927, 2008
Characterization of Gene Expression Profiles of 3 Different Human Oral Squamous Cell Carcinoma Cell Lines With Different Invasion and Metastatic Capacities N. Fazil Erdem, DDS, PhD,* Eric R. Carlson, DMD, MD,† and David A. Gerard, PhD‡ Purpose: The gene expression of 3 oral squamous cell carcinoma (OSCC) human cell lines, BHY, HN,
and HSC-3, were studied based on their reported ability to invade adjacent bone or metastasize to cervical lymph nodes and/or distant organs. Materials and Methods: The characteristics of each cell line were confirmed on scid mice using micro-positron emission tomography (PET)/computerized tomography (CT) imaging techniques. Complimentary DNA (cDNA) microarray techniques were used to determine the gene expression profile differences between each of the three OSCC cell lines. Results: BHY, HN, and HSC-3 cell lines expressed 139, 214, and 128 up-regulated genes; and 117, 262, and 117 down-regulated genes, respectively. The clusterization of data showed that there are 13 genes that are up-regulated and 83 genes that are down-regulated in all 3 OSCC cell lines. Collection of genes organized by pathway may cause aggregate evaluation of anomalies. Thus the pathway analysis performed for each cell line based on cDNA microarray results showed BHY, HN, and HSC-3 cell lines to have 8, 10, and 3 up-regulated pathways and 3, 9, and 6 down-regulated pathways, respectively. Conclusions: This study showed that cDNA microarray analysis is an effective tool for mapping molecular signatures. With this technique it is possible to observe the entire genome of a malignant tumor so as to appreciate the simultaneous interactions among thousands of genes. © 2008 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 66:918-927, 2008 Oral squamous cell carcinoma (OSCC) is the most common cancer of the oral cavity, accounting for 90% of all diagnosed oral malignancies and approximately 6% of all human malignancies.1 There is little pub-
lished information on the capability of some OSCCs to invade adjacent bone while other OSCCs do not exhibit this property. In addition, some OSCCs readily metastasize to cervical lymph nodes and/or distant organs, whereas others do not. OSCC represents an anomaly of multiple molecular genetic events in many chromosomes and genes.2 The consequence of this chromosomal damage is cell dysregulation with disruptions in cell signaling, the cell growth cycle, and mechanisms for repairing cell damage and eliminating dysfunctional cells.2,3 The cluster of genes down-regulated in OSCC includes several extracellular matrix (ECM) proteins that regulate epithelial adhesion and collagen fibril formation. Genes that are upregulated in OSCC are associated with ECM function, interferon response, proliferation, and immune response. In addition, genes implicated in invasion, Ras signaling, the cell surface/ECM, and angiogenesis are highly expressed in OSCC.4
Received from the Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of Medicine, and the University of Tennessee Cancer Institute, Knoxville, TN. *Intern. †Professor and Chairman. ‡Associate Professor. This research was supported by the Physician’s Medical Education and Research Foundation (PMERF), University of Tennessee Medical Center, Knoxville. Address correspondence and reprint requests to Dr Carlson: 1930 Alcoa Highway, Suite 335, Knoxville, TN 37920; e-mail: [email protected] © 2008 American Association of Oral and Maxillofacial Surgeons
0278-2391/08/6605-0014$34.00/0 doi:10.1016/j.joms.2007.12.036
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In this study, 3 OSCC human cell lines—BHY, HN, and HSC-3—were chosen for study based on their reported ability to invade adjacent bone or metastasize to cervical lymph nodes and/or distant organs. The normal human nasal epithelial cell line (HNEC) was selected as a control group. The working hypothesis of this study was that OSCCs that demonstrate different actions have different gene and pathway expression profiles; for example, OSCCs that invade adjacent bone will have different expression profiles than OSCCs that metastasize to cervical lymph nodes and/or distant organs. Identifying the gene and pathway expression differences in these 3 cell lines will provide insight into which genes and pathways are responsible for lymph node or distant organ metastasis or bone invasion of OSCC. Identifying genes or their related proteins that are associated with tumor metastasis or invasion will provide a basis for further studies to investigate metastatic progression and local invasiveness of this tumor and to evaluate these genes and proteins as therapeutic targets. In the present study, micro-positron emission tomography (PET)/computed tomography (CT) imaging was performed to investigate the invasion and/or metastatic potential of each cell line in immunocompromised scid mice and to delineate the differences in jawbone invasion and distant organ metastasis of these 3 OSCC cell lines in a biological system.
Materials and Methods CELL LINES AND CULTURES
The HNEC cell line (PromoCell, Heidelburg, Germany), cultivated in Airway Epithelial Cell Growth medium (PromoCell), was used as a control. This cell line was selected because it is the only normal head and neck mucosal cell line currently available for study. Cell lines BHY and HN were obtained from DSMZ, Braunschweig, Germany. The BHY cell line is known to invade the mandibular bone. The HN cell line is known to metastasize to cervical lymph nodes, lungs, and brain but to not invade bone. These cell lines were cultivated in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Cell line HSC-3 was obtained from the Japanese Health Science Research Resources Bank. It is known to metastasize to cervical lymph nodes. Eagle’s minimal essential medium with 10% calf serum was used as a cultivation medium. After 100 g/mL streptomycin and 100 U/mL penicillin were added to the media, all 4 cell lines were incubated in a humidified atmosphere of 95% air and 5% CO2.
IMAGING THE INVASION AND/OR METASTATIC CHARACTERISTICS OF OSCC CELL LINES
Micro-PET/CT imaging was used to image the invasion and/or metastatic capacities of each cell line in 3 scid mice in each group. The OSCC cell lines and the HNEC cell line were introduced into the left masseter muscles of the scid mice by injecting a suspension of 1 ⫻ 107 cells in Matrigel (BD Biosciences, Franklin Lakes, NJ). The control mice received only Matrigel. After 30 days, the mice were given 300 Ci of [18F] fluorodeoxyglucose intraperitoneally, followed by an intravenous injection of vascular CT contrast agent 30 minutes later. The animals were sacrificed by an overdose of isoflurane inhalation and imaged by microPET, acquired using the Focus 220 imaging system, followed by a micro-CT scan using a MicroCAT II imager (Siemens Molecular Imaging, Knoxville, TN). TOTAL RNA EXTRACTION FROM THE CELL LINES
The total RNA extraction was performed using an RNeasy Midi Kit (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. The quantity and quality of total RNA were checked by spectrophotometry and by the expression levels of aldolase and -actin genes in each cell line. cDNA MICROARRAY ANALYSIS
The PolyATtract mRNA isolation system (Promega, Madison, WI) was used to extract the mRNA from the total RNA. The cDNA synthesis system (Promega) was used to synthesize cDNA from mRNA. Each tumor cell line was compared with the HNEC cell line using the cDNA microarray technique. Operon Human version 3.0 microarray slides (Operon Biotechnologies, Huntsville, AL) were used. Labeled tumor cell lines and HNEC cell line cDNAs were hybridized on microarray slides using the hybridization buffer. All slides were scanned in a GenePix 4000 scanner (Axon Instruments, Union City, CA). cDNA microarray experiments were repeated 4 times for each cell line to obtain a more reliable analysis. The microarray images were processed using GenePix Pro software (Molecular Devices, Sunnyvale, CA). Both bulk normalization and Lowess normalization were applied to analyze the data. The microarray data were confirmed by real-time polymerase chain reaction (PCR) using the 3 most highly up-regulated genes from each cell. Self-organizing maps and hierarchy clustering were performed to gain more insight into gene function and interaction. To narrow down specific genes, analysis of variance was used to obtain the estimates and confidence intervals for all genes in each experiment set. Based on the expression levels of the genes in the microarray analysis, different
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GENE EXPRESSION PROFILES OF ORAL SQUAMOUS CELL CARCINOMA CELL LINES
FIGURE 1. Left sagittal PET/CT image of a mouse with BHY cell line injection. The arrow shows the mandibular bone resorption due to invasion of the bone by OSCC. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
pathways were analyzed on each OSCC cell line using the Kyoto Encyclopedia of Gene and Genomes (KEGG).
Results IMAGING THE INVASION AND/OR METASTATIC CHARACTERISTICS OF THE OSCC CELL LINES
The PET/CT images of HNEC and the Matrigel-only groups demonstrated no evidence of tumor formation in any of the mice. All 3 mice in the BHY cell line group showed invasion of the OSCC to the mandibular bone (Fig 1); however, only 2 mice in the HN group exhibited metastasis to the lymph nodes and distant organs, such as the lungs and kidneys (Fig 2). The other mouse in the HN group had cervical lymph node metastasis of OSCC. All 3 mice in the HSC-3
group demonstrated lymph node metastasis (Fig 3). The imaging study results matched the characteristics of the OSCC cell lines that have been reported to date. This information is very useful, because it demonstrates that the cultured cell lines maintained their biologic characteristics. As such, it suggests that the gene expression profiles of the 3 cell lines were maintained. Stated differently, based on these findings, we can deduce that the culturing process did not alter the gene expression profiles of the BHY, HN, and HSC-3 cell lines. cDNA MICROARRAY ANALYSIS OF 3 OSCC CELL LINES
After the normalization process, each OSCC cell line ended up with different up-regulated and down-regulated genes. The BHY cell line expressed
FIGURE 2. Left sagittal PET/CT image of a mouse with HN cell line injection. The yellow arrow shows lung metastasis, the light blue arrow shows kidney metastasis, and the white arrow shows the cervical lymph node metastasis of OSCC. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
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FIGURE 3. Axial PET/CT image of a mouse with HSC-3 cell line injection. The white arrow shows cervical lymph node metastasis. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
139 up-regulated genes and 117 down-regulated genes, the HN cell line expressed 214 up-regulated genes and 262 down-regulated genes, and the HSC-3 cell line expressed 128 up-regulated genes and 117 down-regulated genes. Clusterization of data identified 13 genes that were up-regulated and 83 genes that were down-regulated in all 3 OSCC cell lines (Figs 4, 5). There were 57 up-regulated genes and 42 down-regulated genes in common in the BHY and HN cell lines (Fig 6), 24 up-regulated genes and 49 down-regulated genes in common in the BHY and HSC-3 cell lines (Fig 7), and 24 upregulated genes and 44 down-regulated genes in common in the HN and HSC-3 cell lines (Fig 8).
The real-time PCR analysis results confirmed the cDNA microarray analysis results (Fig 9). The upregulation values of the 3 genes from each cell line are given in Table 1. The only questionable up-regulation value in the cDNA microarray analysis is the expression level of the MAGEA1 gene in the HSC-3 cell line, with an error bar of 0.89; all of the other genes have reasonable error bars (Table 1). Based on the expression levels of the genes in the microarray analysis, different pathways were analyzed on each OSCC cell line using the KEGG. The BHY cell line had 8 up-regulated and 3 down-regulated pathways, the HN cell line had 10 up-regulated and 9 down-regulated pathways, and HSC-3 cell line had 3 up-regulated and 6 down-regulated pathways (P ⬍ .05) (Table 2). The 3 OSCC cell lines had some upregulated and down-regulated pathways in common. Pathways regulating actin cytoskeleton and MAPKsignaling were up-regulated in all 3 cell lines; however, the focal adhesion pathway was up-regulated only in the BHY and HN cell lines. The ECM–receptor interaction pathway was down-regulated in all 3 cell lines.
Discussion FIGURE 4. Clusterization of the 13 genes that were up-regulated in all 3 OSCC cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
Our findings demonstrate that OSCC represents multiple molecular genetic events occurring in many chromosomes and genes. The genes that demonstrated alterations in this study have roles in ECM
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FIGURE 5. Clusterization of the 83 genes that were down-regulated in all 3 OSCC cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
degradation, cell adhesion, epidermal development and differentiation, signal transduction, transcriptional regulation, cell growth and proliferation, proteolysis, basement membrane degradation, and cytoskeleton degradation. Recently, Choi et al5 reviewed more than 20 articles that incorporated cDNA microarray analysis in the study of head and neck SCC and identified 84 genes with common alterations, 32 of which were up-regulated, 33 of which were down-regulated, and 19 of which had conflicting expression data. Several major biological systems or pathways are altered in head and neck SCC, including cell cycle control, matrix metalloproteinase (MMP), and inflammatory response systems. Genes encoding for ECM and integral membrane proteins, proteins involved in epidermal development and differentiation, and cell adhesion molecules most frequently have altered expression in head and neck SCC.5
In the present study, we identified 13 genes that were up-regulated in all 3 OSCC cell lines studied. Twelve of these are known genes, but 1 has not been described previously. Basically, these genes function in mitotic progression and chromosome segregation, apoptosis inhibition, cell growth, transduction, intercellular transmission of polarity information during tissue morphogenesis and/or in differentiated tissues, embryonal development, tumor transformation and progression, and condensation of nucleosome chains into higher-order structures. We also identified 83 genes that were down-regulated in all 3 OSCC cell lines. Most of these genes have vital functions in cell biology, including cell– cell or cell– basement membrane junction, intercellular signaling, regulation of cell motility, cell growth and differentiation, wound healing, maintenance of cell shape, immune response against tumor cells, and signal transduction cascades.
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FIGURE 6. The 57 up-regulated genes and 42 down-regulated genes in both the BHY and HN cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
The HN cell line exhibited the greatest degree of gene alteration of the 3 OSCC cell lines studied. The up-regulated genetic structure of the BHY cell line was more similar to the up-regulated genetic structure of the HN cell line. The up-regulated genetic structure of the HSC-3 cell line showed more similarity to the up-regulated genetic structure of the BHY cell line. The down-regulated genetic structure of the BHY cell line was closer to the down-regulated genetic structure of HSC-3 cell line. Pathway analyses of each OSCC cell line were conducted to gain insight into the mechanisms of invasion, lymph node metastasis, and distant organ metastasis of OSCC. Based on the P values (P ⬍ .05) of the genes identified, the up-regulated and down-regulated pathways of the 3 OSCC cell lines are listed in Table 2. The pathway regulating the actin cytoskeleton was up-regulated in all 3 OSCC cell lines. Besides provid-
ing a structural framework around which cell shape and polarity are defined, this pathway has dynamic properties that provide the driving force for cells to move and to divide. Overactivation of this system can cause bone metastasis in breast cancer.6 This pathway is a target of most anticancer drugs. Activation of the transforming growth factor- signaling pathway is common in malignancies. This pathway stimulates tumor progression by its pleiotropic activities on both cancer cells and on nonmalignant stromal cells of the tumor.7 This pathway was upregulated only in the BHY cell line. The hedgehog signaling pathway involves the control of cell proliferation and cell differentiation during morphogenesis of the endochondral skeleton of vertebrates.8 It has been shown that blockage of this pathway suppresses the growth of breast carcinoma cells.9 Up-regulation of this pathway is commonly found
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GENE EXPRESSION PROFILES OF ORAL SQUAMOUS CELL CARCINOMA CELL LINES
FIGURE 7. The 24 up-regulated genes and 49 down-regulated genes in both the BHY and HSC-3 cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
in OSCC.10 This pathway was up-regulated in the BHY cell line and down-regulated in the HN cell line, and thus may be involved in bone invasion by OSCC. The focal adhesion pathway plays a role in mechanical adhesion of cells to the ECM, which is important for cell growth, survival, and migration. PC3, a prostate cancer cell line, has been found to invade mature bone and highly express the focal adhesion pathway.11 To the best of our knowledge, there has been no study correlating this pathway with the bone invasion of OSCC. This pathway was up-regulated in the BHY and HN cell lines. The Janus kinase–signal transducers and activators of transcription (Jak-Stat) signaling pathway was another pathway up-regulated in the BHY cell line. The Jak-Stat signaling pathway can be activated by uPA– uPAR interaction.12 Activation of this pathway is known to be involved in bone metastasis by prostate cancer.13 Another role of this pathway is in the activation of MMP, which is involved in bone invasion by OSCC. The up-regulation of MMP in the BHY cell line may be related to activation of this pathway.
The mitogen-activated protein kinase (MAPK) signaling pathway transduces a wide variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation, and apoptosis. It also plays a role in the regulation of MMP gene expression, especially of MMP-1, MMP-3, and MMP-9.14 Inhibition of the MAPK signaling pathway could potentially serve as a therapeutic target to specifically prevent tumor cell invasion. This pathway was up-regulated in all 3 cell lines, suggesting that it may be one of the major pathways related to OSCC invasion and metastasis. The cell cycle pathway is a series of events in a eukaryotic cell between one cell division and the next. Up-regulation of the cell cycle pathway is common in many cancers, especially those with high metastatic rates. Both cyclins and cyclin-dependent kinase genes (the major genes that regulate the cell cycle pathway) were up-regulated in the HN cell line. Cyclins and cyclin-dependent kinases may be associated with distant metastasis of OSCC.
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FIGURE 8. The 24 up-regulated genes and 44 down-regulated genes in both the HN and HSC-3 cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
The calcium signaling pathway was another pathway activated in the HN cell line. It has been shown that prostate cancer frequently metastasizes to bone. Elevated extracellular Ca⫹2 can enhance proliferation of skeletal metastases.15 An increase in the Ca⫹2 signaling pathway plays an important role in cancer progression and metastasis.16 The axon guidance pathway plays an important role in the development of the nervous system. Semaphorin 4D protein, which is responsible for the development of the central nervous system and regulation of axonal growth, is highly expressed in cell lines derived from head and neck SCCs.17 A similar pattern has been observed in malignant cells from prostate, colon, breast, and lung cancer tissues.18 The axon guidance pathway was highly upregulated only in the HN cell line; this suggests that up-regulation of this pathway may occur in advanced stages of OSCC and may have a role in metastasis of OSCC. The Toll-like receptor signaling pathway is closely related to the invasion and metastasis of cancer. It is a novel pathway that engages the apoptotic machinery.19 This pathway was up-regulated only in the HSC-3 cell line. The ECM–receptor interaction pathway was down-regulated in all 3 OSCC cell lines. Degrada-
tion of the ECM is an essential step in the invasion and metastasis of cancer.20 Down-regulation of this pathway may aid in migration of tumor cells to distant organs.21 Circadian rhythm is essentially a 24-hour cycle in the physiological processes of living beings.22 This pathway was down-regulated only in the HN cell line; thus, down-regulation of this pathway may play a role in distant metastasis. The Wnt signaling pathway proteins form a family of highly conserved and secreted signaling molecules that regulate cell– cell interactions during embryogenesis. It was down-regulated only in the HN cell line. Leethanakul et al23 found that most head and neck SCCs overexpress some members of the Wnt signaling pathway; however, Yeh et al24 concluded that Wnt pathway–related genes play a very limited role in the development of OSCC. Deregulation of the Wnt signaling pathway has been shown to occur by several different mutational mechanisms in human cancers.25 The notch signaling pathway is a gene regulatory pathway involved in multiple differentiation processes. This pathway is a highly conserved pathway for cell– cell communication. It is involved in the regulation of cellular differentiation, proliferation, and specification. JAGGED1, a notch receptor li-
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FIGURE 9. Real-time PCR analysis results. The blue columns represent the BHY cell line, the red columns represent the HN cell line, and the green columns represent the HSC-3 cell line. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
gand, is significantly more highly expressed in metastatic prostate cancer compared with localized prostate cancer or benign prostatic tissues.26 This pathway was down-regulated in the HN cell line. The gap junction and cell adhesion pathways were down-regulated only in the HSC-3 cell line. These pathways function in the formation of cell– cell junctions and ion movement between cells and hence play a role in tissue homeostasis.27 The growth rate of tumor cells is negatively correlated with the strength of intercellular communication;
Table 1. UP-REGULATION AND ERROR BAR VALUES OF THE 3 MOST UP-REGULATED GENES IN EACH OSCC CELL LINE ACCORDING TO REAL-TIME PCR ANALYSIS RESULTS
Cell Line: Gene
Up-Regulation Value
Error Bar Value
BHY: MAGEA9 BHY: Q96AL8 BHY: GFI1 HN: NMES1 HN: MAGEA1 HN: Q96AL8 HSC-3: MAGEA8 HSC-3: MAGEA1 HSC-3: EREG
3.34 (P ⬍ .0001) 5.18 (P ⬍ .0001) 7.06 (P ⬍ .0001) 833 (P ⬍ .0001) 3.58 (P ⬍ .0001) 10.67 (P ⬍ .0001) 1.63 (P ⬍ .0001) 0.90 (P ⬎ .0001) 3.71 (P ⬍ .0001)
0.33 0.155 0.62 0.59 0.89 0.615 0.145 0.885 0.41
Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
thus, alteration of gap junction may cause pathology.28 Down-regulation of these pathways may cause tumor cells to move freely and penetrate into lymphatic vessels, thereby encouraging lymph node metastasis. In conclusion, up-regulation of the Jak-Stat signaling pathway and the hedgehog signaling pathway may be involved in bone invasion of OSCC. In addition, the MAPK signaling pathway and actin cytoskeleton pathway were up-regulated in all 3 OSCC cell lines, suggesting that these are common pathways up-regulated in OSCC. The ECM–receptor interaction pathway was the only pathway that was down-regulated in all 3 OSCC cell lines and thus can be considered a common pathway down-regulated in OSCC. Up-regulation of the cell cycle pathway and the calcium-signaling pathway may be the cause of distant organ metastasis of OSCC. Up-regulation of the Toll-like receptor signaling pathway and down-regulation of the gap junction pathway was observed only in the HSC-3 cell line, possibly indicating that these pathways play a role in cervical lymph node metastasis of OSCC. Our findings also demonstrate that cDNA microarray analysis is one of the strongest tools for mapping molecular signatures, because it allows evaluation of the whole genome and provides a good picture of the interactions among thousands of genes simultaneously.
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Table 2. UP-REGULATED AND DOWN-REGULATED PATHWAYS IN THE BHY, HN, AND HSC-3 CELL LINES (P < .05)
Cell Line BHY
Up-Regulated Pathway
HN HN HN
Regulation of actin cytoskeleton Transforming growth factor- signaling Dentatorubropallidoluysian atrophy Hedgehog signaling Focal adhesion Jak-Stat signaling Parkinson’s disease MAPK signaling MAPK signaling Regulation of actin cytoskeleton Antigen processing and presentation Parkinson’s disease Focal adhesion Type I diabetes mellitus
HN HN HN
Cell cycle Calcium signaling Axon guidance
HN
Maturity onset diabetes of the young Regulation of actin cytoskeleton MAPK signaling
BHY BHY BHY BHY BHY BHY BHY HN HN HN
HSC-3 HSC-3 HSC-3 HSC-3
Toll-like receptor
HSC-3 HSC-3
Down-Regulated Pathway Olfactory transduction Taste transduction ECM–receptor interaction
Hedgehog signaling Circadian rhythm Olfactory transduction Wnt signaling Notch signaling ECM–receptor interaction Insulin signaling Taste transduction Amyotrophic lateral sclerosis ECM–receptor interaction Olfactory transduction Taste transduction Huntington’s disease Gap junction Cell adhesion
Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
References 1. Yamamoto K, Konishi N, Inui T, et al: Genomic alterations in oral squamous cell carcinoma cell lines detected by two-dimensional gel analysis. Oral Oncol 34:508, 1998 2. Scully C, Field JK, Tanzawa H: Genetic aberration in oral or head and neck squamous cell carcinoma (SCCHN), 1: Carcinogen metabolism, DNA repair and cell cycle control. Oral Oncol 36:256, 2000 3. Scully C, Field JK, Tanzawa H: Genetic aberration in oral or head and neck squamous cell carcinoma, 2: Chromosomal aberrations. Oral Oncol 36:311, 2000 4. Ginos MA, Page GP, Michalowicz BS, et al: Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck. Cancer Res 64:55, 2004 5. Choi P, Chen C: Genetic expression profiles and biologic pathway alterations in head and neck squamous cell carcinoma. Cancer 104:1113, 2005 6. Li Y, Tondravi M, Liu J, et al: Cortactin potentiates bone metastasis of breast cancer cells. Cancer Res 61:6906, 2001
7. Akhurst RJ, Derynck R: TGF- signaling in cancer: A doubleedged sword. Trends Cell Bio 11:44, 2001 8. St Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 13:2072, 1999 9. Kubo M, Nakamura M, Tasaki A, et al: Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res 64:6071, 2004 10. Snijders AM, Schmidt BL, Fridlyan J, et al: Rare amplicons implicate frequent deregulation of cell fate specification pathway in oral squamous cell carcinoma. Oncogene 24:4232, 2005 11. Zheng D, Woodard AS, Fornaro M, et al: Prostatic carcinoma cell migration via ␣v3 integrin is modulated by a focal adhesion kinase pathway. Cancer Res 59:1655, 1999 12. Ossowski L, Aguirre-Ghiso JA: Urokinase receptor and integrin partnership: Coordination of signaling for cell adhesion, migration and growth. Curr Opin Cell Biol 12:613, 2000 13. Chott A, Sun Z, Morganstern D, et al: Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. Am J Pathol 155:1271, 1999 14. Westermarck A, Kahari V: Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13:781, 1999 15. Liao J, Schneider A, Datta NS, et al: Extracellular calcium as a candidate mediator of prostate cancer skeletal metastasis. Cancer Res 66:9065, 2006 16. Amuthan G, Biswas G, Ananadatheerthavarada HK, et al: Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene 21:7839, 2002 17. Basile JR, Castilho RM, Williams VP, et al: Semaphorin 4D provides a link between axon guidance processes and tumorinduced angiogenesis. Proc Natl Acad Sci 103:9017, 2006 18. Christensen C, Ambartsumian N, Gilestro G, et al: Proteolytic processing converts the repelling signal Sema3E into an inducer of invasive growth and lung metastasis. Cancer Res 65:6167, 2005 19. Aliprantis AO, Yang R, Weiss DS, et al: The apoptotic signaling pathway activated by Toll-like receptor-2. EMBO J 19:3325, 2000 20. Ertel A, Verghese A, Byers SW, et al: Pathway-specific differences between tumor cell lines and normal and tumor tissue cells. Mol Cancer 5:1, 2006 21. Dysvik B, Vasstrand EN, Lovlie R, et al: Gene expression profiles of head and neck carcinomas from Sudanese and Norwegian patients reveal common biological pathways regardless of race and lifestyle. Clin Cancer Res 12:1109, 2006 22. Bjarnason GA, Jordan CK, Sothern RB: Circadian variation in the expression of cell-cycle proteins in human oral epithelium. Am J Pathol 153:613, 1999 23. Leethanakul C, Patel V, Gillespie J, et al: Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 19(28):3220, 2000 24. Yeh KT, Chang JD, Lin TH, et al: Correlation between protein expression and epigenetic and mutation changes of Wnt pathway-related genes in oral cancer. Int J Oncol 23: 1001, 2003 25. Kudo Y, Kitajima S, Ogawa I, et al: Invasion and metastasis of oral cancer cells require methylation of E-cadherin and/or degradation of membranous -catenin. Clin Cancer Res 10:5455, 2004 26. Santagata S, Demichelis F, Riva A, et al: JAGGED1 expression is associated with prostate cancer metastasis and recurrence. Cancer Res 64:6854, 2004 27. Thomas GJ, Speight PM: Cell adhesion molecules and oral cancer. Crit Rev Oral Biol Med 12:479, 2001 28. Livny O, Kaplan I, Reifen R, et al: Lycopene inhibits proliferation and enhances gap-junction communication of KB-1 human oral tumor cells. J Nutr 132:3754, 2002
Characterization of Gene Expression Profiles of 3 Different Human Oral Squamous Cell Carcinoma Cell Lines With Different Invasion and Metastatic Capacities N. Fazil Erdem, DDS, PhD,* Eric R. Carlson, DMD, MD,† and David A. Gerard, PhD‡ Purpose: The gene expression of 3 oral squamous cell carcinoma (OSCC) human cell lines, BHY, HN,
and HSC-3, were studied based on their reported ability to invade adjacent bone or metastasize to cervical lymph nodes and/or distant organs. Materials and Methods: The characteristics of each cell line were confirmed on scid mice using micro-positron emission tomography (PET)/computerized tomography (CT) imaging techniques. Complimentary DNA (cDNA) microarray techniques were used to determine the gene expression profile differences between each of the three OSCC cell lines. Results: BHY, HN, and HSC-3 cell lines expressed 139, 214, and 128 up-regulated genes; and 117, 262, and 117 down-regulated genes, respectively. The clusterization of data showed that there are 13 genes that are up-regulated and 83 genes that are down-regulated in all 3 OSCC cell lines. Collection of genes organized by pathway may cause aggregate evaluation of anomalies. Thus the pathway analysis performed for each cell line based on cDNA microarray results showed BHY, HN, and HSC-3 cell lines to have 8, 10, and 3 up-regulated pathways and 3, 9, and 6 down-regulated pathways, respectively. Conclusions: This study showed that cDNA microarray analysis is an effective tool for mapping molecular signatures. With this technique it is possible to observe the entire genome of a malignant tumor so as to appreciate the simultaneous interactions among thousands of genes. © 2008 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 66:918-927, 2008 Oral squamous cell carcinoma (OSCC) is the most common cancer of the oral cavity, accounting for 90% of all diagnosed oral malignancies and approximately 6% of all human malignancies.1 There is little pub-
lished information on the capability of some OSCCs to invade adjacent bone while other OSCCs do not exhibit this property. In addition, some OSCCs readily metastasize to cervical lymph nodes and/or distant organs, whereas others do not. OSCC represents an anomaly of multiple molecular genetic events in many chromosomes and genes.2 The consequence of this chromosomal damage is cell dysregulation with disruptions in cell signaling, the cell growth cycle, and mechanisms for repairing cell damage and eliminating dysfunctional cells.2,3 The cluster of genes down-regulated in OSCC includes several extracellular matrix (ECM) proteins that regulate epithelial adhesion and collagen fibril formation. Genes that are upregulated in OSCC are associated with ECM function, interferon response, proliferation, and immune response. In addition, genes implicated in invasion, Ras signaling, the cell surface/ECM, and angiogenesis are highly expressed in OSCC.4
Received from the Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of Medicine, and the University of Tennessee Cancer Institute, Knoxville, TN. *Intern. †Professor and Chairman. ‡Associate Professor. This research was supported by the Physician’s Medical Education and Research Foundation (PMERF), University of Tennessee Medical Center, Knoxville. Address correspondence and reprint requests to Dr Carlson: 1930 Alcoa Highway, Suite 335, Knoxville, TN 37920; e-mail: [email protected] © 2008 American Association of Oral and Maxillofacial Surgeons
0278-2391/08/6605-0014$34.00/0 doi:10.1016/j.joms.2007.12.036
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In this study, 3 OSCC human cell lines—BHY, HN, and HSC-3—were chosen for study based on their reported ability to invade adjacent bone or metastasize to cervical lymph nodes and/or distant organs. The normal human nasal epithelial cell line (HNEC) was selected as a control group. The working hypothesis of this study was that OSCCs that demonstrate different actions have different gene and pathway expression profiles; for example, OSCCs that invade adjacent bone will have different expression profiles than OSCCs that metastasize to cervical lymph nodes and/or distant organs. Identifying the gene and pathway expression differences in these 3 cell lines will provide insight into which genes and pathways are responsible for lymph node or distant organ metastasis or bone invasion of OSCC. Identifying genes or their related proteins that are associated with tumor metastasis or invasion will provide a basis for further studies to investigate metastatic progression and local invasiveness of this tumor and to evaluate these genes and proteins as therapeutic targets. In the present study, micro-positron emission tomography (PET)/computed tomography (CT) imaging was performed to investigate the invasion and/or metastatic potential of each cell line in immunocompromised scid mice and to delineate the differences in jawbone invasion and distant organ metastasis of these 3 OSCC cell lines in a biological system.
Materials and Methods CELL LINES AND CULTURES
The HNEC cell line (PromoCell, Heidelburg, Germany), cultivated in Airway Epithelial Cell Growth medium (PromoCell), was used as a control. This cell line was selected because it is the only normal head and neck mucosal cell line currently available for study. Cell lines BHY and HN were obtained from DSMZ, Braunschweig, Germany. The BHY cell line is known to invade the mandibular bone. The HN cell line is known to metastasize to cervical lymph nodes, lungs, and brain but to not invade bone. These cell lines were cultivated in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Cell line HSC-3 was obtained from the Japanese Health Science Research Resources Bank. It is known to metastasize to cervical lymph nodes. Eagle’s minimal essential medium with 10% calf serum was used as a cultivation medium. After 100 g/mL streptomycin and 100 U/mL penicillin were added to the media, all 4 cell lines were incubated in a humidified atmosphere of 95% air and 5% CO2.
IMAGING THE INVASION AND/OR METASTATIC CHARACTERISTICS OF OSCC CELL LINES
Micro-PET/CT imaging was used to image the invasion and/or metastatic capacities of each cell line in 3 scid mice in each group. The OSCC cell lines and the HNEC cell line were introduced into the left masseter muscles of the scid mice by injecting a suspension of 1 ⫻ 107 cells in Matrigel (BD Biosciences, Franklin Lakes, NJ). The control mice received only Matrigel. After 30 days, the mice were given 300 Ci of [18F] fluorodeoxyglucose intraperitoneally, followed by an intravenous injection of vascular CT contrast agent 30 minutes later. The animals were sacrificed by an overdose of isoflurane inhalation and imaged by microPET, acquired using the Focus 220 imaging system, followed by a micro-CT scan using a MicroCAT II imager (Siemens Molecular Imaging, Knoxville, TN). TOTAL RNA EXTRACTION FROM THE CELL LINES
The total RNA extraction was performed using an RNeasy Midi Kit (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. The quantity and quality of total RNA were checked by spectrophotometry and by the expression levels of aldolase and -actin genes in each cell line. cDNA MICROARRAY ANALYSIS
The PolyATtract mRNA isolation system (Promega, Madison, WI) was used to extract the mRNA from the total RNA. The cDNA synthesis system (Promega) was used to synthesize cDNA from mRNA. Each tumor cell line was compared with the HNEC cell line using the cDNA microarray technique. Operon Human version 3.0 microarray slides (Operon Biotechnologies, Huntsville, AL) were used. Labeled tumor cell lines and HNEC cell line cDNAs were hybridized on microarray slides using the hybridization buffer. All slides were scanned in a GenePix 4000 scanner (Axon Instruments, Union City, CA). cDNA microarray experiments were repeated 4 times for each cell line to obtain a more reliable analysis. The microarray images were processed using GenePix Pro software (Molecular Devices, Sunnyvale, CA). Both bulk normalization and Lowess normalization were applied to analyze the data. The microarray data were confirmed by real-time polymerase chain reaction (PCR) using the 3 most highly up-regulated genes from each cell. Self-organizing maps and hierarchy clustering were performed to gain more insight into gene function and interaction. To narrow down specific genes, analysis of variance was used to obtain the estimates and confidence intervals for all genes in each experiment set. Based on the expression levels of the genes in the microarray analysis, different
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FIGURE 1. Left sagittal PET/CT image of a mouse with BHY cell line injection. The arrow shows the mandibular bone resorption due to invasion of the bone by OSCC. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
pathways were analyzed on each OSCC cell line using the Kyoto Encyclopedia of Gene and Genomes (KEGG).
Results IMAGING THE INVASION AND/OR METASTATIC CHARACTERISTICS OF THE OSCC CELL LINES
The PET/CT images of HNEC and the Matrigel-only groups demonstrated no evidence of tumor formation in any of the mice. All 3 mice in the BHY cell line group showed invasion of the OSCC to the mandibular bone (Fig 1); however, only 2 mice in the HN group exhibited metastasis to the lymph nodes and distant organs, such as the lungs and kidneys (Fig 2). The other mouse in the HN group had cervical lymph node metastasis of OSCC. All 3 mice in the HSC-3
group demonstrated lymph node metastasis (Fig 3). The imaging study results matched the characteristics of the OSCC cell lines that have been reported to date. This information is very useful, because it demonstrates that the cultured cell lines maintained their biologic characteristics. As such, it suggests that the gene expression profiles of the 3 cell lines were maintained. Stated differently, based on these findings, we can deduce that the culturing process did not alter the gene expression profiles of the BHY, HN, and HSC-3 cell lines. cDNA MICROARRAY ANALYSIS OF 3 OSCC CELL LINES
After the normalization process, each OSCC cell line ended up with different up-regulated and down-regulated genes. The BHY cell line expressed
FIGURE 2. Left sagittal PET/CT image of a mouse with HN cell line injection. The yellow arrow shows lung metastasis, the light blue arrow shows kidney metastasis, and the white arrow shows the cervical lymph node metastasis of OSCC. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
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FIGURE 3. Axial PET/CT image of a mouse with HSC-3 cell line injection. The white arrow shows cervical lymph node metastasis. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
139 up-regulated genes and 117 down-regulated genes, the HN cell line expressed 214 up-regulated genes and 262 down-regulated genes, and the HSC-3 cell line expressed 128 up-regulated genes and 117 down-regulated genes. Clusterization of data identified 13 genes that were up-regulated and 83 genes that were down-regulated in all 3 OSCC cell lines (Figs 4, 5). There were 57 up-regulated genes and 42 down-regulated genes in common in the BHY and HN cell lines (Fig 6), 24 up-regulated genes and 49 down-regulated genes in common in the BHY and HSC-3 cell lines (Fig 7), and 24 upregulated genes and 44 down-regulated genes in common in the HN and HSC-3 cell lines (Fig 8).
The real-time PCR analysis results confirmed the cDNA microarray analysis results (Fig 9). The upregulation values of the 3 genes from each cell line are given in Table 1. The only questionable up-regulation value in the cDNA microarray analysis is the expression level of the MAGEA1 gene in the HSC-3 cell line, with an error bar of 0.89; all of the other genes have reasonable error bars (Table 1). Based on the expression levels of the genes in the microarray analysis, different pathways were analyzed on each OSCC cell line using the KEGG. The BHY cell line had 8 up-regulated and 3 down-regulated pathways, the HN cell line had 10 up-regulated and 9 down-regulated pathways, and HSC-3 cell line had 3 up-regulated and 6 down-regulated pathways (P ⬍ .05) (Table 2). The 3 OSCC cell lines had some upregulated and down-regulated pathways in common. Pathways regulating actin cytoskeleton and MAPKsignaling were up-regulated in all 3 cell lines; however, the focal adhesion pathway was up-regulated only in the BHY and HN cell lines. The ECM–receptor interaction pathway was down-regulated in all 3 cell lines.
Discussion FIGURE 4. Clusterization of the 13 genes that were up-regulated in all 3 OSCC cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
Our findings demonstrate that OSCC represents multiple molecular genetic events occurring in many chromosomes and genes. The genes that demonstrated alterations in this study have roles in ECM
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FIGURE 5. Clusterization of the 83 genes that were down-regulated in all 3 OSCC cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
degradation, cell adhesion, epidermal development and differentiation, signal transduction, transcriptional regulation, cell growth and proliferation, proteolysis, basement membrane degradation, and cytoskeleton degradation. Recently, Choi et al5 reviewed more than 20 articles that incorporated cDNA microarray analysis in the study of head and neck SCC and identified 84 genes with common alterations, 32 of which were up-regulated, 33 of which were down-regulated, and 19 of which had conflicting expression data. Several major biological systems or pathways are altered in head and neck SCC, including cell cycle control, matrix metalloproteinase (MMP), and inflammatory response systems. Genes encoding for ECM and integral membrane proteins, proteins involved in epidermal development and differentiation, and cell adhesion molecules most frequently have altered expression in head and neck SCC.5
In the present study, we identified 13 genes that were up-regulated in all 3 OSCC cell lines studied. Twelve of these are known genes, but 1 has not been described previously. Basically, these genes function in mitotic progression and chromosome segregation, apoptosis inhibition, cell growth, transduction, intercellular transmission of polarity information during tissue morphogenesis and/or in differentiated tissues, embryonal development, tumor transformation and progression, and condensation of nucleosome chains into higher-order structures. We also identified 83 genes that were down-regulated in all 3 OSCC cell lines. Most of these genes have vital functions in cell biology, including cell– cell or cell– basement membrane junction, intercellular signaling, regulation of cell motility, cell growth and differentiation, wound healing, maintenance of cell shape, immune response against tumor cells, and signal transduction cascades.
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FIGURE 6. The 57 up-regulated genes and 42 down-regulated genes in both the BHY and HN cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
The HN cell line exhibited the greatest degree of gene alteration of the 3 OSCC cell lines studied. The up-regulated genetic structure of the BHY cell line was more similar to the up-regulated genetic structure of the HN cell line. The up-regulated genetic structure of the HSC-3 cell line showed more similarity to the up-regulated genetic structure of the BHY cell line. The down-regulated genetic structure of the BHY cell line was closer to the down-regulated genetic structure of HSC-3 cell line. Pathway analyses of each OSCC cell line were conducted to gain insight into the mechanisms of invasion, lymph node metastasis, and distant organ metastasis of OSCC. Based on the P values (P ⬍ .05) of the genes identified, the up-regulated and down-regulated pathways of the 3 OSCC cell lines are listed in Table 2. The pathway regulating the actin cytoskeleton was up-regulated in all 3 OSCC cell lines. Besides provid-
ing a structural framework around which cell shape and polarity are defined, this pathway has dynamic properties that provide the driving force for cells to move and to divide. Overactivation of this system can cause bone metastasis in breast cancer.6 This pathway is a target of most anticancer drugs. Activation of the transforming growth factor- signaling pathway is common in malignancies. This pathway stimulates tumor progression by its pleiotropic activities on both cancer cells and on nonmalignant stromal cells of the tumor.7 This pathway was upregulated only in the BHY cell line. The hedgehog signaling pathway involves the control of cell proliferation and cell differentiation during morphogenesis of the endochondral skeleton of vertebrates.8 It has been shown that blockage of this pathway suppresses the growth of breast carcinoma cells.9 Up-regulation of this pathway is commonly found
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GENE EXPRESSION PROFILES OF ORAL SQUAMOUS CELL CARCINOMA CELL LINES
FIGURE 7. The 24 up-regulated genes and 49 down-regulated genes in both the BHY and HSC-3 cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
in OSCC.10 This pathway was up-regulated in the BHY cell line and down-regulated in the HN cell line, and thus may be involved in bone invasion by OSCC. The focal adhesion pathway plays a role in mechanical adhesion of cells to the ECM, which is important for cell growth, survival, and migration. PC3, a prostate cancer cell line, has been found to invade mature bone and highly express the focal adhesion pathway.11 To the best of our knowledge, there has been no study correlating this pathway with the bone invasion of OSCC. This pathway was up-regulated in the BHY and HN cell lines. The Janus kinase–signal transducers and activators of transcription (Jak-Stat) signaling pathway was another pathway up-regulated in the BHY cell line. The Jak-Stat signaling pathway can be activated by uPA– uPAR interaction.12 Activation of this pathway is known to be involved in bone metastasis by prostate cancer.13 Another role of this pathway is in the activation of MMP, which is involved in bone invasion by OSCC. The up-regulation of MMP in the BHY cell line may be related to activation of this pathway.
The mitogen-activated protein kinase (MAPK) signaling pathway transduces a wide variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation, and apoptosis. It also plays a role in the regulation of MMP gene expression, especially of MMP-1, MMP-3, and MMP-9.14 Inhibition of the MAPK signaling pathway could potentially serve as a therapeutic target to specifically prevent tumor cell invasion. This pathway was up-regulated in all 3 cell lines, suggesting that it may be one of the major pathways related to OSCC invasion and metastasis. The cell cycle pathway is a series of events in a eukaryotic cell between one cell division and the next. Up-regulation of the cell cycle pathway is common in many cancers, especially those with high metastatic rates. Both cyclins and cyclin-dependent kinase genes (the major genes that regulate the cell cycle pathway) were up-regulated in the HN cell line. Cyclins and cyclin-dependent kinases may be associated with distant metastasis of OSCC.
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FIGURE 8. The 24 up-regulated genes and 44 down-regulated genes in both the HN and HSC-3 cell lines. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
The calcium signaling pathway was another pathway activated in the HN cell line. It has been shown that prostate cancer frequently metastasizes to bone. Elevated extracellular Ca⫹2 can enhance proliferation of skeletal metastases.15 An increase in the Ca⫹2 signaling pathway plays an important role in cancer progression and metastasis.16 The axon guidance pathway plays an important role in the development of the nervous system. Semaphorin 4D protein, which is responsible for the development of the central nervous system and regulation of axonal growth, is highly expressed in cell lines derived from head and neck SCCs.17 A similar pattern has been observed in malignant cells from prostate, colon, breast, and lung cancer tissues.18 The axon guidance pathway was highly upregulated only in the HN cell line; this suggests that up-regulation of this pathway may occur in advanced stages of OSCC and may have a role in metastasis of OSCC. The Toll-like receptor signaling pathway is closely related to the invasion and metastasis of cancer. It is a novel pathway that engages the apoptotic machinery.19 This pathway was up-regulated only in the HSC-3 cell line. The ECM–receptor interaction pathway was down-regulated in all 3 OSCC cell lines. Degrada-
tion of the ECM is an essential step in the invasion and metastasis of cancer.20 Down-regulation of this pathway may aid in migration of tumor cells to distant organs.21 Circadian rhythm is essentially a 24-hour cycle in the physiological processes of living beings.22 This pathway was down-regulated only in the HN cell line; thus, down-regulation of this pathway may play a role in distant metastasis. The Wnt signaling pathway proteins form a family of highly conserved and secreted signaling molecules that regulate cell– cell interactions during embryogenesis. It was down-regulated only in the HN cell line. Leethanakul et al23 found that most head and neck SCCs overexpress some members of the Wnt signaling pathway; however, Yeh et al24 concluded that Wnt pathway–related genes play a very limited role in the development of OSCC. Deregulation of the Wnt signaling pathway has been shown to occur by several different mutational mechanisms in human cancers.25 The notch signaling pathway is a gene regulatory pathway involved in multiple differentiation processes. This pathway is a highly conserved pathway for cell– cell communication. It is involved in the regulation of cellular differentiation, proliferation, and specification. JAGGED1, a notch receptor li-
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FIGURE 9. Real-time PCR analysis results. The blue columns represent the BHY cell line, the red columns represent the HN cell line, and the green columns represent the HSC-3 cell line. Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
gand, is significantly more highly expressed in metastatic prostate cancer compared with localized prostate cancer or benign prostatic tissues.26 This pathway was down-regulated in the HN cell line. The gap junction and cell adhesion pathways were down-regulated only in the HSC-3 cell line. These pathways function in the formation of cell– cell junctions and ion movement between cells and hence play a role in tissue homeostasis.27 The growth rate of tumor cells is negatively correlated with the strength of intercellular communication;
Table 1. UP-REGULATION AND ERROR BAR VALUES OF THE 3 MOST UP-REGULATED GENES IN EACH OSCC CELL LINE ACCORDING TO REAL-TIME PCR ANALYSIS RESULTS
Cell Line: Gene
Up-Regulation Value
Error Bar Value
BHY: MAGEA9 BHY: Q96AL8 BHY: GFI1 HN: NMES1 HN: MAGEA1 HN: Q96AL8 HSC-3: MAGEA8 HSC-3: MAGEA1 HSC-3: EREG
3.34 (P ⬍ .0001) 5.18 (P ⬍ .0001) 7.06 (P ⬍ .0001) 833 (P ⬍ .0001) 3.58 (P ⬍ .0001) 10.67 (P ⬍ .0001) 1.63 (P ⬍ .0001) 0.90 (P ⬎ .0001) 3.71 (P ⬍ .0001)
0.33 0.155 0.62 0.59 0.89 0.615 0.145 0.885 0.41
Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
thus, alteration of gap junction may cause pathology.28 Down-regulation of these pathways may cause tumor cells to move freely and penetrate into lymphatic vessels, thereby encouraging lymph node metastasis. In conclusion, up-regulation of the Jak-Stat signaling pathway and the hedgehog signaling pathway may be involved in bone invasion of OSCC. In addition, the MAPK signaling pathway and actin cytoskeleton pathway were up-regulated in all 3 OSCC cell lines, suggesting that these are common pathways up-regulated in OSCC. The ECM–receptor interaction pathway was the only pathway that was down-regulated in all 3 OSCC cell lines and thus can be considered a common pathway down-regulated in OSCC. Up-regulation of the cell cycle pathway and the calcium-signaling pathway may be the cause of distant organ metastasis of OSCC. Up-regulation of the Toll-like receptor signaling pathway and down-regulation of the gap junction pathway was observed only in the HSC-3 cell line, possibly indicating that these pathways play a role in cervical lymph node metastasis of OSCC. Our findings also demonstrate that cDNA microarray analysis is one of the strongest tools for mapping molecular signatures, because it allows evaluation of the whole genome and provides a good picture of the interactions among thousands of genes simultaneously.
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Table 2. UP-REGULATED AND DOWN-REGULATED PATHWAYS IN THE BHY, HN, AND HSC-3 CELL LINES (P < .05)
Cell Line BHY
Up-Regulated Pathway
HN HN HN
Regulation of actin cytoskeleton Transforming growth factor- signaling Dentatorubropallidoluysian atrophy Hedgehog signaling Focal adhesion Jak-Stat signaling Parkinson’s disease MAPK signaling MAPK signaling Regulation of actin cytoskeleton Antigen processing and presentation Parkinson’s disease Focal adhesion Type I diabetes mellitus
HN HN HN
Cell cycle Calcium signaling Axon guidance
HN
Maturity onset diabetes of the young Regulation of actin cytoskeleton MAPK signaling
BHY BHY BHY BHY BHY BHY BHY HN HN HN
HSC-3 HSC-3 HSC-3 HSC-3
Toll-like receptor
HSC-3 HSC-3
Down-Regulated Pathway Olfactory transduction Taste transduction ECM–receptor interaction
Hedgehog signaling Circadian rhythm Olfactory transduction Wnt signaling Notch signaling ECM–receptor interaction Insulin signaling Taste transduction Amyotrophic lateral sclerosis ECM–receptor interaction Olfactory transduction Taste transduction Huntington’s disease Gap junction Cell adhesion
Erdem, Carlson, and Gerard. Gene Expression Profiles of Oral Squamous Cell Carcinoma Cell Lines. J Oral Maxillofac Surg 2008.
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7. Akhurst RJ, Derynck R: TGF- signaling in cancer: A doubleedged sword. Trends Cell Bio 11:44, 2001 8. St Jacques B, Hammerschmidt M, McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 13:2072, 1999 9. Kubo M, Nakamura M, Tasaki A, et al: Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res 64:6071, 2004 10. Snijders AM, Schmidt BL, Fridlyan J, et al: Rare amplicons implicate frequent deregulation of cell fate specification pathway in oral squamous cell carcinoma. Oncogene 24:4232, 2005 11. Zheng D, Woodard AS, Fornaro M, et al: Prostatic carcinoma cell migration via ␣v3 integrin is modulated by a focal adhesion kinase pathway. Cancer Res 59:1655, 1999 12. Ossowski L, Aguirre-Ghiso JA: Urokinase receptor and integrin partnership: Coordination of signaling for cell adhesion, migration and growth. Curr Opin Cell Biol 12:613, 2000 13. Chott A, Sun Z, Morganstern D, et al: Tyrosine kinases expressed in vivo by human prostate cancer bone marrow metastases and loss of the type 1 insulin-like growth factor receptor. Am J Pathol 155:1271, 1999 14. Westermarck A, Kahari V: Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13:781, 1999 15. Liao J, Schneider A, Datta NS, et al: Extracellular calcium as a candidate mediator of prostate cancer skeletal metastasis. Cancer Res 66:9065, 2006 16. Amuthan G, Biswas G, Ananadatheerthavarada HK, et al: Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells. Oncogene 21:7839, 2002 17. Basile JR, Castilho RM, Williams VP, et al: Semaphorin 4D provides a link between axon guidance processes and tumorinduced angiogenesis. Proc Natl Acad Sci 103:9017, 2006 18. Christensen C, Ambartsumian N, Gilestro G, et al: Proteolytic processing converts the repelling signal Sema3E into an inducer of invasive growth and lung metastasis. Cancer Res 65:6167, 2005 19. Aliprantis AO, Yang R, Weiss DS, et al: The apoptotic signaling pathway activated by Toll-like receptor-2. EMBO J 19:3325, 2000 20. Ertel A, Verghese A, Byers SW, et al: Pathway-specific differences between tumor cell lines and normal and tumor tissue cells. Mol Cancer 5:1, 2006 21. Dysvik B, Vasstrand EN, Lovlie R, et al: Gene expression profiles of head and neck carcinomas from Sudanese and Norwegian patients reveal common biological pathways regardless of race and lifestyle. Clin Cancer Res 12:1109, 2006 22. Bjarnason GA, Jordan CK, Sothern RB: Circadian variation in the expression of cell-cycle proteins in human oral epithelium. Am J Pathol 153:613, 1999 23. Leethanakul C, Patel V, Gillespie J, et al: Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 19(28):3220, 2000 24. Yeh KT, Chang JD, Lin TH, et al: Correlation between protein expression and epigenetic and mutation changes of Wnt pathway-related genes in oral cancer. Int J Oncol 23: 1001, 2003 25. Kudo Y, Kitajima S, Ogawa I, et al: Invasion and metastasis of oral cancer cells require methylation of E-cadherin and/or degradation of membranous -catenin. Clin Cancer Res 10:5455, 2004 26. Santagata S, Demichelis F, Riva A, et al: JAGGED1 expression is associated with prostate cancer metastasis and recurrence. Cancer Res 64:6854, 2004 27. Thomas GJ, Speight PM: Cell adhesion molecules and oral cancer. Crit Rev Oral Biol Med 12:479, 2001 28. Livny O, Kaplan I, Reifen R, et al: Lycopene inhibits proliferation and enhances gap-junction communication of KB-1 human oral tumor cells. J Nutr 132:3754, 2002