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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 6146-6154
Copyright © 2004 by The Endocrine Society

Microarray Analysis of Metastasis-Associated Gene Expression Profiling in a Murine Model of Thyroid Carcinoma Pulmonary Metastasis: Identification of S100A4 (Mts1) Gene Overexpression as a Poor Prognostic Marker for Thyroid Carcinoma

Minjing Zou, Konrad S. Famulski, Ranjit S. Parhar, Essa Baitei, Futwan A. Al-Mohanna, Nadir R. Farid and Yufei Shi

Departments of Genetics (M.Z., E.B., Y.S.) and Biological and Medical Research (R.S.P., F.A.A.-M.), King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia; Department of Medicine (K.S.F.), University of Alberta, Edmonton, Alberta, Canada T6G 2S2; and Osancor Biotech Inc. (N.R.F.), Watford, Herts WD1 3BY, United Kingdom

Address all correspondence and requests for reprints to: Yufei Shi, MBC 3, Department of Genetics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. E-mail: yufei{at}kfshrc.edu.sa.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Tumor cell invasion and metastasis are the hallmark of malignant neoplasm. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. To gain new insights into this complex process in thyroid carcinoma, we established a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lung by sequential passage of a human anaplastic thyroid cancer cell line (ARO) through the lung of a nude mouse. Global patterns of gene expression were analyzed in cells of the parental ARO and the ARO-met2, using Atlas human cancer 1.2 array with 1176 cancer-related genes. In total, 184 genes were differentially expressed more than 1.5 times, and 64 genes were differentially expressed over two times. Among those 64 genes, 43 were overexpressed, and 21 genes were underexpressed. Many genes whose increased expression was thought to be related to tumor progression were identified, such as c-Met, ezrin, integrin, motility-related protein-1, cadherin, and S100A4. The most highly expressed gene is the S100A4 (8-fold higher than control), which is a member of a small calcium binding protein family and is involved in the cell proliferation and cancer progression. The S100A4 overexpression in the ARO-met2 cells was later confirmed by Northern blot and real-time reverse transcriptase-PCR. Analysis of 49 thyroid tumor specimens by real-time reverse transcriptase-PCR (eight benign goiters, 36 papillary, and five anaplastic carcinomas) revealed that S100A4 overexpression was present in most advanced thyroid carcinomas and lymph node metastases, and was associated with poor prognosis. None of the benign goiters was found to have S100A4 overexpression. These data suggest that S100A4 could be used as a prognostic marker for thyroid carcinoma. Given that S100A4 is involved in tumor progression and metastasis, it may be a potential target for therapeutic intervention.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
TUMOR CELL INVASION and metastasis define malignancy and are major causes of cancer morbidity and mortality (1, 2). Multiple pathogenic steps are involved in tumor invasion and metastasis. These include the proliferation and detachment of tumor cells from the primary neoplasm, invasion of the surrounding extracellular matrix, angiogenesis, vascular or lymphatic dissemination, and eventually, homing of the tumor cells and proliferation at the new site (3). Due to its complexity, cancer metastasis remains undefeated and accounts for 25% of deaths in the developed world. Understanding the mechanism of metastasis at the molecular level is essential to devise new modalities of cancer therapy.

It has been recognized that neoplasms are biologically heterogeneous and contain subpopulations of cells that have different metastatic properties. The process of metastasis is selective for cells that preexist in the parental neoplasm, and the outcome of metastasis depends on multiple interactions of metastatic cells with host’s immune and nonimmune defenses, implicating the involvement of dozens of different genes (4). This provides the challenging task of identifying critical genes controlling the metastatic process to use as targets for either diagnosis or therapeutic intervention.

Although the survival rate of patients with well-differentiated thyroid cancer exceeds the rate for most other cancers, the development of metastasis continues to be the most significant cause in thyroid cancer mortality (5, 6). To gain new insights into this complex process and identify metastasis-controlling genes in thyroid carcinoma, we established a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lungs of nude mice. Global patterns of gene expression were compared between the parental ARO and ARO-met2 by microarray analysis. Candidate genes were identified by searching for a minimum of 2-fold differential increase in mRNA.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Thyroid tumor specimens

All tumor tissues were obtained at surgery and were immediately frozen in liquid nitrogen and stored at –70 C until processed. Informed consent was obtained from all individuals involved in this study, and the research project was approved by both the Basic Research Committee and the Research Ethical Committee of the Institution. The clinical staging of thyroid cancer was based on the TNM (tumor-lymph node-metastasis) classification introduced in 1987 by the international Union Against Cancer (7). Forty-nine thyroid tumors were studied: eight benign multinodular goiters, 36 papillary, and five anaplastic carcinomas. In six of the 36 patients with papillary carcinoma, matched tissues of normal thyroid, papillary thyroid carcinoma (PTC), and its lymph node metastasis were obtained. Tissues from lymph node metastasis were confirmed by a pathologist to contain at least 80% of tumor tissue.

Animals

Female BALB/c (nu/nu) nude mice 6 to 8 wk of age were used as model hosts for the production of experimental pulmonary metastasis of thyroid tumor. ARO-met2 was derived from two cycles of pulmonary metastasis of ARO cells, a human anaplastic thyroid carcinoma cell line. Briefly, mice were injected iv with 2 x 105 ARO cells. On d 60 after tumor cell injection, colonies of pulmonary metastases were removed, expanded in cell culture, and injected back to mice for another cycle of pulmonary selection (Fig. 1Go).



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  		FIG. 1. Establishment of thyroid carcinoma cell line with high metastatic potential. Female BALB/c (nu/nu) nude mice were used as model hosts for the production of experimental pulmonary metastasis of thyroid tumor. Mice were injected iv with 2 x 105 ARO cells, a human anaplastic thyroid carcinoma cell line. On d 60 after tumor cell injection, colonies of pulmonary metastases were removed, expanded in cell culture, and injected back to mice for another cycle of pulmonary selection. ARO-met2 was derived from two cycles of pulmonary metastasis of ARO cells.

 
Cell culture

ARO and ARO-met2 were propagated in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in humidified atmosphere containing 5% CO2.

RNA extraction, Northern hybridization, and microarray analysis

Total RNA was extracted by quanidinium thiocyanate-phenol-chloroform method (8). The integrity of RNA was verified by denaturing gel electrophoresis, and Northern hybridization was carried out as described previously (8). Clontech’s Altas human cancer cDNA expression array (cancer 1.2 array with 1176 cancer-related genes) was used to compare gene expression profile between ARO and ARO-met2 cell lines. 32P-labeled cDNA probes were synthesized from both ARO and ARO-met2 RNA, respectively, by reverse transcribing each RNA population, and hybridized to the Atlas cancer cDNA array according to the manufacturer’s specification. Expression was visualized by autoradiography and analyzed using AtlasVision 3.0 software.

Quantitative real-time reverse transcriptase (RT)-PCR analysis

Total RNAs from normal thyroid, thyroid tumor, lymph node metastasis, and cell lines were isolated as described above. Two micrograms of each total RNA was reverse-transcribed using the Promega RT system (Promega, Madison, WI). LightCycler DNA Master SYBR Green 1 kit was used for quantitative real-time PCR analysis according to the manufacturer’s protocols (Roche, Mannheim, Germany). The cDNA mix was diluted 10-fold, and 2 µl of the dilution was used for real-time PCR analysis. PCR primers for the 440-bp S100A4 cDNA fragment were 5'-TCTTTCTTGGTTTGATCCTG-3' (sense) and 5'-GCATCAAGCACGTGTCTGAA-3' (antisense). The sense primer spans over the 938-bp intron 1 so that the contaminated genomic DNA will not generate expected the 440-bp cDNA fragment. The mRNA level of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and a 300-bp PCR product was amplified using the following two primers: 5'-ACAGTCAGCCGCATCTTCTT-3' (sense) and 5'-TTGATTTTGGAGGGATCTCG-3' (antisense). The PCR conditions are 95 C for 30 sec followed by 40 cycles of amplification (95 C for 0 sec, 48 C for 5 sec, and 72 C for 10 sec). The resulting concentration of S100A4 PCR products was normalized by comparison with GAPDH and was used to determine the mRNA level of S100A4 among thyroid tumor specimens.

Statistical analysis

The significant difference of S100A4 gene expression between specimen groups was done using the unpaired Student’s t test. Differences were considered statistically significant when the P < 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Establishment of a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lung

In an attempt to establish a thyroid cancer cell line with high metastatic potential, we injected iv 2 x 105 ARO cells into the tail veins of nude mice. Metastatic foci were later isolated from the lung, cultured in vitro, and injected back into nude mice for another round of pulmonary metastasis (Fig. 1Go). The resulting cell line ARO-met2 was morphologically different from its parental ARO in culture. As shown in Fig. 2Go, ARO-met2 cells appear to be larger, have enhanced spread phenotype, and tend to fuse together. Sequence analysis of mitochondria DNA coding regions on both ARO and ARO-met2 revealed that both cell lines have same mutation (C to A changing Leu to Met) in ND5 located at nucleotide 13,831, confirming that ARO-met2 is a human-derived cell line (ARO-derived). ARO-met2 cells also have higher metastatic capacity in vivo. Significantly higher number of pulmonary metastasis foci (average 25 foci/10 microscopic field 20x) can be seen after injection of 105 ARO-met 2 cells compared with five metastasis foci/10 field in the lung after injection of 105 ARO cells (Fig. 3Go). Therefore, a murine model of thyroid carcinoma pulmonary metastasis was established using ARO-met2 in nude mice.



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  		FIG. 2. Morphological changes of ARO-met2. Cells were cultured 72 h after trypsinization and analyzed by phase contrast microscope. ARO-met2 cells appear to be larger, have enhanced spread phenotype, and tend to fuse together. ARO cells with different magnifications of 100x (A), 200x (B), and 400x (C). ARO-met2 cells with different magnifications of 100x (D), 200x (E), and 400x (F).

 


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  		FIG. 3. Histology of pulmonary metastasis of ARO-met2. Lungs from nude mice were removed 6 wk after iv injection of 2 x 105 ARO cells. They were fixed in 10% neutral formalin buffer, embedded in paraffin, and sectioned at 5 µm. Sections were stained by hematoxylin and eosin. A and D are histology of a normal lung with 100x and 200 x magnification, respectively. B and C and E and F are histology of two pulmonary metastasis foci with 100x and 200x magnification, respectively.

 
Gene expression profile in ARO-met2

Global patterns of gene expression were analyzed in ARO-met2 cells compared with the parental ARO cells, using Atlas human cancer 1.2 array with 1176 cancer-related genes. The array was performed on two identical blots and was repeated once with similar results. Internal controls to correct the abundance of mRNA and interblot normalization was done using nine housekeeping genes: GAPDH, tublin, actin, ubiquitin, phosphalipase A2, HPRT, 40S ribosomal protein S9, 23-kDa basic protein, and HLA-C4 {alpha}-subunit. The signals of those control genes were averaged, and the mean was used to normalize the target genes. In total, 184 genes were differentially expressed more than 1.5 times and 64 genes were over two times. Among those 64 genes, 43 were overexpressed, and 21 genes were underexpressed when compared with the ARO cells (Table 1Go). The genes identified can be classified into distinct subsets. These include transcriptional regulators, oncogenes, signaling molecules, growth/cell cycle, invasion/metastasis, extracellular and adhesion proteins, and metabolism. Most differentially expressed genes are involved in cell proliferation, DNA replication and repair, cell adhesion and cytoskeleton, cell motility, and metabolism (Table 1Go). Many genes whose increased expression thought to be related to tumor progression and metastasis were identified such as c-Met, ezrin, EphA2, TRK-T3, bikunin, integrin {alpha} 6, motility-related protein-1 (or CD9 antigen), cadherin, and S100A4. The most highly expressed gene is the S100A4 (8-fold higher than control), which is a member of small calcium binding protein family and is involved in the cell proliferation and cancer progression (9). TRK-T3 is a thyroid-specific oncogene, isolated from a human papillary thyroid tumor, and derived from oncogenic rearrangement of the NTRK1 gene, which encodes one of the receptors for the nerve growth factor. The chromosomal rearrangements juxtapose the tyrosine kinase domain of the NTRK1 receptor to the N terminal domain of the TFG gene. The 68-kDa TRK-T3 oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform NIH3T3 cells (10).


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  		TABLE 1. Up- or down-regulated genes in ARO-met2 cells

 
Genes whose decreased expression is related to tumor progression and metastasis were also identified such as early growth response protein 1 and insulin-like growth factor binding protein-6. Early growth response protein 1 has been reported to have tumor suppressor function (11), whereas insulin-like growth factor binding protein-6 inhibits cell growth by binding to IGF-II, preventing IGF-II from interacting with the IGF-I receptor to stimulate cellular proliferation (12, 13).

To verify microarray data, we did Northern blot analysis of S100A4 (Fig. 4AGo) and real-time RT-PCR analysis of expression of eight genes (Ezrin, NEDD5, EPHB6, MET, CD9, EB1, RAD21, and S100A4) in both ARO and ARO-met2 cells (Fig. 4BGo). The primer sequences used are shown in Table 2Go. As shown in Fig. 4Go, the levels of differential gene expression are in agreement with the cDNA microarray.



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  		FIG. 4. Northern analysis of S100A4 gene expression (A) and quantitative RT-PCR analysis of eight differential gene expression (B and C) in ARO and ARO-met2 cell lines. A, Differential expression of S100A4 gene between the two cell lines was verified by Northern blot hybridization of 300-bp S100A4 cDNA probe to 20 µg of total RNA extracted from both cell lines (upper panel). The actual RNA loading was monitored by ethidium bromide staining of RNA loaded for Northern analysis (lower panel). B, Eight different genes were selected for real-time RT-PCR analysis to confirm the microarray data of differentially expressed genes between the two cell lines. Total RNA was isolated from ARO and ARO-met2. After RT, real-time PCR amplification was performed using primer pair specific for each gene (Table 2Go). Data are expressed as fold of changes of three separate experiments (mean ± SEM) between ARO and ARO-met2 after normalization to that of GAPDH (ratio of S100A4 to GAPDH). C, RT-PCR products of nine different genes (GAPDH serves as an internal control) were shown on the 1.5% agarose gel.

 

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  		TABLE 2. Primers used to amplify cDNA fragments of eight different genes

 
Analysis of thyroid tumor specimens for S100A4 gene expression by quantitative real-time RT-PCR and Northern blot analysis

Given that S100A4 was highly expressed in ARO/met2, we wondered whether its expression was associated with aggressiveness of thyroid cancer. Therefore, we analyzed 49 thyroid tumor specimens by real-time RT-PCR for S100A4 gene expression (eight benign goiters, 36 papillary, and five anaplastic carcinomas). As shown in Fig. 5Go, the average S100A4 level was 0.17 ± 0.09 in goiters (n = 8), 0.26 ± 0.08 in stage 1 carcinomas (n = 8), 1.73 ± 0.64 (n = 9) in stage 2 carcinomas, 3.35 ± 0.82 (n = 14) in stage 3 carcinomas, 3.76 ± 0.75 (n = 5) in stage 4 carcinomas, and 5.42 ± 1.38 (n = 5) in anaplastic carcinomas. Clearly with disease progression, S100A4 mRNA increased gradually. The highest level of S100A4 expression was seen among advanced stages of thyroid cancer (stages 3 and 4 and anaplastic carcinoma). None of the goiters and stage 1 carcinomas were found to have S100A4 overexpression. The difference in S100A4 expression between stages 1 and 2 and stages 2 and 3 was statistically significant (unpaired Student’s t test, P < 0.01). There is no significant difference in S100A4 expression among advanced stages of cancers (stage 3, 4, and AC). We also performed Northern blot analysis of 25 thyroid tumor specimens with different disease stages (Fig. 5BGo), and the results are consistent with real-time RT-PCR data. Furthermore, we compared S100A4 expression from six patients with matched tissues of normal thyroid, PTC, and its lymph node metastasis by real-time RT-PCR. As shown in Fig. 6Go, significantly higher S100A4 expression was seen in metastatic tumors compared with primary tumors (P < 0.01) even though S100A4 overexpression was present in primary tumors when compared with normal thyroid tissue (P < 0.01). These data suggest that S100A4 overexpression was present in advanced stages of thyroid carcinoma and lymph node metastases and was, thus, related to poor prognosis.



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  		FIG. 5. Comparison of S100A4 expression among different stages of thyroid tumors. A, S100A4 mRNA levels were quantitated by real-time RT-PCR, and the housekeeping gene GAPDH was served as an internal RNA control. Data were expressed as fold of changes in S100A4 expression among different tumor stages after normalization to that of GAPDH expression in each sample (ratio of S100A4 to GAPDH). A column scatter graph was used to show each data point in a group, with a line at the mean. Goiter, benign multinodular hyperplasia; PC-1, -2, -3, and -4, differentiated thyroid cancer stages 1, 2, 3, and 4, respectively. AC, Anaplastic thyroid cancer. B, Representative Northern blot analysis of S100A4 expression among different stages of thyroid tumor. Twenty micrograms of total RNA was extracted from tumor samples and hybridized with 300-bp S100A4 cDNA probe (upper panel). The actual RNA loading was monitored by ethidium bromide staining of RNA loaded for Northern analysis (lower panel). Goiter, 3 and 11; PC-1, 1, 7, and 8; PC-2, 4, 5, 12, and 15; PC-3, 2 and 9; PC-4, 10 and 14; and AC, 6 and 13.

 


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  		FIG. 6. Comparison of S100A4 expression in tissues from normal thyroid, PTC, and PTC lymph node metastasis. Total RNAs were isolated from six patients with matched tissues of normal thyroid, PTC, and its lymph node metastasis. S100A4 mRNA levels among different tissues were determined by quantitative real-time RT-PCR. The fold of changes in S100A4 expression among six matched tissue samples (means ± SEM of three separate experiments) was shown after normalization to that of GAPDH expression.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
There are two well-recognized approaches to establish tumor metastasis in mice: one is through tail vein injection of tumor cells that cause experimental metastasis, the other is through sc injection of tumor cells resulting in spontaneous metastasis. Although iv injection model offers more rapid development of metastasis, the spontaneous metastasis model offers better selection of metastatic cells. The tumor cells have to go through invasion process twice: one at the injection site, the other one at the distant site (metastatic site). Apparently, for tumor cells to colonize in the lung, they must have the ability to break through the basement membrane of blood vessels and invade into the extracellular matrix to establish metastasis. The disadvantage of this approach is that it takes longer time for metastasis to develop, and the sc tumor burden is sometimes too large (as in our case) to allow the metastasis to develop due to ethical consideration. Because control ARO cells were in culture for the same period of time as the injected ARO cell line before metastasis experiment, the potential for a component of in vitro rather than in vivo selection would be minimal. The gene expression profiling was performed comparing ARO and ARO-met2, which derived from ARO cell line with high metastatic potential. The disadvantage of this approach is that contributions to metastasis by stromal cells and transcripts induced by the interaction of tumor cells with their environment are lost. It has been suggested that cancer invasion and metastasis is a product of tumor-host microenvironment, and host contributes important molecules to promote tumor growth and metastasis (3, 4). However, this approach is outweighed by numerous advantages. Tumor cell lines often exhibit a reproducible metastatic phenotype that can be retested at any stage of the analysis. Moreover, the high degree of homology in ARO and ARO-met2 would make data analysis easier, whereas contamination with normal thyroid tissue in primary tumor material could lead to misleading conclusions.

Several metastasis-related genes have been identified, and most of them are involved in the cell adhesion, motility, and cytoskeleton. The involvement of integrins in thyroid cancer metastasis is not surprising given their roles in cell invasion and migration (14). However, there are some other interesting members such as cadherin 3 (P-cadherin), S100A4, motility-related protein-1, ezrin, and NEDD5, whose overexpression has not previously been reported in thyroid carcinoma. For example, cadherins are transmembrane Ca2+-dependent homophilic adhesion receptors that include E-, N-, and P-cadherin and have important roles in cell recognition and cell-cell adhesion (15). Cadherins are localized in specialized cell-cell adhesion site called adherence junctions. At these sites, cadherins establish linkages with the actin-containing cytoskeleton. It has been found that increased P-cadherin expression is associated with progression to late stage of ovarian cancer (16). Ezrin functions as a protein-tyrosine kinase substrate in microvilli and is a member of the ERM and merlin family of genes (17, 18). This cytoplasmic peripheral membrane protein serves as an intermediate between the plasma membrane and the actin cytoskeleton. It promotes cell motility by linking the actin cytoskeleton to the plasma membrane through the membrane-spanning ECM receptor CD44 and plays a key role in cell surface structure, adhesion, migration, and organization (17, 18). The protein is also a downstream effector of the RHO kinase signaling pathway (19, 20) and frequently overexpressed in metastatic tumor cells (21, 22). The importance of the RhoC, the Rho/Rho kinase (ROCK), and myosin light-chain kinase signaling pathways in cell mobility, actin cytoskeleton, and metastasis has been demonstrated recently (20, 21, 22, 23). Overexpression or activation of the RAC, Rho/Rho-associated kinase (ROCK), or myosin light-chain kinase signaling pathways has been correlated with tumor invasion and metastasis (23, 24, 25, 26).

Cancer cell motility involves integrin signaling, focal contact formation, cell-cell adhesion through cadherins and other adhesion receptors, and actomyosin-dependent contractility. The importance of motility during metastasis has been reviewed recently (26, 27). In our study, we have identified overexpression of many genes involved in the cell adhesion, motility, and cytoskeleton in ARO-met2 cells, indicating their involvement in thyroid cancer metastasis.

It is known that matrix metalloproteinases (MMPs) play a critical role in tumor cell invasion and metastasis (28). Interestingly, the mRNA level of MMPs such as MMP-2 and MMP-9 was not increased significantly in ARO-met2, suggesting that their expression in both parental and metastatic tumors may be sufficient to allow the tumor cells to metastasize.

S100A4 (also called mts1, p9Ka, calvasculin, CAPL, and pEL98) is a member of a family of at least 19 S100 calcium binding proteins that all have in common a functional EF-hand domain [the EF hand motif has two cooperatively interacting domains, each of which consists of two perpendicularly placed {alpha}-helices (termed E and F) connected by a Ca2+ binding loop] that mediates their activity (9). The S100A4 gene was originally cloned by differential screening experiments in which cDNAs were compared between cells before and after growth stimulation or transformation by oncogenes (29, 30, 31) as well as gene isolation from metastatic tumor cell lines (32). S100A4 has been reported to promote metastasis and is overexpressed in many types of cancer with aggressive phenotype (33, 34, 35, 36, 37, 38). In our present study, we have shown increased S100A4 expression in ARO-met2 cells, thyroid carcinoma specimens with advanced disease stage, and their lymph node metastases, indicating that S100A4 is involved in thyroid cancer progression and metastasis. It may be a useful prognostic marker for thyroid carcinoma.

Several groups have investigated gene expression profiles in thyroid tumors (39, 40, 41, 42). They have shown distinct gene expression patterns among follicular adenoma, follicular carcinoma, and papillary carcinoma. Gene expression profiles involving follicular thyroid cancer metastasis have been reported by two groups (43, 44). The paper by Chen et al. (43) investigated gene expression profile of metastasis tissue and primary tumor from a patient with follicular thyroid cancer. Except for CD 9, which is overexpressed in both metastatic thyroid tissue and ARO-met2, the up-regulated genes reported in the metastatic follicular cancer were not found in our study, suggesting that different mechanisms may be involved in anaplastic thyroid cancer metastasis. S100A4 overexpression could not be evaluated in their study because S100A4 gene was not present in their array used, which contained only 588 genes. We are not sure whether S100A4 gene was included in the array used by Ying et al. (44) where they studied gene expression profile in a mouse model of follicular thyroid carcinoma during tumor progression. Again, the metastasis/invasion-related genes identified were different from those of our current study. The overall gene expression patterns involving thyroid cancer metastasis are also quite different from those reported in thyroid carcinogenesis (39, 40, 41, 42), supporting the concept that different mechanisms are involved in those processes. Interestingly, Met protooncogene was found to be overexpressed in both follicular and PTCs compared with adenoma or normal thyroid tissue (39, 40). It also overexpressed in highly metastatic thyroid carcinoma as shown in our study. Considering Met product is a tyrosine-kinase receptor for hepatocyte growth factor and activation of this receptor by hepatocyte growth factor can induce proliferation, motility, adhesion, and invasion in tumor cells, it is likely that Met is not only involved in the thyroid carcinogenesis but thyroid cancer metastasis as well (45, 46, 47, 48, 49).

In summary, we have successfully established a thyroid carcinoma cell line with high metastatic potential (ARO-met2) from its parental cell line ARO. Microarray analysis of gene expression profile between these two cell lines reveals a distinct class of genes involved in tumor metastasis that have not been observed by previous microarray analysis on thyroid tumorigenesis. Many of the genes identified are involve in cell adhesion and motility. Further analysis of S100A4 expression in thyroid tumor specimens demonstrated that S100A4 overexpression was associated with disease progression and may be a useful prognostic marker for thyroid carcinoma. Given that S100A4 is involved in tumor progression and metastasis, it may also be a potential target for therapeutic intervention.


   Acknowledgments
 
We thank Ms. Soad Saleh for excellent technical support for real-time RT-PCR and Drs. Shaheen Nakeeb and Raafat M. El-Sayed for assistance in maintaining animals.


   Footnotes
 
Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase; PTC, papillary thyroid carcinoma; RT, reverse transcriptase.

Received March 2, 2004.

Accepted August 30, 2004.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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